Poster Session 1
Thursday, 11 April 2019
PS01: Potential role of long noncoding RNA ASO3491 and E2F2 in spermatogenesis of non-obstructive azoospermia
Yun Xing Fu; Rong Hu
Background: To explore the expression characteristics of long noncoding RNA (lncRNA) and mRNA in human testicular tissue of non-obstructive azoospermia (NOA) patients and confirm the potential role of lncRNA ASO3491 and E2F2 in spermatogenesis of NOA.
Methods: Testicular tissue samples from patients with NOA (n=3), the control (n=3) and obstructive azoospermia (n=3) were collected and the lncRNA and mRNA expression profile was assessed by means of microarray. Candidate lncRNAs and mRNAs were chosen for further validation with real-time quantitative reverse-transcription polymerase chain reaction. We predicted the target genes of significant differentially expressed lncRNAs and then performed gene ontology and pathway analysis. Finally, the levers of serum E2F2 and seminal plasma E2F2 were detected from patients with NOA (n=40), the normal control (n=40) and the patients with OA (n=35) by ELISA.
Results: lncRNAs (ASO3491,ENSG00000262188.1 and XLOC_010514) and mRNAs (ENST00000428216,ENST00000392112,ENST00000361729, ENST00000262848,ENST00000392038,ENST00000376726) were expressed unusually in testicular tissue of NOA.
qRT-PCR were similar to those found by means of microarray analysis.
ASO3491 down-regulated the expression of E2F2 in (http://www.targetscan.org/vert_60/) and DIANA LncBaseV2 (https://www.lncrna2target.org/index.jsp).
The microarray profiling and qRT-PCR outcome indicated the expression of ASO3491 was statistically significant increase and E2F2 was statistically significant decrease compared the control group and OA group.
The levers of E2F2 were no statistically significant difference in serum from all groups. The levers of E2F2 in seminal plasma were no statistically significant difference in the OA group compared NOA group and the normal. There were statistically significant decrease in the NOA group compared the normal group.
Conclusion: ASO3491 suppressed E2F2 in spermatogenesis of non-obstructive azoospermia. This breakthrough will provid a new biomarker for the NOA and a new therapy for infertility of NOA.
Funding: National Natural Science Foundation of China (No. 81660257), the Ningxia Natural Science Foundation(NZ16125), 2016 Autonomous Region Leader in Science and Technology Innovation. First-Class Discipline Construction Founded Project of NingXia Medical University and the School of Cincical Medicine (NXYLXK2017A05).
PS02: Effect of selective estrogen receptor modulator (SERM) and tribulus Terrestris L. in an azoospermic male with hyperprolactinemia after treatment with dopamine agonist
Tiara Kirana, Mom and Child Care Clinic; Edwin Pradana
Background: A case of fertility success after combination treatment of a Selective Estrogen Receptor Modulator (Clomiphene Citrate) and Tribulus Terrestris L. (Vitan) in an azoospermic male with hyperprolactinemia after already treated with dopamine agonist (Bromocriptine).
Methods: Case Report.
Results: A 32-year-old man presented with 5 years primary male infertility and sexual dysfunction. Semen analysis with centrifugation showed azoospermia result. Morning blood test result showed Total Testosterone (TT) 275 ng/dL (249-836 ng/dL, in men 20-49 y.o), Prolactin 54.2 ng/mL (2.1-17.7 ng/mL, in men >18y.o), FSH 2.4 mIU/mL (1.4-18.1 mIU/mL, in men 18-70 y.o). Erection Hardness Score (EHS) 1-2 and no morning erection.
Treatment with 1.25 mg of a dopamine agonist for 30 days, decreased the level of Prolactin to 13.7 ng/mL. An improved sexual function with EHS 2-3. However, the level of TT only slightly increased to 384.4 ng/dL. The dopamine agonist then was stopped and switched to SERM 25 mg, daily for 3 weeks followed by a week of rest, and Tribulus Terrestris L. 250 mg twice daily for 3 months. Semen analysis revealed sperm concentration of 83 million per milliliter, sperm motility A 90% and 2% normal morphology. The morning blood test result showed TT 624.9 ng/dL, Estradiol 27 pg/mL. Sexual function improved significantly with EHS 4 and routine morning erection. All treatment then discontinued.
One month after the treatment stopped, without any intervention in the female partner, naturally conceive achieved.
Conclusion: Azoospermia with sexual dysfunction is typical in men with hyperprolactinemia. However, even though the prolactin already in normal level with treatment, the TT level did not elevate significantly. Therefore, an intervention with SERM combined with Tribulus Terrestris L. may have the effect to increase intratesticular testosterone and spermatogenesis also maintain the balance ratio of testosterone-estradiol.
PS03: Antisperm antibody positivity and clinical correlation in couples attending infertility clinic at Ahmadu Bello University teaching hospital, Zaria Nigeria
Iloabuchi Izuka, Ahmadu Bello University Teaching Hospital, Zaria Nigeria; Adebiyi Adesiyun; Solomon Avidime; Bop Musa; Umma Bawa
Background: Presence of antisperm antibody in either the male or the female, or both is linked to infertility. Test for antisperm antibody is not routine in the evaluation of infertile couples in most low resource countries like Nigeria. Making a decisive aetiologic diagnosis of infertility and proffer appropriate treatment is paramount in the management of childlessnes. This study determined the correlation of ASA positivity and clinical characteristics of couples with infertility.
Methods: A cross-sectional study. Serum and tissue fluid (semen and cervical mucus) were tested for ASA using ELISA method. Socio-demographic characteristics of infertile couples, type of infertility, duration of infertility and risk factors to developing ASA were sought for in the males and females member of couples attending infertility clinic.
Results: Of the 104 males and 108 females that had ASA analysed, 56 out of the total of 212 had ASA positivity. There was an association between ASA positivity and younger female age. Age of male, ethnicity, religion and educational level and type of infertility (primary, secondary) did not show significant association. There was linear relationship between duration of infertility and ASA positivity; with increased duration of infertility ASA positivity decreased (linear by linear association value was significant at 0.020). In females, of all the risk factors, coitus during menstruation will have a significant association at a lower confidence interval (CI) of 90%, P-value=0.1. In male, there was significant association of ASA positivity and smoking of all the risk factors tested for.
Conclusion: Younger female age, smoking and at a lower CI coitus during menses showed significant relationship with ASA positivity.
Funding: This research was privately funded.
PS04: Comparative study of peroxidase test and leishman stain in the diagnosis of leucocytospermia at a tertiary hospital Northwest Nigeria
Hauwa Shuaib Umar, Ahmadu Bello University Teaching Hospital, Zaria Nigeria; Adebiyi Adesiyun; Saad Ahmad; Umma Bawa
Background: Genital tract infection is the commonest cause of male and female infertility in sub-Saharan Africa. Peroxidase test as recommended by WHO in diagnosing leucocytospermia is largely not available and affordable in our setting. Leishman test is an alternative diagnostic method although with less precision. This study compared the diagnosis of leucocytospermia using the peroxidase test and Leishman test.
Methods: A cross-sectional study. Semen analysis including peroxidase test and Leishman test was done on all semen samples.
Patients were spouse of women attending infertility clinic. There was no intervention in this study. Main outcome measure was percentage of leucocytospermia diagnosed by peroxidase and leishman test in semen with significant round cell count
Results: Of the 136 semen, 47 (35%) semen was diagnosed with leucocytospermia by peroxidase test and 19 (14) by Leishman test. Semen of 42 (31%) clients had significant round cell count and 94 (69%) had non-significant round cell count. Out of the 42 with significant round cells, 39 (83%) were found to be leucocytospermic by peroxidase stain, while 11 (57.9%) were leucocytospermic by the Leishman stain and both were statistically significant (P<0.05).
Conclusion: Peroxidase test is more accurate than Leishman test in the diagnosis of leucocytospermia, although Leishman test may serve as a suitable alternative in resource constrained settings.
Funding: This study was privately funded.
PS05: Comparison of pregnancy outcomes of fresh or frozen sperm from patients of different types of azoospermia
Meng Dong, &#; Jichun Tan
Background: To investigate the effects of fresh and frozen sperm on pregnancy outcome in patients with different types of azoospermia (OA or NOA).
Methods: A retrospective investigation was made based on 1628 azoospermic patients who underwent ICSI cycles in Shengjing Hospital Affiliated to China Medical University from January 2010 to October 2017. According to testicular spermatogenesis, sperm acquisition and storage methods, patients were are divided into six groups, A1: OA, PESA, fresh sperm, 582 cycles; A2: OA, PESA, frozen sperm, 234 cycles; B1: OA, TESA, fresh sperm, 225 cycles B2: OA, TESA, frozen sperm, 126 cycles; C1: NOA, TESA, fresh sperm, 176 cycles; C2: NOA, TESA, frozen sperm, 285 cycles. The laboratory results and pregnancy outcomes of each group were compared.
Results: There were no significant differences in fertilization rate, cleavage rate and high quality embryo rate among the six groups. The clinical pregnancy rates of in each group were 40.38%, 43.59%, 40.00%, 39.68%, 31.82%, and 28.07%, respectively. The clinical pregnancy rates in A1, A2, B1, and B2 were significantly higher than that in C2 (P<0.05). They are higher in A1 and A2 than that in C1 (P<0.05). The implanting rates in A1, A2, B1, B2, C1, C2 were 30.70±21.17, 31.91±21.94, 30.03±20.45, 29.73±20.65, 22.09±18.37, 19.89±16.95, A1, A2, respectively. The implantation rates in A1, A2, B1 and B2 were significantly higher than those in C1 and C2 (P<0.05). The live birth rates in A1, A2, B1, B2, C1 and C2 were 34.88%, 36.32%, 33.78%, 31.75%, 25.57%, 22.81%, respectively. The live birth rates in A1 and A2 were significantly higher than that in C1. Those in A1, A2 and B1 were also significantly higher than that in C2 (P<0.05). There were no significant differences in biochemical pregnancy rate, embryonic diapaus rate, ectopic pregnancy rate, abortion rate, stillbirth rate and fetal malformation rate among the six groups (P>0.05).
Conclusion: The fresh or frozen sperm from patients of different types of azoospermia did not affect the fertilization rate and embryo development potential, but could affect the pregnancy outcome.
PS06: Salvage microsurgical varicocelectomy for grade 1 varicocele has a potential to be an effecive treatment choice
Shinnosuke Kuroda, Department of Urology, Reproduction Center, Yokohama City University Medical Center; Kimitsugu Usui; Kohei Mori; Kengo Yasuda; Takuo Asai; Teppei Takeshima; Takashi Kawahara; Yasuhide Miyoshi; Yasushi Yumura
Background: It is controversial that varicocelectomy should be performed to low grade varicocele. However, depending on the semen analysis, clinical courses such as lack of improvement of semen analysis by taking medication, repetitive failure of assisted reproducive technology, we present varicocelectomy as a treatment choice for the patients with grade 1 varicocele. The aim of this study is to evaluate the efficacy of varicocelectomy for the patents with grade 1 varicocele.
Methods: We retrospectively analyzed the male infertility patients with grade 1 varicocele who visited Reproduction Center in Yokohama City University Medical Center between April 2013 and July 2017, and could be followed up more than 6 months after the treatment. The patients were divided in two groups; the patients treated by only oral antioxidant and Chinese medicine, and the patients who underwent microsurgical subinguinal varicocelectomy with oral medication. The groups were compared with respect to the patients’ characteristics, the pre- and post- treatment semen parameters using univariate analysis.
Results: Twenty four patients kept taking medication, and 14 patients underwent surgery with medication. The observation period was significantly longer in surgery group (478.4 d vs. 255.8 d, P<0.01). Six months after the start of treatment, the motile sperm concentration were 4.939±6.257 ×106/mL in medication group and 9.853±16.8 ×106/mL in surgery group, respectively (P=0.26). The amount of change in the motile sperm concentration was significantly higher in surgery group (−0.94±5.82×10^6/mL vs. 4.33±5.46×10^6/mL, P=0.029). The wives of 7 patients in medication group (29.2%) and 7 patients in surgery group (50%) became pregnant during the observation period.
Conclusion: Six months after the treatment, motile perm concentration was significantly improved in surgery group than in medication group. Microsurgical varicocelectomy is considered as an effective choice for the patients with grade 1 varicocele especially after the failure of oral medication.
PS07: Gut microbiome by metagenomic sequencing in patients with chronic prostate syndrome
Ming Liao, Guangxi Medical University; Yuanliang Xie; Yang Chen; Zengnan Mo
Background: The Chronic Prostate Syndrome (CPS) was characterized by its unknown etiology, complicated phenotypes and dissatisfactory treatment. Identification of its characteristic gut microbiome shed new light on its diagnostic or treatment strategies.
Methods: A total of 68 participants from the first affiliated hospital of Guangxi Medical University were selected, including 34 healthy volunteers from routine physical examination and 34 patients from department of reproductive center and urology clinics. The Chronic Prostatitis Symptom Index and International Prostate Symptom Score (IPSS) were applied. The gut microbiome were sequenced by metagenomic technique, and the MetaPhlAn v2.0 was used for phylogenetic analysis, HUMAnN2 for metabolic analysis, the pamk function for optimal clustering.
Results: The CPS patients presented higher IPSS scores than the healthy volunteers (P<0.001). The phylogenetic analysis of 319 species showed the relative abundance of Escherichia_coli was significantly higher in CPS patients (P=0.013), and its mean abundance reached 1.2%, almost 10 times greater than the healthy volunteers. The metabolic analysis of 233 KEGG pathways showed that gut microbiome in the CPS patients involved a distinctive pattern of metabolic pathways: the pathways of higher abundance included drug metabolism, clavulanic acid biosynthesis, Fc gamma R-mediated phagocytosis; while the pathways of lower abundance covered protein export, RNA degradation, oxidative phosphorylation. The optimal clustering showed a small part of CPS patients presented similar pattern of metabolic pathways, but relatively higher abundance of Escherichia_coli, indicating the pain symptoms in these patients were probably related to gut microbiome rather than its metabolic pathways.
Conclusion: The gut microbiome in CPS patients presented characteristic pattern of metabolic pathways. The metagenomic sequencing of gut microbiome was potentially applicable for precisely selecting the CPS patients for satisfactory treatment.
Funding: This research was supported in part by grants from the National Natural Science Foundation of China (#81272853, #81472414, and #81370857), Guangxi scientific research and technology development project (Guikegong 2013BC26299 and 1355005-3-17), and the Guangxi Natural Science Foundation (2015GXNSFBB139008, 2014GXNSFBA118201, and 2013GXNSFFA019002).
PS08: Expression profile analysis in testis reveal the critical roles of long non-coding RNAs as molecular biomarkers for non-obstructive azoospermia
Xuan Zhou, Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanfang Hospital/The First School of Clinical Medicine, Southern Medical University; Song Quan; Xuan Zhou; Tong Zhu
Background: Non-obstructive azoospermia (NOA) is one of the most severe male-related reproductive disorders, which results from spermatogenic failure. Increasing evidence indicates that long non-coding RNAs (lncRNAs) play pivotal roles in spermatogenesis, however, little is known about the function of lncRNAs in the underlying etiology of NOA which are not systematically understood.
Methods: In this study, the lncRNA and mRNA profiles in the testis of NOA and controls with normal spermatogenesis were generated by microarray analysis. The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to reveal the potential functions of disease-related genes. In addition, the lncRNA ande mRNA co-expression networks were constructed to investigate the relationship between the highly conserved lncRNA and NOA-associated genes for further functional analysis of lncRNAs. Finally, quantitative real-time polymerase chain reaction (qPCR) were used to validate the results of microarray.
Results: Our data showed that thousands of lncRNAs and hundreds of mRNAs that were differentially expressed (≥2.0-fold-change, P< 0.05) among NOA compared with controls. Several GO terms including spermatogenesis, male gamete generation, cell cycle, were enriched in gene lists, suggesting a potential correlation with NOA. Pathway analysis demonstrated that apoptosis pathway, lysosome and phagosome pathway play important roles in spermatogenesis. Co-expression network analysis indicated that 94 highly conserved lncRNAs and 158 mRNAs were included in the co-expression network. Finally, three candidate lncRNAs were verified by qPCR and indentified for further study.
Conclusion: The results indicate that these significanty altered lncRNAs may lead to impaired spermatogenesis, paving the way for deciphering the pathogenesis of NOA. Furthemore, our findings provided clues for further diagnosis and investigation of male-related reproductive disorders.
PS09: Protective effects of Prdx4 on Spermatogenesis by endoplasmic reticulum stress
Weiwei Ma, Nanjing Medical University; Yan Meng; Zhengjie Yan; Mengting Hu; Jiayin Liu; Yugui Cui
Background: Oxidative stress is one of the main causes of the functional disorder of testis and male infertility. Prdx4 located in endoplasmic reticulum, as a member of the Peroxiredoxins, play an essential role in the redox balance and oxidative folding. We hypothesized that two kinds of Prdx4 play protective effects on spermatogenesis in testis by suppressing oxidative stress. This study was designed to investigate the possible effect of Prdx4 on spermatogenesis using the Prdx4-KO mice.
Methods: Mice were divided into Prdx4-KO and control groups, and sacrificed in young and middle age, respectively. Prdx4-gene knockout using CRISPR/Cas9 technology in mice. The testicles/body weight were compared. The apoptosis and active caspase 3 were tested. Subcellular structures were observed by electron microscopy. The oxidative stress level in testis was measured by Immunohistochemistry. The levels of endoplasmic reticulum stress level were quantified by real-time PCR.
Results: The apoptosis index was significantly increased in Prdx4-KO mice. In mice aged 8 weeks, Prdx4-KO mice had the decreased testicles/body weight (P<0.05), the increased oxidative stress level, and the endoplasmic reticulum stress level (all P<0.05). Under the electron microscope, spermatogenic cells in the Prdx4-KO mice showed the swelling phenomena in nucleus, and chromatin towards agglutinated. In mice aged 9 months, Prdx4-KO mice had the decreased testicles/body weight (P<0.05) and epididymal sperm count (both P<0.05). Besides above phenomena, the residual body was increased in Sertoli cells. The swelling and expansion of endoplasmic reticulum and the mitochondrial lysis were also found in Leydig cells.
Conclusion: Prdx4 knockout mice developed testis atrophy and the declined epididymal sperm count, which suggested the protective effects of Prdx4 by the inhibition of the endoplasmic reticulum stress.
PS10: CHK1 regulates sperm motility via modulating intracelluar Ca2+concentration
Guangxin Yao, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai; Xiaobin Zhu; Yuchuan Zhou; Aijun Zhang; Yonglian Zhang
Background: Good sperm motility is essential for normal male fertility and a large proportion of infertile males suffer from problems with sperm motility. Demonstration of the mechanisms that regulate sperm motility will be of great benefit for the treatment of this group of patients. Since sperm is virtually devoid of transcription and translation due to highly condensed genomic DNA and lack of endoplasmic reticulum, gene expression accounts little for sperm motility regulation. Instead, signaling pathways and protein posttranslational modifications must play a central role in this process. Signaling transduction is mediated via the sequential phosphorylation of target protein by various corresponding kinases. Thus, screening the key kinases governing sperm motility by specific inhibitors will help us to uncover new target to improve semen quality and develop male contraceptive medicine.
Methods: Sixty inhibitors were selected from Selleck company’s Protein Kinase Inhibitor Library and incubated with sperm in a final concentration of 10μM, respectively. Sperm motility was monitored by computer-aided sperm analysis.
Results: Three chemicals from the Protein Kinase Inhibitor Library significantly inhibit both mouse and human sperm motility, but the effect of only one inhibitor targeting CHK1 can be validated by other inhibitors targeting the same kinase, which indicates that CHK1 was the bona fide key kinase for sperm motility. Further study demonstrates that all of the four different inhibitors targeting CHK1 can inhibit sperm motility in a time and concentration dependent manner. Western blot and indirect fluorescent immunostaining study imply that CHK1 exists in sperm and localizes in the redundant nuclear envelop, which is known as one of the main calcium stores in sperm. Monitoring the intracellular calcium concentration in sperm reveals that the Ca2+concentration significantly reduced when CHK1 activity was inhibited.
Conclusion: Our results indicate that CHK1, a nuclear protein and an essential component of the DNA damage checkpoint pathway, retains in the redundant nuclear envelop during spermatogenesis and regulates sperm motility via modulating the intracellular Ca2+concentration.
Funding: This research was funded by “Open Project Fund from Key Laboratory of Reproduction Regulation of NHC” (No. KF2018-04).
PS11: Comparison of outcome of patients with AZFc microdeletions and non-obstructive Azoospermia (NOA) undergoing ICSI-ET with ejaculated versus testicular sperms
Li Zhang, Medical Center for Human Reproduction, Dept. of Ob/Gyn, Peking University Third Hospital; Jiaming Mao; Li Xue; Ping Liu
Background: We performed a retrospective cohort study to investigate the effect of sperm retrieval (ejaculated vs. testicular sperms) on the outcomes of ICS-ET in the treatment of AZFc microdeletions and NOA.
Methods: We retrospectively analyzed the outcoms of 293 patients with AZFc microdeletions and NOA who underwent 449 cycles between September 2015 to September 2018. Inclusion criteria: (1) Patients with AZFc microdeletion and NOA; (2) Couples have been treated with ICSI. Exclusion criterion: (1) Wives were or older than 35 years and infertility caused by females; (2) Patients with Klinefelter syndrome, orchitis, cryptorchidism and chromosomal abnormalities other than AZFc microdeletions. Patients were divided into four groups: group A, testicular sperms for ICSI in patients with AZFc microdeletions; group B, ejaculated sperms for ICSI in patients with AZFc microdeletions; group C, testicular sperms for ICSI in NOA patients; group D, ejaculated sperms for ICSI in NOA patients.
Parameters were the fertilization rate (FR), 2PN cleavage rate (2PNCR), blastocyst formation rate (BFR), quality embryo rate (QER), implantation rate (IR), pregnancy rate (PR), live-birth rate (LBR), miscarriage rate (MR), cumulative PR (CPR), cumulative LBR (CLBR) and cumulative MR (CMR).
Results: The FR and BFR were significantly higher in group B compared with group A (57.04 vs. 29.77 and 39.51 vs. 21.37%). The 2PNCR and QER were higher in group A compared with group B (98.95 vs. 96.6 and 71.78 vs. 36.63%). The FR and QER were higher a little in group D compared with group C (57.8 vs. 52.55 and 38.16 vs. 38.07%). Four groups had the simliar outcomes in fresh cycles. In frozen-thawed cycles, the IR, PR and LBR were higher in group B and C compared with group A and D (38 and 31.5 vs. 7.41 and 21.67, 50 and 45.33 vs. 11.76 and 30.77, 38.89 and 25.33 vs. 0 and 7.67%, respectively). The CPR and CLBR were significantly higher in group B and C compared with group A (56.9 and 61.83 vs. 27.03, 43.1 and 36.64 vs. 14.86%, respectively).
Conclusion: Comparing testicular sperms, patients with AZFc microdeletions using ejaculated sperms can improve the outcomes in frozen-thawed cycles, while NOA patients with testicular sperms had a batter outcome in frozen-thawed cycles. Above results suggest that ejaculated sperms is a more optimal treatment for patients with AZFc microdeletions while NOA patients with testicular sperms could be a better choice to get better outcome after ICSI-ET.
PS12: Relationship between humanin levels in human seminal plasma and sperm and sperm quality
Meng Rao, Department of Reproduction and Genetics, The First Affiliated Hospital of Kunming Medical University; Ya Wen; Shuhua Zhao; Li Tang
Background: Humanin has reportedly been expressed in testis and sperm, but no study has yet reported its presence in human seminal plasma (SP). The aim of this study was to investigate the presence of humanin in human SP, and to determine the correlation between humanin levels in SP/sperm and sperm quality.
Methods: Semen samples for SP/sperm humanin level measurement were collected from 185 patients who attended our andrology clinic for fertility evaluation. The localization of humanin in sperm was evaluated using an immunofluorescence method and SP/sperm humanin levels were measured with ELISA. Correlations between SP/sperm humanin levels and sperm parameters were analyzed.
Results: Humanin was expressed in the midpiece of the sperm. Humanin concentrations in the SP ranged from 13.6 to 285.1 pg/mL, with a median of 98.11 pg/mL. The SP humanin concentration in normospermia was significantly higher than that in oligospermia (P<0.001), asthenospermia (P=0.007) and oligoasthenospermia (P<0.001). Spearman analysis showed a positive and significant correlation between SP humanin concentration and sperm concentration (r=0.70, P<0.001), progressive sperm motility (r=0.32, P<0.001) and rate of normal sperm morphology (r=0.34, P<0.001). Sperm humanin level was significantly and positively associated with progressive sperm motility (r=0.71, P<0.001). In addition, a significantly lower level of humanin was found in progressive sperm than in non-progressive/immotile sperm (P=0.03).
Conclusion: SP and sperm humanin levels were significantly and positively correlated with sperm quality, especially sperm motility. Further studies of the origin of SP humanin and its role in spermatogenesis should be conducted.
Funding: This study was supported by the National Natural Science Foundation of China (No. 81801416), the Technology & Innovation Team of Reproduction and Genetics from Kunming Medical University (No. CXTD201708), and the Scientific Funding from the First Affiliated Hospital of Kunming Medical University (No. 2017BS008).
PS13: Correlation of parameters of unliquefiable semen with sperm DNA fragmentation
Minglian Zhou, Reproductive Medical Center, Lianyungang Maternal and Child Health Care Hospital Lianyungang; Linqing Pan
Background: To analyze the correlation of parameters of unliquefiable semen with sperm DNA fragmentation index (DFI) in infertile males and to explore the clinical significance of DFI in assessing male infertility.
Methods: Seventy patients with unliquefiable semen and 40 fertile males’ semen were collected. According to WHO standard method, those semen were measured with traditional semen examination, morphology analysis and sperm chromatin dispersion test (SCD) and analyzed the relationship between sperm concentration, forward motile sperm, motility, normal morphology and DFI.
Results: The sperm concentration, forward motile sperm, motility, normal morphology of infertile male were significantly lower than that of fertile male (40.36±15.42×106/mL vs. 79.03±39.61×106/mL, 29.17±6.13% vs. 47.15±7.23%, 7.45±3.03% vs. 22.19±2.06%, 29.17±8.46% vs. 14.36±3.78% respectively, P <0.05), while DFI of infertile male was significantly higher than that of fertile male (30.24±7.59% vs. 15.25±4.82%, P <0.05). The correlation coefficients between DFI and sperm concentration, forward motile sperm, motility, normal morphology were −0.503,−0.683,−0.799, respectively.
Conclusion: The sperm concentration, forward motile sperm, normal morphology of infertile male were decreased compared with control, while DFI was increased. These parameters are also one of the reasons leading to infertility in non-liquefied patients. These parameters may be one reason leading to male infertility.
PS14: DNAH2 is identified as a novel pathogenic gene of multiple morphological abnormalities of the sperm Flagella (MMAF)
Yang Li; Yan-Wei Sha; Xiong Wang; Wen-sheng Liu
Background: Teratozoospermia, also known as teratospermia, is one of the most common phenotypes of male infertility 1,2. It is defined by over 85% of sperm with abnormal morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) is one type of teratozoospermia 1 and characterized by a variety of flagellar defection including absent, short, curled, bent or angulated flagella 3. This sperm flagella abnormal phenotype was previously reported as dysplasia of the fibrous sheath or short/stump tail syndromes and occurs to be higher than 1 per 10,000–20,000 births 4-6. Patients with this phenotype could not spontaneously pregnancy or fertilization in vitro, but high success rate of intracytoplasmic sperm injection could been observed in some individuals with MMAF.
Methods: Whole genome sequencing and Sanger sequencing, Papanicolaou staining, Transmission Electron Microscopy, Western blot analysis, Immunostaining.
Results: By screening the gene variants of 38 MMAF patients using who-exome sequence, we identified five new mutations in the dynein axonemal heavy chain 2 (DNAH2) gene in four individuals, include 2 unrelated cases and 2 brother in single family, and provide a novel genetic pathogeny for human MMAF phenotype. We identified 5 mutations in DNAH2 gene in 4 patients with MMAF from 3 families, including 2 unrelated individuals and 2 brothers. TEM analysis showed severely disarranged axonemal structure with mitochondrial sheath defection. WB and IF analysis showed significantly decreased DNAH2 protein level in patient’s spermatozoa.
Conclusion: DNAH2 may be a novel pathogenic gene of Multiple Morphological Abnormalities of the Sperm Flagella (MMAF).
Funding: The open project of Key Laboratory of Male Reproduction and Genetics, National Health and Family Planning Commission (Grant No. KF201704); the Medicine and Health Science Technology Development Project of Shandong Province [Grant No. 2016WS0704]; Natural Science Foundation of Shandong Province [Grant No. ZR2017LH012]; National Natural Science Foundation of China [Grant No. 31171375, No. 81871200]; the Science Technology Guidance Project of Fujian Province [Grant No. 2017D018]; the Linqiaozhi Funding Supporting Youth Project of Xiamen Maternity and Child Care Hospital [Grant No. FYLQZ2015004].
PS15 - (Chinese language abstract – see Chinese supplement)
Wang Jing, The Zhongda Affiliated Hosopital with Southeast University
PS16: Correlations between sperm head vacuoles and semen quality, sperm DNA fragmentation
Xueyu Li, Guangxi Maternal and Child Health Hospital; Jinhui Shu; Hong Zhou; Ruoyun Lin
Background: This study investigates the correlations between sperm head vacuoles and semen quality, sperm DNA fragmentation.We aim to reveal the potential relationships between sperm head vacuoles and male infertility.
Methods: A total of 174 infertile men were involved in our study, and severe oligoasthenospermiawere excluded. Semen samples were collected bymasturbationafter 2–7 days of sexual abstinence. (1) Semen parameters assay: The semen volume, sperm concentration, motility, vitality, semen liquefaction, and roundcells were measured according to the fifth edition of WHO Laboratory Manual for the Examination and Processing of Human Semen. Sperm Morphology was stained with the Diff-Quik Staining kit for each sample, and the normal sperm morphological rate was counted. (2) Sperm head vacuoles detection: the spermatozoa with more than two small vacuoles inthe acrosomal region, the sperm head vacuoles that was greater than 20% of sperm head orthe spermatozoa with vacuoles in the postacrosomalregion were defined as abnormal head vacuoles. The rate of abnormal head vacuoles was calculated. (3) Detection of sperm DNA fragmentation: The spermatozoa expressed as big halo, middle halo, small halo, halo-free and degenerated was counted by the sperm chromatin dispersion (SCD) test. At least 500 spermatozoon were counted for each sample and the DNA fragmentation index (DFI) was calculated. (4) Statistical analysis: The associations between the abnormal sperm head vacuoles and semen parameters, sperm morphology or DFI were assessed by spearman’s rank correlation coefficient. P<0.05 was regarded as statistically significant.
Results: The abnormal sperm head vacuoles showed a negative correlation withsperm concentration (r=−0.241, P=0.001), sperm progressive motility (r=−0.274, P=0.000) andnormal sperm morphology (r=−0.275, P=0.000). However, the abnormal sperm head vacuoleshad a negative correlation with the rate of DNA fragment (r=0.234, P=0.02) and percentage of motionless sperm (r=0.193, P=0.011). Additionally, there were no correlations between the abnormal sperm head vacuoles and age, abstinence time, semen volume, round cell concentration, nonprogressive sperm motility (all P>0.05).
Conclusion: The increased sperm head vacuoles that accompanied with a decrease of semen quality and an increase of DNA fragmentation may be one of the most important reasons for male fertility.
PS17: Uropathogenic E. coli infection compromises blood-testis barrier by disturbing mTORc1-mTORc2 balance
Yongning Lu, Zhongshan hospital, Fudan University; Miao Liu; Bin Yan; Suying Liu; Xi Dong
Background: Infections and inflammation in the testis is widely accepted as an important etiological factor of male infertility. The blood-testis barrier (BTB) plays a critical role in maintaining immune privilege statue of the testis. Our preliminary study shows that BTB is very likely compromised during bacterial infection in an orchitis model induced by uropathogenic E. coli (UPEC). However, it is still largely unknown about underlying mechanism of BTB impairment caused by bacterial infection. Very recently, it is found that mTOR pathway plays an essential role in regulating the BTB timely “opening” and “closing” in the cycle of seminiferous epithelium. Whether mTOR pathway is also involved in BTB dysfunction following bacterial infection is yet to be elucidated. This study aims to explore the role of mTORc1-mTORc2 balance in UPEC induced orchitis and BTB destruction.
Methods: UPEC induced epididymo-orchitis rats model was established as previous described. For in vitro infection model, Sertoli cells was treated with filtered UPEC culture medium. mTORc1 inhibitor rapamycin was used additionally in an attempt to alleviate cell junctions destruction. Subsequently, BTB structures were evaluated using transmission electron microscopy and biotin assay; anti-sperm antibody in the serum of rat model was detected by ELISA; cell junctions protein expression patterns were demonstrated using immunofluorescent assay and mTOR pathway activation were observed using western blotting.
Results: Firstly, it was found that UPEC virulent factors disturbed Sertoli cell junctions by down-regulation of ß-catenin, Cx-43, F-actin, N-cadherin, Occludin, and ZO-1. BTB was disrupted in UPEC infected rats. Secondly, anti-sperm antibody was only detected in the blood samples from orchitis animals. Furthermore, our results indicated that mTORc1 over-activation and mTORc2 suppression contribute to the disturbance of BTB “open” and “closing” balance. More importantly, using rapamysin, a specific mTORc1 inhibitor, can significantly restore the expression of all cell junctions proteins and seems to have protective effect on BTB following UPEC infection.
Conclusion: Our study demonstrates the connection of mTOR signaling pathway abnormal activation and BTB impairment in UPEC induced epididymo-orchitis. mTORc1 inhibitor rapamycin treatment may be a option to alleviate BTB damage.
Funding: Research was funded by National Natural Science Foundation of China (NSFC) project 81300473.
PS18: The attractive effect of DNA fragmentation index on assisted reproductive treatment outcome: a large sample size and well-controlled retrospective study
Cuncan Deng, Reproductive Medicine Research Center, The Sixth Affliated Hospital, Sun Yat-sen University; Ting-ting Li; Yun Xie; Cai Meihong; Yingchun Guo; Liang Xiaoyan; Guihua Liu
Background: The effect of DNA fragmentation index (DFI) on assisted reproductive treatment (ART) outcome remains controversial.
Methods: We conducted a retrospective analysis of infertile couples undergoing IVF or ICSI treatments at our reproductive center from May 2012 to April 2018. Only infertile couples caused by cervical factors, male factors, pelvic factors, fallopian tube factors or sexual disturbances were included in the study. Inclusive couples are divided into four subgroups according to the age of the female, including the subgroup less than 30 years old, 30 to 35 year old subgroup, 35 to 40 year old subgroup and subgroup more than 40 years old. The sperm DNA fragmentation was evaluated by SCSA assay and the threshold was set at 30%.
Results: 4841 cycles were included, 4117 in low DFI group and 724 in high DFI group. The average age of female and male in high DFI group was slightly higher than low DFI group (P<0.05)in each subgroups. Baseline factors were the same between groups. There was no statistical difference in oocytes number, 2PN fertilization rate, cleavage rate, number of available embryos (P>0.05). There were no significant differences in biochemical pregnancy rate (417/676 VS. 33/61, P>0.05), clinical pregnancy rate (386/412 vs. 32/33, P>0.05), miscarriage rate (48/386 vs. 4/32, P>0.05) between low DFI group and high DFI group. The good quality embryo rate of high DFl group were significantly than low DFl group in couples received both IVF and ICSI in subgroup less than 30 years old (IVF:+0.40 vs. 0.16+0.33, P<0.05; ICSI: 0.05+0.20 vs. 0.16+0.35, P<0.05), the 30 to 35 year old subgroup (IVF: 0.28 vs.+0.35, P<0.05; ICSI: 0.05+0.20 vs. 0.16+0.34, P<0.05).
Conclusion: We concluded that DFI had adverse impacts on embryo quality in both IVF and ICSI but didn’t affect miscarriage rate and clinical pregnancy rate.
PS19: Outcome analysis of ICSI in patients
Yang Xiaoyu, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; Jiayin Liu; Wei Wu
Background: Four phenotypes can be considered as pure teratozoospermia with 100% abnormal sperm and with a constant uniform pattern of anomalies, such as globozoospermia, large-headed multiflagellar spermatozoa or acephalic spermatozoa, multiple morphological anomalies of the the flagella. To investigate the outcome of ntracytoplasmic sperm injection (ICSI) in the treatment of specific pure teratozoospermia.
Methods: Retrospective analysis of clinical data of specific pure teratozoospermia (globozoospermia, large-headed multiflagellar spermatozoa, acephalic spermatozoa, multiple morphological abnormalities of sperm flagella (MMAF) treated with ICSI from August 2008 to August 2008 was carried out. The clinical data (age, BMI, sperm parameters, MII oocytes, fertilization rate, high-quality embryos rate, clinical pregnancy rate, spontaneous abortion rate) were compared.
Results: Thirty-six patients of these specific pure teratozoospermia underwent 36 ovul ation and ICSI cycles and 51 embryo transfer cycles. A total of 22 clinical pregnancie s were recorded. At present, 19 live births and 2 spontaneous abortions have occurred.
There was no significant difference in age, BMI and number of mature oocytes betwe en the four groups (P>0.05). The sperm concentration (58.32±33.80*106/mL) and spe rm motility(40.85±16.86%) of round-headed spermatozoa were significantly higher than those of the other three groups (P<0.01), and the fertilization rate (18.9%) was significantly lower than those of the other three groups (P<0.01). The rate of high qu ality embryos (26.1%) was significantly lower than that of the other three groups (P<0.01). The clinical pregnancy rate of globozoospermia (25%) and large-headed multifla gellar spermatozoa (0) was significantly lower than that of acephalic spermatozoa (100%) and MMAF (75%) (P<0.01). There was no significant difference in spontaneous abortion rate among groups.
Conclusion: Here is a relatively good pregnancy outcome in patients with acephalic spermatozoa and MAMF. The fertilization rate of globozoospermia is low or completely failureeven if the egg is activated. The fertilization rate of large-headed multiflagellar spermatozoa is normal, but embryo quality and clinical pregnancy rate are poor.
PS20: Effect of healthy dietary patterns on semen quality: An updated systematic review and meta-analysis
Jialyu Huang, Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; Jiaying Lin; Hongyuan Gao; Renfei Cai; Yanping Kuang
Background: Dietary patterns represent how foods and nutrients are consumed in combination, and therefore reflect real-world dietary practices. Several studies have examined the association between dietary patterns and semen quality, but the results have been conflicting.
Methods: This study was conducted and reported in accordance with the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. Two authors independently searched four databases (PubMed, EMBASE, Web of Science and the Cochrane Library) for observational studies of the association between dietary patterns and semen quality parameters from inception to June 2018. The quality of included studies was evaluated by the Agency for Healthcare Research and Quality (AHRQ) scale. Heterogeneity between studies was assessed with Cochran’s Q test and quantified with I-squared values. Data were pooled to calculate standard mean differences (SMDs) with corresponding 95% confidence intervals (CIs) by using the random-effects or fixed-effects model according to heterogeneity.
Results: A total of 7 high-quality studies (score ≥8) involving 1873 participants were included. The 10 identified dietary patterns were categorized into unhealthy (i.e. “traditional Dutch” and “Western”) and healthy (i.e. “health conscious”, “prudent” and “Mediterranean”) patterns based on food factor loading. The meta-analysis showed that healthy dietary patterns were associated with a significant increase in sperm concentration (SMD=0.20, 95% CI: 0.07-0.33, P=0.003), count (SMD=0.27, 95% CI: 0.07-0.46, P=0.008) and progressive motility (SMD=0.46, 95% CI: 0.24-0.69, P<0.001), but not associated with semen volume (SMD=−0.20, 95% CI: −0.82-0.42, P=0.525), sperm morphology (SMD=−0.01, 95% CI: −0.14-0.12, P=0.890) and total motility (SMD=−0.10, 95% CI: −0.55-0.36, P=0.680).
Conclusion: The results of our meta-analysis showed that healthy dietary patterns could improve part of semen quality parameters, and suggested the beneficial effects of switching to healthy dietary patterns in infertile male partners.
Funding: This study was funded by the National Key Research and Development Program of China (SQ2018YFC100163) and National Natural Science Foundation of China (81771533, 81571397).
PS21: The impact of ejaculatory abstinence on semen analysis parameters of asthenozoospermia and oligospermia
Yelin Jia, West China Second University Hospital; Huanxun Yue; Wenrui Zhao; Fuping Li
Background: Many factors impact semen parameters. Abstinence time is one of the factors affecting semen quality. The World Health Organization (WHO) recommends semen samples should be collected >2 days and <7 days after the latest ejaculation. This study evaluates the ejaculate abstinence affect semen analysis parameters of asthenozoospermia and oligospermia patients in Andrology clinic of West China Second University Hospital.
Methods: A retrospective review of semen parameters of all asthenozoospermic and oligospermic patients in Andrology clinic of West China Second University Hospital. All data were conducted from January 1, 2017 to December 20, 2018. The parameters which evaluated in this study were volume, pH, concentration, sperm count, motility, round cells, normal morphology rate and viability. A total of 7013 semen samples’ parameters were collected. Spears’ correlation Coefficient was conducted to analysis the data.
Results: Significant correlations were observed between abstinence and volume, concentration, motility, round cells, normal morphology rate and viability in oligospermic patients. Spears’ correlation Coefficient suggested that in oligospermic patients the abstinence markedly positively correlated with semen volume and normal morphology rate. The abstinence negatively correlated with motility and viability. In asthenozoospermic patients, the abstinence slightly negatively correlated with motility and viability. Significant correlations were observed between abstinence and volume, sperm count, motility, viability in asthenozoospermic patients.
Conclusion: The impact of abstinence on asthenozoospermic and oligospermic patients are complex. Shorten the abstinence time is likely help oligospermic patients conduct semen with higher motility and viability. Motility and viability may negatively affected by the extension of abstinence time in asthenozoospermic patients.
Funding: Not funded.
PS22: The serumtestosterone/estrogen ratio and semen quality
XianYou Gan, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region; Hong Zhou; XiaoBin Ling; KongRong Xu
Background: The serum testosterone/estrogen (T/E2) ratio has been recently identified as a good indicator for predicting normal spermatogenesis. However, how to define the range of T/E2 ratio in fertile males, and how to evaluate its predictive value is still a difficult and worth exploring question. Although there were a few studies concerned it, but the sample size was small, and lack of the data from Asian or Chinese. Based on this, a large sample of asians or Chinese study is needed.
Methods: All the objects were the first physician visit in our center, without any drug administration since three months ago. They underwent semen analyses and hormonal assessments at the same time. semen analyses were performed according to the WHO’s fifth edition procedure, hormonal assessments including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), testosterone (T) and prolactin. Hyperprolactnemia and hypogonadotropin azoospermia were ruled out. A total of 1575 men were included in this study. Based on semen examination results, they were divided into seven groups:nomal (Group A, n=381), non-obstructive azoospermia (NOA, Group B, n=62), obstructive azoospermia (OA,Group C, n=62), severe oligoasthenozoospermia or oligoasthenoteratozoospermia (Group D, n=211), oligoasthenozoospermia or oligoasthenoteratozoospermia (Group E, n=187), asthenozoospermia or asthenoteratozoospermia (Group F, n=616), teratospernia (Group G, n=56). The serum T (nmol/L)/E2 (pmol/L) ratio of individual was calculated according to the hormonal assessmen results. The differences were evaluated using Kruskal-Wallis H test and Nemenyi test.
Results: Among the 381 men with normal semen, the mean T/E2 ratio was 0.158, the 5th percentile was 0.074, the 50th percentile was 0.146, and the 95th percentile was 0.288. Comprared to group A (nomal semen), T/E2 ratio of group B (NOA) and D (severe oligoasthenozoospermia) were significanltly different (P=0.008, P=0.00, respectively), but there was no difference of group C, E, F and G (P=1.00). There were significanltly difference of T/E2 ratio between group B and D (P=0.00).
Conclusion: The serum T/E2 ratio can be identified as a indicator for predict spermatogenesis, but not a sensitive one. When T/E2 ratio is abnormal, it may indicate that spermatogenesis function has been in a serious state.
Early Pregnancy Loss
Thursday, 11 April 2019
PS23: Autophagy regulates abnormal placentation induced by folate deficiency in mice
Xin Yin, Chongqing Medical University; Rufei Gao; Xin Yin; Rufei Gao; Yanqing Geng; Xuemei Chen; Xueqing Liu; Yubin Ding; Yingxiong Wang; Junlin He
Background: Folate deficiency has been linked to a wide range of pregnant disorders. Most researches about folate-deficiency focused on embryo itself, little attention has been paid to the possible effects of placenta. Folate deficiency might result in abnormal morphological structures on D10 in mice, decreasing HTR-8/SVneo cell viability and invasive capability. Autophagy was related to placental development and gestational diseases and had a function within neuron cells with folate deficiency.
Methods: Serum folate levels in pregnant mice were measured using an electro-chemiluminescence immunoassay. Enzyme-linked immunosorbent assays were used to measure serum hormones. The markers related to the expression of placentation and autophagy in the placenta was examined by real-time PCR, western blot, immunohistochemistry and immunofluorescence. Autophagosomes and autophagic flux were examined using transmission electron microscopy and dual-fluorescence mRFP-eGFP-LC3, respectively. The apoptosis and invasion capability of HTR8/SVneo cells were determined using flow cytometry analysis and a transwell assay.
Results: The spongy layer and labyrinth zone of the placenta, endocrine function and placental differentiation genes were all abnormal in folate-deficient mice on embryonic day 10, 12 and 14. Similar results were found in human placenta explants cultured in folate-deficient medium. The aberrant number of autophagosomes and the deviant expression of autophagy-related markers showed a disordered autophagy in placentas under conditions of folate-deficiency in vivo and in vitro. Enhanced autophagy flux was detected in HTR8/SVneo cells treated with folate-deficient medium. Importantly, the placentation impairment in mice and human placenta explants could be recovered by inhibiting placental autophagy using 3-Methyladenine (3-MA) in mice on D10. In addition, the apoptosis and invasive capability of HTR8/SVneo cells were obviously suppressed by folate deficiency, which was notably elevated by 3-MA.
Conclusion: We demonstrated, for the first time, that impaired placental development was caused by a folate-deficient diet throughout placentation in mice and that autophagy could regulate the abnormal placentation induced by folate deficiency in mice and human.
Funding: The National Natural Science Foundation of China (No.31571554).
The Research and Innovation Projects of Chongqing Municipal Education Commission (No. CYS17168).
PS24: EIF5A1 promotes trophoblast migration and invasion via ARAF-mediated activation of the integrin/ERK signal pathway
Jing Zhang, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University; Yi Lin; Hui-Qin Mo
Background: Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function is complex and the mechanisms resulting in dysregulation remain to be elucidated. Eukaryotic translation initiation factor 5A1 (EIF5A1), is a protein that needs to be hypusinated at position 50 (Lys50) after translation. To date, the identified functions of EIF5A1 include RNA binding, translation elongation and termination, promoting cancer progression. Our previous studies have shown that EIF5A1 levels are lower in uterine lymphocytes derived from non-obese diabetic mice (fertility impaired) than in wild-type mice; inhibition hypusination of EIF5A1 with GC7 leads to uterine natural killer cell dysfunction and embryo loss in mice. However, it is unknown whether EIF5A1 participates in the regulation of trophoblast biology.
Methods: Immunohistochemical staining and immunofluorescence were used to measure EIF5A1 expression levels in villous tissues. Wound-healing and Matrigel transwell assays were performed to evaluate the migratory and invasive abilities of trophoblast in vitro. Villous explants model was used to observe trophoblast migration ex vivo. GC7 and EIF5A1K50A plasmid [bearing a single point mutation (K50→A50)] was applied to abolish hypusination of EIF5A1. iTRAQ and bioinformatics analysis were conducted to identify the signal pathways regulated by EIF5A1. RNA-binding protein immunoprecipitation assay was performed to study whether EIF5A1 binds to ARAF mRNA.
Results: In this study, we found EIF5A1 expression levels were decreased in cytotrophoblasts in RM villous tissues compared to healthy controls. Overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1, inhibiting its hypusination with GC7 or EIF5A1K50A suppresses these activities. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblast ex vivo, whereas knockdown of EIF5A1 or GC7 treatment has the opposite effect. Mechanistically, hypusinated EIF5A1 promotes trophoblast migration and invasion via controlling the translation of ARAF and mediates the activation of the integrin/ERK pathway. ARAF expression level is positively correlated with EIF5A1 in villous tissues.
Conclusion: Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal–fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.
PS25: High insulin could restrain angiogenesis in endometrial decidualization through autophagy
Wenqi Chen, Chongqing Medical University; Siyu Lu, Rufei Gao
Background: In the past, the mechanism of insulin was mainly focused on metabolism. Previous studies have shown that embryonic development is abnormal under the direct action of insulin, but the effect of insulin on the endometrium is rarely mentioned in previous studies. Our previous studies found that high insulin result in impaired endometrial receptivity and abnormal decidualization. Previous researchs have shown that hyperinsulinemia play a crucial role in angiogenesis on other tissues and organs. Therefore, we speculate that high levels of insulin may be involved in the regulation of angiogenesis in endometrial decidualization. This study aims to investigate the effect of insulin on angiogenesis in endometrial decidualization.
Methods: 1. Model construction (1) A high insulin mouse model was constructed by subcutaneous injection of insulin. (2) Female mice were mated with normal males or vasectomized males to cause pregnancy (D1) or pseudo-pregnancy (PD1). (3) To construct the artificially induced decidualization model, we injected sesame oil into one side uterus on the morning of PD4, whereas the other side uterus served as control group.
2. Detection of endometrial decidualization marker molecules (BMP2 and PRL).
3. Detection of vascular network and lumen formation (CD34).
4. Detection of angiogenesis-related factor (VEGFA, VEGFR2, ANG-1 and TIE2).
5. Detection of autophagy marker (LC3, p62, Atg5, Beclin1, ULK1 and p-ULK1).
Results: (1) The insulin pregnancy mouse model was successfully constructed (ELISA). (2) In high insulin group, the mRNA and protein expression of BMP2 and PRL were significantly decreased on D6, D7 and D8. (3) Vascular sinus size is reduced and the lumen production capacity of HUVECS cells is blocked. (4) Angiogenesis-related factor (VEGFA, VEGFR2, ANG-1 and TIE2) are changed in multiple models. (5) Autophagy marker (LC3, Atg5, Beclin1 and p-ULK1) are decreased on D6, D7 and D8.
Conclusion: Endometrial angiogenesis is a complex process that is regulated by a variety of factors. This study suggests that under the action of high insulin, the expression of endometrial angiogenesis-related markers is disordered, and the extension and expansion of vascular sinus are inhibited. Autophagy may be involved in the process of angiogenesis disorders during endometrial decidualization caused by high insulin, but its specific mechanism remains to be further studied.
PS26: Molecular detection of uterine Innate lymphoid cells in the immunological mouse model of pregnancy loss
Xiangli Pang, Reproductive Medicine Center, Renmin Hospital of Wuhan University; Jing Yang
Background: Innate lymphoid cells (ILCs) are newly identified members of the innate lymphocyte family, which can function as adaptive T cells and act as critical modulators of inflammatory processes within different tissues and immune diseases. The role of uterine ILCs (uILCs) has recently been elucidated alongside changes associated with normal pregnancy. However, the proportions of uterine ILCs and their role in unsuccessful pregnancy remain unclear.
Methods: We analyzed the characterization of uILC subsets and the expression of signature cytokines associated with ILCs in a mouse model of unsuccessful pregnancy induced by LPS, and we describe the dynamic changes they undergo during this process.
Results: We found that mice exposed to LPS display significantly higher levels of uNK cells, and uILC3s. However, a lower proportion of uILC2s and uILC1s were detected in abortion mice. In addition, we found that abortion mice display markedly higher expression of IFN-γ and IL-A17, and lower levels of IL-5. No significant differences in the expression of IL-13 and IL-22 were observed.
Conclusion: The findings suggest that uILCs play distinct non-redundant roles during pregnancy, and uILCs may affect maternal-fetal tolerance via IL-17A, IL-5, and IFN-γ production.
Funding: This project was supported by National Natural Science Foundation of China (grant number 81401255, 81771618, 81571513) and National Key Research and Development Program of China (2018YFC1002804).
PS27: Haplotype-based association of two SNPs in miR-423 with unexplained recurrent pregnancy loss in a Chinese Han population
Xueqin Wang, Department of Reproductive Medicine, Qingdao Municipal Hospital; Hu Wang; Lu Zhang; Haining Liu; Jing Gao; Yongyong Wang; Xu Ma; Hongfei Xia
Background: MicroRNAs (miRNAs) regulate diverse cellular processes such as cell differentiation, proliferation and apoptosis. Mutations and SNPs in miRNAs results in various pathological conditions such as inflammation, viral infections, neurodegeneration, autoimmunity and so on. It remains unknown whether miR-423 rs6505162 and rs8067576 is associated with human recurrent pregnancy loss (RPL) in Chinese Han population. In this study, we explore the association of miR-423 rs6505162 and rs8067576 with RPL and further investigate the possible mechanism.
Methods: Blood samples of 316 RPL and 309 controls were collected. MiR-423 rs6505162 and rs8067576 genotypes were analyzed using polymerase chain reaction (PCR) and direct sequencing. MTT and Edu assays were used to detect cell proliferation and apoptosis were analyzed by FACS Calibur system. Migration and invasion were evaluated by transwell assays. MiR-423 target genes were predicted by online software and validated by luciferase reporter assay, qRT-PCR and western blotting.
Results: Our study found that one SNP rs6505162 C>A in miR-423 coding region was associated with the increase risk of human unexplained RPL (URPL), but no differences were found in another SNP rs8067576 A>T. However, in two-locus haplotype analysis, miR-423-CC/TT haplotype was associated with an increased risk of URPL. The level of mature miR-423 was obviously down-regulated in cells transfected with miR-423-CC/TT haplotype. miR-423-CC/TT haplotype inhibited HTR-8/SVneo cells proliferation and migration and promoted cells apoptosis. Further experiments identified that mesoderm development candidate 1 (MESDC1) was a functionally relevant target of miR-423, and its expression was reversely regulated by miR-423. More importantly, dual-luciferase assay indicated miR-423-CC/TT haplotype decreasing miR-423 expression, could up-regulated MESDC1 expression.
Conclusion: Our data suggest that miR-423-CC/TT haplotype in pre-miR-423 may aggravate the risk of developing URPL and influence the level of mature miR-423 and its target gene MESDC1.
Funding: This work was supported by grants from The National Key Research and Development Program of China (2016YFC1000307) and the National Natural Science Foundation of China (No. 81771590).
PS28: The exploration of the experiences of African American women and involuntary pregnancy loss: a literature review
Sarah Langley, LeadHer International, LLC
Background: The death of a child is one of the most difficult experiences to face. Research data and surveys concluded that African American women experience involuntary pregnancy loss due to biological factors such as obesity, diabetes, and deficiency of collagen. Yet, little is known about the major themes that emerge from the experience of the involuntary pregnancy loss among African American women. Through a literature review of 15 publications, researchers discussed the themes surrounding grief experienced and coping strategies used by African American women regarding involuntary pregnancy loss.
Methods: The qualitative literature review drew from fifteen articles in search of emerging themes surrounding the experiences and coping strategies of African American women and involuntary pregnancy loss.
Results: Themes surrounding the experiences of post involuntary pregnancy loss and the coping strategies adopted by grieving women are subjective and individualistic. Yet, the experiences of involuntary pregnancy loss vary and need to be captured in how they may differ between African American and women of other races. Regarding coping strategies, some women of other races include a series of exercises and counseling therapies to cope with involuntary pregnancy loss, while African American women concentrate on their religious credence or attending places of worship as a method of counseling and way to cope. Other predominate themes like cultural biases, inequalities, norms, and subject-related mortalities among African Americans in the U.S. needs further exploratory research regarding African American women and involuntary pregnancy loss in order to create a culturally sensitive framework of clinical prevention and interventions for them.
Conclusion: Knowledge remains limited about the experience and coping strategies of African American women and involuntary pregnancy loss, and further research is needed. In this literature review, themes emerged regarding the experiences and coping strategies by African American women suffering from the pregnancy loss. A culturally sensitive framework of clinical prevention and interventions may educate interested stakeholders on the various cultural, biological, and social factors that impact African American women’ responses and coping strategies to involuntary pregnancy loss, to better support African American women’s emotional, mental, and physical health.
PS29: Analysis of the occurrence of 45XO embryonic abortions in recurrent and sporadic miscarriage
Zhang Ruixiao, Henan Provincial People’s Hospital; Zhang Limei; Wang Qiong
Background: To analyze the incidence of 45XO embryonic abortions, and to investigate its relationship with the recurrent miscarriage.
Methods: Patients recruited were those with at least one embryonic chromosomal result after early miscarriage (≤12 wk of gestation) in the First Affiliated Hospital of Sun Yat-Sen University from January 2010 to September 2017. They were divided into groups of sporadic miscarriage (SM) and recurrent miscarriage (RM).
Results: 1326 cases of embryonic chromosomal results and their relevant medical history were analyzed, 82 cases were 45XO (6.18%). The frequency of 45XO embryonic abortions in SM and RM patients were 6.05% and 6.30% respectively (P>0.05), and the age of patients with 45XO embryonic abortions in SM and RM patients were 29.55(y) and 29.75(y) respectively (P>0.05). The average age of RM patients with 45XO embryonic abortions was younger than that in normal embryonic abortions (31.64 vs. 29.75, P<0.001).
Conclusion: 45XO embryonic abortions in RM patients ocurred commonly in young women with an incidence of ~6%. The frequency and average age of patients with 45XO embryonic abortions in SM and RM patients were similar, indicating 45XO embryonic abortions might had no relationship with the recurrent miscarriage.
PS31: To investigate the clinical effect of aspirin, low molecular heparin, metacortandracin, aspirin unite low molecular heparin in abortion patients, The effect of aspirin, low molecular heparin, metacortandracin is significant
Hu Jingjing, The First Affiliated Hospital of Anhui Medical University; Cao Yunxia; Wang Chao; Wei Zhaolian
Background: To investigate the clinical effect of spirin, low molecular heparin, metacortandracin, aspirin unite low molecular heparin in patients with unexplained recurrent abortion.
Methods: A total of 1013 women with unexplained recurrent abortion who accept artificial cycle freezing embryo transfer were enrolled in Reproductive Medicine Center, the First Affiliated of Anhui Medical University from January, 2014 to January, 2018. 596 patients in control group, according to the treatment regimen 417 patients in experimental group divided into parts(aspirin, low molecular heparin, metacortandracin, aspirin unite low molecular heparin). F test and χ2 test were used to analyze and compare the statistical significance of the difference between the two groups of patients.
Results: Biochemical pregnancy rate and clinical pregnancy rate were significant statistical differences in control group and experimental group (P<0.05). There were no significant differences in the different anticoagulant groups (P>0.05).
Conclusion: The effect of aspirin, low molecular heparin, metacortandracin in patients with unexplained recurrent abortion is significant.
PS32: Construction and prediction value of ultrasonic prediction model for adverse pregnancy outcome
Xinhua Wang, Binzhou Medical University Hospital; Xiaohui Deng; Yanlin Wang; Xinghua Diao; Xianghui Zhang
Background: Research shows that 15% - 30% of pregnancies have spontaneous abortion at the early stage. At present, there is a lack of a unified and highly sensitive ultrasound prediction model for reference at home and abroad.
This study attempts to synthesize four ultrasound indexes, establish a prediction model based on scores, unify the measurement of gestational age and methods, clarify the time point of examination, improve the accuracy of prediction.
Methods: (1) Research object 208 cases of IVF/ICSI were randomly selected. The first vaginal ultrasonography is performed at the time point 33-35 days after transplantation. (2) Method: Gestational sac size, the proportion of the embryo and gestational sac, yolk sac size, and fetal cardiac activity were measured. (3) Establishment of ultrasound prediction model. The above data were assigned according to the ongoing pregnancy rate (up to 14 wk), and the score grew in parallel to the pregnancy rate. Then, all patients were grouped by their different scores.
Results: (1) Predictive value of ultrasound single index for early pregnancy outcome. (2) Establishment of ultrasound prediction model and its predictive value for adverse outcomes in early pregnancy. Patients with 5 scores had a low ongoing pregnancy rate of 33.33% (4/12), and patients with 6 scores had an ongoing pregnancy rate of 50.00% (8/16), patients with 7 scores had an ongoing pregnancy rate of 60.00% (12/20). Surprisingly, patients with 8 scores had an ongoing pregnancy rate of 96.3% (104/108). In addition, we found that the ongoing pregnancy rate was 100% (36/36) in patients with nine scores. Conversely, there was no ongoing pregnancy in patients with four scores. Patients with a history of recurrent spontaneous abortion (n=32) also had a surprising ongoing pregnancy rate of 100%, when they gained 8-9 scores, and if patients could gain 8-9 scores, success rate was also 100% in the 35–40 years age range, which was still at a high success rate (66.67%) in patients older than age 40 years.
Conclusion: (1) Every ultrasound parameter plays an important role in evaluating Embryo potential. (2) This scoring system is strongly associated with ongoing pregnancy over 14 wk. (3) For patients with a history of recurrent abortion or advanced age, the predictive model also has a certain predictive value.
PS33: Non-coding RNAs regulate GTPBP10 via competitive endogenous RNA (ceRNA) mechanism in recurrent abortion
Jiao Chen, Reproductive Medicine Center, Renmin Hospital of Wuhan University; Liang Ao; Jing Yang
Background: To investigate the immune regulation through a ceRNA network in macrophages of recurrent abortion.
Methods: Whole-transcriptome sequencing and bioinformatics analysis for macrophages in decidual tissue of recurrent spontaneous abortion and artificial abortion cases were performed to identify differently expressed non-coding RNAs, and screen a possible ceRNA, and q-PCR was performed to verify the relationships of ceRNA network in much more samples; biological functions of ceRNA were studied in macrophages cell line RAW with overexpression or knockdown of genes; a recurrent abortion mouse model was constructed to verify the presence of ceRNA and explore the regulation of ceRNA on GTPBP10.
Results: The expression of immune-related miR-410-5P and EMT-related lncRNA AOC4P in macrophages of decidual tissue in recurrent abortion were significantly different from that of normal human by RNA-seq (P<0.05). We found that the gene GTPBP10 was targeted by miR-410-5P and lncRNA-AOC4P by predict program; lncRNA-AOC4P could induce the expression of GTPBP10, while lncRNA-AOC4P down regulate miR-410-5P, and miR-410-5P down regulate GTPBP10 by q-PCR and Western blotting (P<0.05); lncRNA-AOC4P and miR-410-5P could both target GTPBP10, while lncRNA-AOC4P could bind to miR-410-5P by RIP and luciferase reporter systems; lncRNA-AOC4P could promote the polarization of M2 macrophages by regulating GTPBP10 by flow cytometry; EMT phenomenon was found in macrophages cell line RAW with high expression of GTPBP10 co-cultured with stromal cells; the rate of miscarriage was increased in pregnant mice without GTPBP10.
Conclusion: lncRNA-AOC4P could regulate GTPBP10 as a competitive endogenous RNA of miR-410-5P to promote the polarization of M2 macrophages and the occurrence of EMT to maintain pregnancy.
Funding: Natural Science Foundation of China (No.81771618).
PS34: The intrauterine perfusion of granulocyte-colony-stimulating factor before frozen-thawed embryo transfer in patients with two or more implantation failures
Lihong Wei; Zhong Lin
Background: Previous studies have explored the effect of granulocyte colony stimulating factor (G-CSF) administration on the outcome of assisted reproductive technology (ART), and came into controversial conclusions. The present study to investigate the effect of the intrauterine perfusion of granulocyte colony-stimulating factor in patients undergoing frozen-thawed embryo transfer (FET) after at least two implantation failures (TIFs).
Methods: A prospective, randomized, single-blind study. The sample included patients undergoing FET who had experienced at least two TIFs. The intervention groups received an intrauterine infusion of granulocyte colony-stimulating factor (G-CSF). The placebo group was given an intrauterine injection of physiological saline before the embryo transfer. The control group did not receive an intrauterine injection. The clinical pregnancy rates, abortion rates, and live birth rates were compared between the three groups.
Results: The clinical pregnancy rates were 53.84%, 53.84%, and 35.60%, the abortion rates were 7.14%, 42.86%, and 42.85%, and the live birth rates were 50.00%, 30.77%, and 20.34%, respectively, in the G-CSF, placebo, and control groups. The clinical pregnancy rates of the intervention groups and the placebo group were all significantly higher than that in the control group (P<0.05). But the abortion rates of the G-CSF were significantly lower than those of the other two groups (P<0.05). The live birth rates of the G-CSF were significantly higher than those of the other two groups (P<0.05).
Conclusion: Intrauterine infusion G-CSF and physiological saline before frozen-thawed embryo transfer all significantly improves the clinical pregnancy rates after TIFs. However, intrauterine perfusion G-CSF can reduce abortion rates,the physiological saline can not do it. So the intrauterine infusion of G-CSF before frozen-thawed embryo transfer significantly improve the live birth rates after TIF. While intrauterine perfusion with physiological saline cannot improve the live birth rates after TIF.
Funding: This work was supported by the Guangxi Zhuang Autonomous Region Health and Family Planning Research Project (Z2015177); the Guangxi Liuzhou Science Research and Technology Development Project (2016G020216).
PS35: Association between karyotype and embryonic heart rate of day 27-29 in early pregnancy loss after in vitro fertilization-embryo transfer
Yan Ouyang, Reproductive and Genetic Hospital of Citic-Xiangya; Xihong Li; Fei Gong; Ge Lin
Background: The first transvaginal sonography examination is routinely arranged one day 27-29 after in vitro fertilization-embryo transfer (IVF-ET), and the embryoinc heart rate (EHR) could be detected in most normal cases. However, up to 25% of early pregnancy losses (EPLs) occur after the appearance of EHR. Previous studies have found the associations between no EHR detection, bradycardia and EPLs. But the relationship between EHR and different karyotypes were seldom studied.
Methods: This retrospective study included 159 singleton pregnancies which experienced EPLs after IVF-ET from 2012-2016. The EPLs were divided into normal karyotype (A, n=76) and abnormal karyotype (B, n=83) groups according to the karyotype results. 3715 cases with normal ongoing singleton pregnancies during the same period consisted the control group.
Results: Except the maternal age of group B was much higher (P<0.05), other characteristics were similar among 3 groups (P>0.05). The EHR differed significantly among 3 groups, and group B had the lowest EHR (106.2±14.8 vs. 101.1±16.2 vs. 115.5±7.0 bpm, P<0.001). The 3 groups were significantly different in the EHR distribution (P<0.001). Most cases in group A (56.6%) and B (55.4%) had an EHR among 101-120 bpm, but 111-130 bpm was the most common findings in group C (77.1%).
Autosomal trisomy was the most common abnormality in group B. Trisomy 15 (12.0%), trisomy 14 (9.6%) and monosomy X (9.6%) were the top 3 abnormalities. There were significant differences in the EHR among EPLs with different karyotypes (P<0.005). Trisomy 13 (115.7±8.7 bpm) and normal karyotype (115.5±7.0 bpm) had the fastest EHR, while trisomy 8 (72.5±10.1 bpm) and 12 (85.3±23.3 bpm) had the lowest EHR. 82.6% (19/23) cases with viable abnormal karyotype had an EHR over 100 bpm (trisomy 13, 100%; trisomy 18, 33.3%; trisomy 21, 83.3%; monosomy X, 87.5%). Additionally, the EHR in EPLs with viable abnormal karyotype was significantly higher compared to non-viable karyotype (107.7±13.4 vs. 98.6±16.7 bpm, P=0.022), but was similar with that in normal karyotype (P=0.659).
Conclusion: The EHR differs significantly among EPLs with different karyotypes. When a slow EHR is detected on day 27-27 after IVF-ET, there is a higher likelihood of abnormal karyotype. However, the EHR in viable abnormal karyotype is similar with that in normal karyotype.
Funding: The Science and technology project of Health and Family Planning Commission of Hunan Province (No. C20180289).
PS36: The effect of Nedd8-mediated neddylation on the development of trophoblast cell
Xiaohe Sun, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University; Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province; Xiaomei Tong; Songying Zhang; Qiaoqiao Chen
Background: Proper trophblast development is necessary for embryo growth. The proliferating cytotrophoblast (CTB) is recognized as trophoblast progenitor cells, which can further differentiate into syncytiotrophblast (STB) or extravillous trophoblast (EVT). Abnormal differentiation of trophoblast differentiation can cause many pregnancy complications such as early pregnancy loss, fetal growth restriction and preeclampsia. Neddylation is a modulation process tagging NEDD8 onto a substrate protein. The most typical substrates of neddylation are cullins of the cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases which requires cullin neddylation for its activation. The Nedd8 pathway is important for cell proliferation and differentiation. The aim of this study is to explore the role of neddylation in placental development.
Methods: The placental villus of unexplained early recurrent spontaneous miscarriage (RSA) (n=6) and normal control (n=5) are collected. The collected tissue was processed for protein extraction as well as paraffin-embedded tissue section. The level of neddylation was analyzed by Western blot and the distribution of Nedd8 was analyzed by immunofluorescence and immunohistochemistry. MLN4924, a NAE inhibitor, was use for further mechanism studies in HTR8/Sveno and Jeg-3 cell line.
Results: Nedd8 is most extensively expressed in the proliferating vCTB and CCT cell in human early pregnancy placentae. The neddylation process is significantly inhibited in the early pregnancy placentae in RSA patients compared to normal placentae from elective pregnancy terminations. Different expression pattern of Nedd8 was also observed between normal pregnancy and RSA. Further mechanism study using a NAE inhibitor, MLN4924, revealed that Neddylation inhibition causes proliferation inhibition, G2/M arrest in trophoblast cell line as well as Nedd8 protein translocation. Moreover, Neddylation inhibition causes abnormal accumulation and translocation of Notch intracellular domain (NICD) of Notch signal pathway, which has been reported to control the proliferation and differentiation of trophoblast cell.
Conclusion: Nedd8 mediated neddylation is required for normal trophoblast development. Low level of neddylated-Cullins as well as abnormal Nedd8 translocation was observed in the trophoblast of recurrent spontaneous miscarriage. However, further studies are needed to reveal the specific mechanism involved.
PS37: Value of transvaginal sonography in predicting pregnancy outcomes after in vitro fertilization-embryo transfer
Xihong Li, Reproductive and Genetic Hospital of Citic-Xiangya; Yan Ouyang; Ge Lin; Fei Gong
Background: With the development of transvaginal sonography (TVS), more detailed information including embryonic heart rate (EHR) may be obtained precisely during early pregnancy. Sonographic parameters and characteristics such as maternal age (MA) and vaginal bleeding (VD) have been reported to provide a prognosis of pregnancy outcome. However, most studies are focused on natural conceptions.
In this study, all pregnancies were obtained via IVF, and thus the gestational age (GA) was accurate. The aim of this study was to investigate the predictive value of sonographic markers combined with MA and VD in 6-10 gestational weeks after IVF-ET.
Methods: A retrospective study was conducted of 2601 singleton pregnancies, which had positive EHR during the first trimester TVS detection. These pregnancies were divided into the ongoing pregnancy (n=2400) and the early miscarriage (n=201) groups. The sonographic measurements of each patients including gestational sac diameter (GSD), yolk sac diameter (YSD), crown–rump length (CRL) and EHR were collected. MA and VD were also recorded.
Results: The MA of ongoing pregnancy group was significantly smaller compared with the early miscarriage group (30.60±4.31 y vs. 32.67±4.35 y, P<0.05).
The total ongoing pregnancy rate (OPR) was 92.27% after the appearance of EHR during 6-10 gestational weeks. When GSD, CRL, HR were below and YSD beyond (smaller or larger) the normal range, the OPR were 57.22%, 51.00%, 58.90%, 71.60%, respectively. Four normal sonographic markers contributed to an OPR of 97.32%. While the OPR was only 6.90% when all 4 markers were abnormal.
Multivariate logistic regression analysis showed that 4 sonographic markers, MA and VD were all risk factors, and the predictive model was LogitP=−5.161+1.401GSD+1.876CRL+2.341HR+1.150YSD+0.530MA +2.006VD. The predictive efficiency of this equation were as follows: sensitivity of 51.74%, specificity of 99.29%, positive predictive value of 85.95%, negative predictive value of 96.09%, false-positive rate of 0.71%, and diagnostic accurate rate of 95.62%.
Conclusion: In the 6-10 gestational weeks, greater MA, VD, smaller GS, shorter CRL, slower EHR and abnormal YSD are related to adverse pregnancy outcomes. Sonographic markers together with MA, VD had important value in predicting early pregnancy outcome.
Funding: The Science and technology project of Health and Family Planning Commission of Hunan Province (No. C20180289).
PS38: Absence of ZBED3 induces oxidative stress leading to abnormal development of pre-implantation embryos
Xiaodan Shi, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University; Wei Jiang; Ye Yang; Jiayi Wang Xiufeng Ling; Chun Zhao
Background: Embryo implantation depends on the acquisition of embryo implantation ability, the establishment of maternal uterine receptivity and maternal-fetal signal interaction. These processes involve the interaction of many genes. Any abnormal gene may cause the failure of embryo implantation. The study of these genes is conducive to finding out the causes of low implantation rate of embryos and providing theoretical basis for improving clinical pregnancy rate. This study will explore the function of ZBED3 on mouse embryo implantation.
Methods: RT-PCR showed that ZBED3 was expressed in ovaries and uterus in mice, suggesting that ZBED3 may be involved in oogenesis, early embryonic development and embryo implantation. We constructed knockout mice (Zbed3-/-), found Zbed3 null female mice were sub-fertility. In order to explore the stage of embryo loss, we observed embryos at 11.5d and 7.5d of gestation. There was no difference in embryo implantation sites, but the number of embryos in Zbed3-/- decidua decreased by about 50%. Subsequently, we found the formation rate of MII in Zbed3-/- decreased by about 16% compared with WT, and there were no significant differences in the 2 cells rate, 4 cells rate and blastocyst formation rate, but the hatching rate of Zbed3-/- blastocyst was decreased.
Results: In order to explore the mechanism of ZBED3 in blastocyst haching, we collected WT hatched blastocysts and Zbed3-/- unhatched blastocysts for single-cell transcriptome sequencing. The results showed that 1099 genes were down-regulated and 1204 genes were up-regulated in Zbed3-/- involving in cell metabolism, cytoskeleton, mitochondrial oxidative phosphorylation and cell apoptosis. These processes were related to embryonic development. Study showed Sparc, Id3, Lamc1, Flna and Slc7as are related to embryo implantation. In addition, we found that ROS, oxygen-carrying, mitochondrial, transcription, translation, and apoptosis related genes were down-regulated in Zbed3-/-.
Conclusion: In conclusion, we believe that ZBED3 deletion can lead to oxidative stress, abnormal mitochondrial function, increased intracellular nucleic acid and protein oxidation, triggering cell apoptosis, leading to failure of embryo implantation. At the same time, down-regulation of genes related to embryo implantation can also lead to failure of embryo implantation.
Funding: Research was funded by the National Natural Science Foundation of China (81471457).
PS39: Effect of oxytocin receptor antagonist on IVF pregnancy outcome before transplantation
Yao Wen, Department of Obstetrics and Gynaecology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China; Yue Jing; Tang Xianan; Yao HaiXia; Cong Sui; Cai FuYing; Xiao Ying
Background: Previous reviews have indicated inconsistent results on fertility outcomes on oxytocin receptor antagonist use before transplantation.
Methods: A retrospective analysis in January 2015 to June in reproductive center of Tongji hospital. We collected 841 cases infertility women undergoing IVF-ET and divide into two groups according to whether application Atosiban before transplantation. Group A (513 cycles) injected Atosiban 6.75 mg, 0.9 mL, 30 min before embryo transfer. According to the transplantation time, it was divided into A1 group (fresh cycle transplantation) and A2 group (resuscitation cycle transplantation), with 264 and 269 cycles respectively. The control group (328 cycles) did not receive AStosiban or any placebo injection. The control group(328 cycles) did not receive AStosiban or any placebo injection. It also divided into B1 group (fresh cycle transplantation, 106 cycle) and B2 group (resuscitation cycle transplantation, 222 cycle). Pregnancy outcomes were closely followed up after transplantation. T test and chi-square test were used for data statistics, and P<0.05 was considered statistically significant difference.
Results: There was no significant difference in the mean age, infertility years, BMI, basal FSH, AFC (number of follicles in the sinus), endometrial thickness and transferred embryos number between group A and group B. The clinical pregnancy rate and implantation rate of women in group A were 54.6% and 40.4%, respectively, slightly lower than the control group (55.2% and 41.3%), and the continued pregnancy rate of Atosiban group was higher than the control group (52.2% vs. 51.2%), with no significant difference (see table 2). Furthermore, the pregnancy outcomes of patients with and without Atosiban in the fresh cycle and frozen cycle were further compared, and it was found that there were no significant changes in embryo implantation rate, clinical pregnancy rate and ongoing pregnancy rate in the fresh cycle and frozen cycle compared with the control group.
Conclusion: We found that the application of oxytocin receptor antagonists could not improve the outcome of IVF pregnancy in non-RIF women.
PS40: MiRNA-4497 inhibits HTR-8/SVneo cells proliferation, migration and invasion via increasing caspase3, BAX and supressing MMP2/9 signaling pathway by targeting SP1 in recurrent miscarriage
Linqing Pan, Reproductive Medical Center, Lianyungang Maternal and Child Health Care Hospital; Lisha Tang; Linqing Pan; Huaiyun Tang; Leilei Wang; Yun Xiong
Background: MicroRNAs (miRNAs) have emerged as a crucial regulator of gene expression and reported to associate with many human diseases including reproductive disorders, such as recurrent miscarriage (RM). Although the function and regulation of miRNAs in RM have been extensively studied, the precise regulated mechanisms of miRNAs in this important field remained incompletely understood. Herein, we identified an aberrantly expressed miRNA, miRNA-4497, in the early chorionic villi of RM patients. Ectopic expression of miRNA-4497 significantly induced HTR-8/SVneo cells apoptosis and suppressed cells viability, migration and invasion. To further investigate the molecular mechanism of action of miRNA-4497 in early RM, we predicted and studied the targets genes of miRNA-4497.
Methods: Bioinformatic analysis software miRanda were used to predict the target genes of miRNA-4497. MiRNA-4497 mimics and inhibitors were transiently transfected into HTR-8/SVneo cells, and QRT-PCR and Western blot were used to detect the expression of miRNA-4497 candidate targets genes and cell apoptosis, migration and invasion associated genes. Dual-luciferase assay was used to further identify the target of miRNA-4497. Moreover, we also decrease the target gene of miRNA-4497 by specific siRNA.
Results: We predicted 12 candidate target genes of miRNA-4497 by bioinformatics analysis software, in which the expression of SP1 and CXCR5 was downregulated in both mRNA level and protein level (P<0.05), while bone Bamp8a only decreased in mRNA level (P<0.05) when miRNA-4497 was overexpressed in HTR-8/SVneo cells. Moreover, overexpression of miRNA-4497 also increased the expression of apoptotic protein, caspase3 and BAX, suppressed the migration and invasion associated protein, MMP2 and MMP9, in HTR-8/SVneo trophoblastic cells. Additionally, luciferase assay further indicated that SP1 was a direct target of miRNA-4497. We also showed that decreased the expression SP1 inhibited HTR-8/SVneo cell proliferation, migration and invasion which was consistent with the effect of overexpression miRNA-4497.
Conclusion: Our results suggested miRNA-4497 played a key role in regulating trophoblastic cells apoptosis, migration and invasion by targeting SP1 and opened new a view for further understanding the etiology and pathophysiological mechanism of early RM.
PS41: Estrogen induces Indoleamine 2,3-dioxygenase (IDO) expression by TGF-Î² in chorionic villi and decidua of early pregnancy women
Jun Wang, Reproductive Medicine Center, Department of Obstetrics and Gynecology, The Affiliated Hospital of Guizhou Medical University, Guiyang, People’s Republic of China
Background: Indoleamine 2,3-dioxygenase is one of the most important proteins in protect the embryos from the mother’s immune system during pregnancy, however, little is known about how to regulate the expression of the protein at the maternal‐fetal interface. We investigate potential mechanism involved in the regulating the expression of indoleamine 2,3-dioxygenase by estrogen in chorionic villi and decidua.
Methods: Chorionic villi and decidua of early pregnancy women were collected. Block of the chorionic villi and decidua were cultured in the medium contains with different concentration of 17β-estradiol and estriol respectively, and with or without fulvestrant. Western blot analysis and/or immunofluorescent staining were used to detected the expression of transforming growth factor-β and indoleamine 2,3-dioxygenase in chorionic villi and decidua, and in cultured tissues. Statistical analyses were performed using Pearson’s correlation analysis and paired samples t-test.
Results: Both transforming growth factor-β and indoleamine 2,3-dioxygenase expressed in chorionic villi and decidua. The expression of the two proteins increased the most, and transforming growth factor-β exhibited the same expression tendency with indoleamine 2,3-dioxygenase in blocks of chorionic villi and decidua cultured in the medium containing 17β-estradiol at the concentrations of 10 ng/mL, or estriol at the concentrations of 1 ug/mL. This increase can be reversed when fulvestrant was added in the medium at the concentra-tions of 10 ug/mL. indoleamine 2,3-dioxygenase expression increased in a dose dependent manner in blocks of the tissues cultured in the medium containing transforming growth factor-β.
Conclusion: Estrogen near pregnancy concentrations may upregulate the expression of indoleamine 2,3-dioxygenase by Transforming growth factor-β, which implicate that estrogen may prevent allogeneic fetal rejection and may be used as immunomodulator.
Funding: This work was supported by the National Natural Science Foundation of China (http://www.nsfc.gov.cn/GrantNo.81360452).
PS42: The relationship between physical activities during pregnancy and pregnancy outcomes
Haoting Ge, Zhejiang University School of Medicine; Xinhan Zhang; Hao Zhao; Guangdi Chen
Background: Previous reports on the relationship between physical activities during pregnancy and pregnancy outcomes were inconsistent.
Methods: During 2015 to 2017, 1380 pregnant women were included in the study. A face-to-face interview was performed by well-trained staff using constructed questionnaires to collect information about physical activity in the second trimester, demographic characteristics, lifestyles, behaviors and so on. Besides, pregnant outcomes of birth weight, preterm delivery, fetal distress and so on were abstracted through existing medical records. Two widely acceptable methods were used for the classification of maternal physical activity. Chi-square test, analysis of variance (ANOVA), and Logistic regression and multi-class Logistic regression models were applied for statistics analyses.
Results: Totally, 1037 eligible participants included for data analyses. We found that maternal education, working at pregnancy and gestational weeks were different among the low, medium and high levels of physical activity with statistical significance (P<0.05). Physical activity during pregnancy and offspring birth weight were not significantly associated (P>0.05). In contrast, compared with neonates of mothers at the lowest level, those of women with medium level of physical activity at pregnancy had an increase of 65.94 (95% CI=3.47 to 128.40) g of birth weight in the crude linear regression model. Medium (OR=0.44, 95% CI=0.19 to 1.00) and high physical activity (OR=0.46 95% CI=0.21 to 0.99) during pregnancy were related with reduced incidence of premature birth; Moderate physical activity was associated with reduced incidence of premature rupture of membranes (OR=0.62, 95% CI=0.41 to 0.92). However, when the maternal physical activity was assessed by another method, we found significantly negative associations for fetal distress (OR=0.70, 95% CI=0.51 to 0.97) in the sensitivity analysis.
Conclusion: We found reduced risks of premature rupture of membranes, fetal distress and premature birth for the groups of moderate, over moderate and high levels of physical activity, respectively.
Funding: This study was supported by the Program of the National Natural Science Foundation of China (No. 71503235) and the Fundamental Research Funds for the Central Universities (No. 2017XZZX011-01).
PS43: Potential risks of biochemical pregnancy in women after in vitro fertilization embryo transfer
Wan Yang, Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, China; Rui Yang; Haiyan Wang; Rong Li
Background: There is limited literature investigating the potential risks of biochemical pregnancy during in vitro fertilization embryo transfer (IVF-ET).
Methods: A retrospective cohort study. Total 9213 infertile women who received embryo transfer fresh cycles with autologous oocytes from January 2009 to December 2016 at the Center for Reproductive Medicine in Peking University Third Hospital were included. Only the first infertility treatment ended with hCG positive 14 days after embryo transfer was eligible for the study. Cycles were excluded if patients were diagnosed with systemic diseases, abnormal karyotypes or uterine anomalies like asherman syndrome. Patients were divided into two groups: biochemical pregnancy (n=1122) and Embryo implantation (n=8091).
Results: There were no significant differences in female BMI, basal FSH between the groups. The age, basal luteinizing hormone (LH) levels and androgen levels were significantly higher in the biochemical pregnancy group than in the embryo implantation group. A significantly higher number of oocytes were retrieved, thicker endometrium was observed, and the total Gn consumption as well Gn consumption/kg/day was significantly lower, in the embryo implantation group than in the biochemical pregnancy group. Female age, basal LH and androgen level, these three factors in baseline characteristics, were significantly associated with biochemical pregnancy after adjusting for confounders. Adjusted odds ratio suggested an 89.1% increase of biochemical pregnancy in female older than 40 years, 42.6% increase of it in women with higher level of T and A, and 18.2% increase of it in women with basal LH over 4.38 IU/L. These three baseline factors can be used in the following formula to calculate the possibility of biochemical pregnancy: Ln (P/1-P)=0.035×Female age+0.059×basal LH+0.12×Androgen category (1) +0.355×Androgen category (2) −3.271.
Conclusion: Female age, basal LH and androgen level were significantly influential factor on biochemical pregnancy.
Funding: Research was funded by the National 13th Five-Year Plan of China (2016YFC1000201).
PS44: Correlation research between abnormal uterine artery blood flow in the midluteal phase and spontaneous abortion
Kemei Zhang, Reproductive Medicine Centre
Methods: 444 patients treated in the department of reproductive medicine centre of our hospital from Jan 1, 2015 to Dec 31, 2017 were selected as subjects. According to the medical history, they were divided into three groups: group A (control group), group B (one spontaneous abortion) and group C (two spontaneous abortions or more). The blood flow spectrum of uterine artery in the midluteal phase were measured by transvaginal color doppler ultrasound. The arterial blood flow index, including S/D value (the ratio of the systolic peak value and the end-diastolic velocity of blood flow), PI value (pulse index) and RI value (resistance index), and abnormal blood flow spectrum were calculated.
Results: The patient age in group A, group B and group C were 28.89±3.32 y, 29.46±4.01 y and 28.78±3.59 y, respectively. There were no statistical differences among the three groups, P>0.05. The sum of the S/D value on both sides in group A and group B were 13.68±4.95, 14.80±6.02, the sum of bilateral PI value were 4.42±0.95, 4.63±0.93, the sum of bilateral RI values were 1.67±0.11, 1.69±0.10, respectively. There was no statistical difference between the two groups, P>0.05. The proportion of abnormal blood flow spectrum in group A and group B were 6.67% (17/255) and 16.35% (17/104), respectively. There was significant difference between the two groups, P<0.01. The sum of the S/D value on both sides in group A and group C were 13.68±4.95, 15.80±6.76, the sum of bilateral PI value were 4.42±0.95, 4.75±1.06, the sum of bilateral RI values were 1.67±0.11, 1.70±0.12, respectively. There was statistical difference between the two groups, P<0.05. The proportion of abnormal blood flow spectrum in group A and group C were 6.67% (17/255) and 23.53% (20/85), respectively. There was significant difference between the two groups, P<0.05.
Conclusion: There is a correlation between abnormal uterine arterial flow in the midlutral phase and spontaneous abortion. The higher the index of uterine artery blood flow, the greater blood flow resistance, which might leads to spontaneous abortion. Meanwhile, the correlation is associated with the time of miscarriages.
Thursday, 11 April 2019
PS45: Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis
Jingjing Shi, Center for Reproductive Medicine, Shandong University, China; Qing Gao; Cao Yongzhi; Jiang Fu
Background: The DENND1A has been identified as a guanine nucleotide exchange factor (GEF) for small GTPase Rab35, which functions in endocytic trafficking to mediate the recycling of selective cargos. Genetic alterations within the DENND1A gene have been implicated in human disease such as polycystic ovary syndrome (PCOS). However, the role of DENND1A in developmental and reproductive processes is largely unknown.
Methods: Using a Dennd1a gene knock-out mouse strain, we examined the development of Dennd1a mutant embryos to investigate how the ablation of Dennd1a affect embryogenesis.
Results: We uncovered that homogeneous Dennd1a-/- mutants died around embryonic day (E) 14.5. The brain of Dennd1a-/- embryos exhibited defects, partially attributed to the dysregulation of cell division and survival in the telencephalon. The transcription of Axin2, a downstream target of canonical Wnt signaling was downregulated in the brain of Dennd1a-/- embryos, concomitant with an elevation of Fgf8 mRNA, which was ectopically expressed in the dorsal midline of telencephalon. During liver morphogenesis, the ablation of Dennd1a impaired hepatic cell proliferation, the differentiation of hepatocyte, and hepatic hematopoiesis. In addition, the loss of Dennd1a also resulted in delayed migration and reduced proliferation of primordial germ cells.
Conclusion: We demonstrate that Dennd1a, a susceptibility gene for PCOS, is essential for embryogenesis, probably through the mediation of endocytic recycling of selective cargos that are involved in cell signaling crucial for the development of multiple embryonic organ systems.
Funding: National Key Research and Development Program of China, Grant number: 2016YFC1000601; Natural Science Foundation of Shandong Province, China, Grant numbers: ZR2016HM79 and ZR2014HM017.
PS46: Expression of target genes in cumulus cells derived from human oocytes with and without blastocyst formation
Tuanping Zhou, The First Affiliated Hospital with Nanjing Medical University; Ri-cheng Chian; Lingbo Cai; Yixin Xu; Kailu Liu
Background: The transcriptional profile of cumulus cells during oocyte maturation provides the information to predict oocyte developmental competence. Our previous study with mouse model indicated that there were 9 different genes expressed in cumulus cells during oocyte maturation related to oocyte development potential.
Methods: The collected human cumulus cells from each oocyte were divided into two groups following tracking whether or not developed to blastocyst stage: (1) the oocytes were developed to blastocyst stage after fertilization in vitro (+B); (2) the oocytes were not developed to blastocyst stage after fertilization in vitro (-B). The expressions of the 9 selected genes (arrb1, atp2c1, cdh5, cntnap1, lgr4, mkln1, rhobtb1, six2, smc2) were examined.
Cumulus cells were obtained from 29 women who were undergoing ICSI treatment cycles. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed on cumulus masses collected before insemination. The paired cumulus cell samples from oocytes that developed into blastocyst (B+) versus not developed to blastocyst (B-) were compared. Each sample was run three times. The significance of differences in mRNA expression of the target genes in independent samples were evaluated by two-tailed student’s t-test, and P value<0.05 was considered significant difference.
Results: There were significant differences in the expressions of arrb1 (P=0.026), cntnap1 (P=0.036) and lgr4 (P=0.006) from mRNA levels between the group B+and the group B-.
Conclusion: Identification of genes, arrb1, cntnap1 and lgr4 expressions in cumulus cells may provide predicting information of oocyte developmental competence before insemination and fertilization, leading to enhanced implantation rates of IVF.
Funding: Project supported by the National Natural Science Foundation of China(Grant No.81270746 and No.81370754), the 973 Program of China (2012CB944703) and the Research Fund of NHFPC (201302013, 201402004). There were no competing interests.
PS47: CNP-mediated pre-IVM improves cumulus function and oocyte quality in mildly stimulated mice
Yiqing Zhao, Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Xiuhua Liao; Adriana E. Krysta; Michael J. Bertoldo; Dulama Richani; Robert B. Gichrist
Background: Oocyte pre-in vitro maturation (IVM) using c-type natriuretic peptide (CNP) is an innovative new IVM system that is currently undergoing clinical evaluation. Understanding its mechanism of action is required for clinical application. This study aimed to determine temporal effects of CNP-mediated pre-IVM on cumulus function and oocyte developmental competence in mildly-stimulated mice.
Methods: Immature cumulus oocyte complexes (COCs) were derived from mildly stimulated (23h PMSG) 28-day old mice. COCs were cultured for 0 (control), 2, 6, 12 or 24 h in pre-IVM medium (CNP+E2+FSH+insulin), prior to IVM/IVF. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus cell proliferation, apoptosis, cumulus matrix expansion, gap junction communication (GJC), and energy metabolism were assessed at various time points during pre-IVM and IVM.
Results: Day-6 blastocyst rate (blastocyst/oocyte) increased with increasing pre-IVM duration: 40.6±2.0%, 45.8±1.2%, 52.2±3.5%, 53.3±5.9% and 59.9±2.5% for 0, 2, 6, 12 and 24h pre-IVM, respectively (P<0.01). DNA content/COC, a marker of cell proliferation, was significantly higher in 24h pre-IVM group compared to 0, 2, and 6, but not the 12h, pre-IVM groups (P<0.001). 24h pre-IVM increased COC ATP and ADP content (P<0.05), but not AMP, ATP/ADP and energy charge. At 6h of IVM culture, expression of Has2 and Tnfaip6 mRNA in COCs pretreated with 24h pre-IVM was significantly higher than control COCs (P<0.01). At the end of IVM, cumulus expansion was significantly improved in COCs exposed to 24h pre-IVM than control COCs (P<0.0001). Cumulus-oocyte GJC was maintained throughout 24h pre-IVM (P<0.0001), and GJC loss was slowed during the subsequent IVM phase, whilst meiotic resumption was accelerated (P<0.05).
Conclusion: CNP-mediated pre-IVM improves the quality of cumulus cells in a temporally-dependent manner, leading to improved oocyte developmental competence in mildly stimulated mice.
PS48: Expression and distribution of the zinc finger protein, SNAI3, in mouse ovaries and pre-implantation embryos
Shujuan Guo, Shandong Normal University; Feifei Shi; Cong Zhang
Background: The Snail gene family includes Snai1, Snai2, and Snai3 that encode zinc finger-containing transcriptional repressors in mammals. The expression and localization of SNAI1 and SNAI2 have been studied extensively during folliculogenesis, ovulation, luteinization, and embryogenesis in mice. However, the role of SNAI3 is unknown. In this study, we determined the expression of SNAI3 in the ovaries of neonatal and gonadotropin-treated immature mice, as well as in GV oocytes and pre-implantation embryos.
Methods: Animals, Ovary, oocyte, and embryo collection, Immunohistochemistry, Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, Immunofluorescence, Statistical analyses.
Results: Localization of SNAI3 during postnatal ovarian follicular development.
Localization of SNAI3 during follicular maturation, ovulation, and luteinization.
Subcellular localization of SNAI3 in oocytes and during early embryonic cleavage.
Conclusion: SNAI3 may plays some roles during folliculogenesis, luteinization, and early embryonic development.
Funding: This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31471399 and 31671199), National Key R&D Program of China (2017YFC1001403), and Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20152515) to CZ. Support was also received by a grant from the Major Program of the National Natural Science Foundation of China (81490743) to Z-JC, and by the Shanghai Commission of Science and Technology (funding number: 17DZ2271100).
PS49: Melatonin reduces 2-cell block via non-receptor pathway in mice
Feifei Shi, Shandong Normal University; Hua Li; Cong Zhang
Background: In vitro embryo production (IVP) plays an indispensable role in livestock production and human assisted reproductive technology (ART). However, the success of IVP and ART has been greatly affected since the embryo developmental block. Although numerous researchers have explored this problem, it remains to be solved. Melatonin (N-acetyl-5-methoxytryptamin) is a “ubiquitously distributed and functionally diverse molecule”. The key physiological function of melatonin is as a broad-spectrum antioxidant.
Methods: Chemicals and reagents
Animal care and embryo collection
RNA isolation and Quantitative Real-time PCR
ROS determination in embryos
Mitochondrial superoxide determination in embryo
Results: Comparison of the effects of three kinds of culture mediums on 2-cell block
Melatonin has beneficial effect on 2-cell block and improves the embryo quality
Melatonin reduces ROS in 2-cell and 4-cell embryos
Melatonin reduces mitochondrial superoxide in 2-cell and 4-cell embryos
Melatonin up-regulates the expression of antioxidant genes and inhibits the expression of pro-apoptotic gene
Melatonin up-regulates the expression of zygotic genes and the inducer of ZGA
The Expression and function of melatonin receptors
The effect of melatonin on gene expression is independent of the melatonin receptors
Conclusion: Melatonin can be used to improve the quality and the production efficiency of the embryos cultured in vitro by removing ROS and activates zygotic gens, thus consequently decreases 2-cell block.
The effect of melatonin on 2-cell block may be independent of the receptors.
Funding: This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31471399 and 31671199), National Key R&D Program of China (2017YFC1001403), and Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20152515) to C. Z. Support was also received by a grant from the Major Program of the National Natural Science Foundation of China (81490743) to Z.-J.C., and by the Shanghai Commission of Science and Technology (funding number: 17DZ2271100).
PS50: Combination with timelapse parameters and soluble HLA-G expression in embryos’ day3 medium for evaluation of the embryos implantation potential in patients undergoing frozen-thawed embryo transfer
Menghui Geng, Nanjing Medical University First Affiliated Hospital; Can Zhang; Yanqiu Hu
Background: The clinical pregnancy rate (CPR) and multiple pregnancy rate (MPR) are two crucial issues in assisted reproductive technology (ART) practice. Selective single embryo transfer (SET) strategy is the best solution to minimize MPR. Whereas, for the embryologists, accurate estimation of implantation potential of embryos is the way not to reduce CPR while SET. As numerous technologies are applied in this field, many novel and non-invasive embryo selection methods have presented. We investigated if the time-lapse imaging (TLI) hierarchical classification model combined with the expression of soluble human leukocyte antigen G (sHLA-G) in embryos’ day3 mediumcan be a valuable strategy to identify those embryos with the best implatation potential.
Methods: This is a retrospective study involving 146 patients who received Frozen-thawed embryo transfer (FET) with single or two embryos. We selected and transferred embryos based on traditional morphological evaluation. In the meantime, we used timelapse instrument to record all embryos’ development. According to the clinical results, we analyzed the TLI morphokinetic parameters (t2, t3, t4, t5, t6, t7, t8, cc2, s2, cc3, s3) then developed a hierarchical classification model which classified the embryos into five groups (A, B, C, D, E). We also examined sHLA-G levels in the day3 (D3) culture fluid by ELISA. We correlated the TLI hierarchical classification and the expression level of sHLA-G in embryos’ D3 medium to find a non-invasive method for assessing the implantation potential of embryos.
Results: The results of multivariate unconditional logistic regression analysis showed that TLI parameters (t5 and s3) and the content of sHLA-G are independent risk factors influencing embryo implantation. The implantation rate decreases from embryo TLI classification A to E. The classification model presented here was capable of assessing the implantation potential. The average sHLA-G concentration and implantation rate of sHLA-G positive group greater than sHLA-G negative group (P=0.000). The expression of sHLA-G in D3 medium of embryos combined with TLI classification model can assess the implantation potential with more accuracy.
Conclusion: We showed that TLI hierarchical classification model combined with the expression of sHLA-G in D3 medium of embryos is a useful tool to identify embryos’ implantation potential.
PS51: Effect of laser assisted hatching on the clinical pregnancy result in vitrification-warmed blastocyst transfer cycles
Mei Li, Shandong University; Shui-Ying Ma; Lian-Jie Li; Hui Zhao; Cheng Li; Hai-Bin Zhao; Qing Li; Kai Xu; Ke-Liang Wu
Background: Assisted hatching (AH) was a widely accepted technology in assisted reproductive technology, but it’s efficiency was controversial. It is thought that freezing/thawing and vitrifying/warming embryos may impair the successful hatching process of the embryo out of its zona pellucida (ZP), so AH was usually used in frozen embryo transfer cycles. However a large data showed that in the frozen-thawed embryo transfer patients, there was no significant difference in pregnancy and live birth rate between the AH group and the non-AH group. Anyway, as an intervention to the blastocyst or cleavage embryo, laser induced hyperthermia was bad for the live cells, but which did contribute to the hatching of the blastocyst. The aim of this study was therefore to evaluate the efficacy of the laser AH on the clinical pregnancy result in vitrification-warmed blastocyst transfer cycles.
Methods: This is a randomized controlled trial. Data collected from one IVF lab of Center for Reproductive Medicine, Shandong University. A total of 4142 patients to be given vitrification-warmed balstocysts transfer were involved in this study. All the patients were randomly divided into two groups: assisted hatching (AH) group (study group) and no assisted hatching (NAH) group (control group). The blastocyst in AH group was given laser AH before transfer and NAH group was not. All the survival balstocysts were transferred into uterus after four to five hour’s culture. The clinical pregnancy rate, early miscarriage rate and live birth rate were calculated and analyzed.
Results: Total 4142 patients gathered included 1974 patients in AH group and 2168 patients in NAH group. There was no significant difference in age composition, endometrial preparation protocols, number of embryo transferred and blastocyst developmental stage between the two groups (P>0.05). The clinical pregnancy rate, and live birth rate in AH group were significantly higher than those in NAH group (59.17% VS. 56.04%, P=0.042; 48.63% VS. 45.43%, P=0.0394). At the same time early miscarriage rate was similar in the two groups (17.12% VS. 17.78%, P>0.05).
Conclusion: Laser AH increases clinical pregnancy results in vitrification-warmed blastocyst transfer cycles. AH does not affect the early miscarriage rate.
Funding: Research was funded in part by the National Natural Science Foundation of China (No. 81401266).
PS52: Serum and follicular fluid fetuin-B levels are correlated with fertilization rates in conventional IVF cycles
Li Fang, Women’s Hospital, Zhejiang University School of Medicine; Ying-Hui Ye
Background: Total fertilization failure or very low fertilization still occurs in 5-10% of in vitro fertilization (IVF) cycles. Upon fertilization, cortical granules are released from the mammalian oocytes, trigger structural changes of the ZP leading to ZP hardening and blockade of further sperm entry. However, small amounts of cortical granules are also released prior to fertilization, during oocyte meiotic maturation. Fetuin-B, a liver-derived plasma protein present in follicular fluid, prevents premature ZP hardening by inhibiting ovastacin, thus keeps the oocyte fertilizable. It has been shown that female Fetub−/− mice are completely and permanently infertile and down-regulation of serum fetuin-B by antisense oligonucleotides leads to transient infertility of female mice. It has been reported that high fetuin-B levels in serum and follicular fluid were associated with fertilization success in IVF. However, it was a pilot study and the sample size is limited.
In this study, we aimed to further explore the relationship between fetuin-B levels and fertilization rates in IVF cycles. And the association of fetuin-B levels to oocyte maturation, embryo development, clinical pregnancy, live birth was also investigated.
Methods: A prospective observational study of conventional IVF treatments including 78 cycles with low fertilization rate (two pronuclei (2PN) rate <30%) (LF group) and 104 cycles performed during the same period with 2PN rate>70% (high fertilization group, HF). Fetuin-B was measured employing a commercial sandwich enzyme-linked immunosorbent assay.
Results: Serum fetuin-B and follicular fluid fetuin-B were positive correlated (r=0.703, P<0.001). Compared with the HF group, the LF group had a significantly lower level of fetuin B concentrations both in serum (5.81±1.53 vs. 7.19±1.42, P<0.001) and follicular fluid (5.06±1.29 vs. 6.16±1.52, P<0.001). And serum fetuin-B level from cycles with polypronuclear (PPN) zygotes was significantly lower when compared with cycles without PPN zygotes (6.82±1.65 vs. 6.10±1.43, P=0.006). However, serum fetuin-B level is not correlated to preimplantation embryo development and clinical pregnancy.
Conclusion: Serum fetuin-B level is correlated to fertilization rate in conventional IVF and it may be used as a predictive marker of fertilization in IVF treatment.
PS53: In vitro culture impairs mitochondrial DNA replication and development of mouse embryos
Miaoxin Chen, Tongji University, China; Siew Wong; Leonie Heilbronn; Rebecca Robker
Background: Evidence suggests that IVF impairs glucose metaboliam in humans and mouse models. Both ovarian hyperstimulation and embryo culture contributed to the adverse outcome in the offspring. This study examined developmental characteristics IVF mouse embryos including embryo morphokinetic development, cell number and differentiation and mtDNA copy number as compared to in vivo conceived embryos.
Methods: C57/B6J mouse blastocysts were generated with natural cycle IVF (nIVF), or with mild stimulation (5 IU) followed by IVF (IVF 5IU) or in vivo fertilisation but in vitro culture from 2 cells (IVC), or with hyperstimulation IVF (IVF 7.5IU). Additional cohorts underwent natural mating without stimulation as the controls (NM), or with mild stimulation (5 IU) and gestation to the blastocyst stage (OS). Embryo morphokinetics were assessed by continuous time-lapse monitoring. Cell lineage allocation to the inner cell mass (Oct4+) or trophectoderm (Cdx2+) was determined by immunohistochemistry. mtDNA copy number was measured by qPCR.
Results: Blastocyst rate was significantly reduced in all of the IVF groups compared to the IVC group which was fertilised in vivo (P<0.001). Time-lapse analysis showed cell division and development was similar between 3 IVF groups. Compared to the IVC group, embryo development was significantly slower in IVF 5IU and IVF 7.5IU groups after HCG injection and slower in all 3 IVF groups after the 3-cell stage (P≤0.03). Reduced total and trophectoderm (TE) cell number was observed in blastocysts from the IVF 5IU and IVC groups compared to OS and NM embryos and further reduced in IVF 7.5IU blastocysts (P<0.01). Gonadotropin treatment did not affect mtDNA copy number of oocytes however embryos from all 3 IVF groups, but not the IVC group, exhibited reduced mtDNA copy number compared to naturally conceived OS and NM embryos.
Conclusion: In vitro culture during embryogenesis reduced TE cells and mtDNA as well as embryo growth and development independently of ovarian stimulation. Importantly, embryos that were fertilized in vivo then cultured from the 2 cell stage were less affected indicating that in vitro culture during just the first cell cycle impairs embryogenesis. High doses of ovarian stimulation also impair embryo developmental potential.
Funding: The study was funded by Diabetes Australia (#Y16G-ROBR).
PS54: Is embryo development morphokinetics affected by different cultural mediums? A timelapse study
Bo Huang, Reproductive Medical Center, Tongji Hospital, Tongji College of Medicine, Huazhong University of Science and Technology; Lei Jin
Background: The embryo culture medium, is an important factor in IVF laboratories. Embryologists used to make their own culture media in their laboratory. Time-lapse system could provide a better comparison among embryos, and find out subtle changes during the early embryonic development. In this study, we aim to find out the influence of different culture mediums on early embryonic cleavage kinetics by time-lapse analysis and the possible relationship between the energy substrate and the cleavage kinetics.
Methods: In the present retrospective cohort study, only embryos cultured in time-lapse incubator were involved. They came from couples performing IVF/ICSI cycles at Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology during 12 months in 2016. However, patients of ≥40 years of age and with a Body Mass Index (BMI) of >30 kg/m2 were excluded. Also, oocyte donation cycles and preimplantation genetic diagnosis cycles were excluded. According to the mediums, couples and embryos were divided to Group I and Group II.
Results: The baseline demographic features and IVF indications of the included patients in Group I and Group II were similar (P>0.05). Also, the main results of OCCs, fertilization, pregnancy and perinatal outcomes of embryos between the two groups did not show any significant difference (P>0.05). According to the time-lapse analysis, all embryos in Group I showed significantly faster cleavage kinetics than Group II (P<0.05). Furthermore, there was better synchrony in division (s3), and a longer length of third cell cycle (cc3) was observed in Group II. For top quality embryos, only t2 and s2 showed a significant difference (P<0.05). Interestingly, for the implanted embryos, Group II showed faster cleavage than Group I, especially, t4 and t7. Also, the synchrony in division (s2) of embryos was better in Group II. In order to clarify the difference between the two groups, main energy substrate contents were quantified. Similar glucose contents were observed. Lactic and pyruvic acid contents were generally higher in Group I. Main amino acid contents and types were similar in both groups, except essential amino acids in fertilization medium of Group I.
Conclusion: The different cleavage kinetics may be related to the content determination data of energy substrate in different culture mediums. Therefore, different culture mediums should be taken into consideration when embryos were chosen by to implant Time-lapse system.
PS55: Limited value to select human embyos by noninvasive preimplantation genetic screening using genome sequencing of blastocoele fluid DNA
Lingbo Cai, The First Affiliated Hospital with Nanjing Medical University; Qiao Zeng; Chao Gao; Wei Wu; Jiandong Shen; Dao wu Wang; Jiayin Liu
Background: Preimplantation genetic screening (PGS) is widely used to select embryos and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation embryos. A niPGS method was presented that was based on sequencing the genomic DNA secreted into the culture medium and blastocoele from the human blastocyst. The niPGS method avoids the need for embryo biopsy and increases the safety of its use. However, the niPGS method has inborn defects from PGS.
Methods: In this prospective cohort study performed in a single IVF center, a total of 182 blastocysts from patients undergoing their first in vitro fertilization and embryo transfer (IVF-ET) cycle in 2017 were analyzed (one blastocyst per patient), after 33 patients withdrew from the study. BF was collected prior to blastocyst vitrification. Clinical pregnancy outcomes were followed up. Cell-free DNA from BF was amplified (WGA) for next generation sequencing (NGS) -based comprehensive chromosome screening (CCS). The results were compared with clinical outcomes from newborn baby karyotyping, Chorionic Villus Sampling (CVS) results from miscarriages, or fetal Noninvasive Prenatal Test (NIPT) results from ongoing pregnancies.
Results: A total of 139 BF samples produced CCS results for analysis (76.37%). Balanced euploidy was observed in 34 samples (24.46%) and aneuploidy in 92 samples (75.54%). A total of 113 patients (76.87%) achieved clinical pregnancy. Of those, 14 (11.38%) miscarried in their first trimester. CVS analysis results revealed partial correlation between miscarriage tissue DNA samples and BF DNA samples. Sex chromosome DNA samples were tightly correlated with both DNA sample types. The 97 NIPT results from women with ongoing pregnancies were completely consistent with the corresponding PGT-A results from 38 babies.
Conclusion: When compared with PGS, NiPGT-A is a lower risk method for the preimplantation screening of embryos, but currently can only identify partial correlations between clinical outcomes and BF ploidy analysis results.
Funding: This work was supported by the China National 973 program (2012CB944902), Jiangsu Provincial Science and Technology Project (BL20122009 and BE2011798), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
PS56: Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos
Yumeng Cao, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China; Ran Huo
Background: The successful initiation of early embryo development is almost entirely dependent on the oocyte for the initial supply of subcellular organelles and macromolecules required for survival and development. Many maternal components are encoded by maternal effect genes, which provide mRNAs or proteins that accumulate in the oocyte during oogenesis, and are present in early embryos before the expression of zygotic genes. The ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) protein recognizes DNA methylation and histone modification and plays a critical role in epigenetic regulation. Recently, UHRF1 was shown to have a role in DNA methylation in oocytes and early embryos. Here, we focus on the role of UHRF1 in addition to DNA methylation in the regulation of oocytes quality.
Methods: Generation of mouse lines.
Assessment of embryo implantation sites.
In vitro fertilization, embryo culture.
Immunofluorescence and confocal microscopy.
Results: In the present study, we showed that besides DNA methylation, maternal UHRF1 also controls histone modification in mouse oocytes, plays a crucial role in determining oocyte quality, and is essential for preimplantation embryo development. Conditional deletion of UHRF1 in growing oocytes resulted in spindle and chromosomal separation abnormalities, and increased DNA damage. These defects were consistent with the poor quality of the oocytes. Although both in vivo and in vitro experiments showed Uhrf1-deficient oocytes could be fertilized, embryos derived from these oocytes failed to reach the blastocyst stage. The consequences of UHRF1 deficiency included decreased H3K9 di-methylation, increased H4K12 acetylation, altered gene expression, genomic and chromatin defects, and the lack of maternal Uhrf1 expression may affect other essential cellular processes.
Conclusion: In summary, our study showed that disruption of murine Uhrf1 severely impaired oocyte quality, suggesting that maternal UHRF1 may be important for oocyte epigenetic modification and DNA integrity, and determining the developmental potential of oocytes.
Funding: This work was supported by NSFC (31471107) and National Key Research and Development Program of China (2017YFC1001301).
PS57: MicroRNA-21 expression in extracellular vesicles involves in the growth of fertilized egg cells and embryonic development in mice: an in vitro study
Chao Lv, Institute of Reproductive & Stem Cell Engineering, School of Basic Medical Science, Central South University; Hong-Mei Xiao
Background: Human preimplantation embryo development is susceptible to high rates of early embryo wastage. In this study, we determined the microRNA-21 (miR-21) expression of extracellular vesicles (EVs) in fertilized eggs and embryo of varying stages and their response to miR-21 microinjection.
Methods: A total of 134 mature female mice and 134 mature male mice were selected, then mouse fertilized eggs in 2-cell stage were collected and divided into the blank group (without microinjection), experimental group (microinjected with miR-21) and negative control (NC) group (microinjected with TE solution). Inverted microscope was used for observing the development of fertilized egg cells. Immunohistochemical staining was used for detecting surface marker expression (CD9 and CD63) on extracellular vesicles between the pregnant and nonpregnant female mice. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for determining the miR-21 expressionamong fertilized eggs at the stages of 2 cells, 4 cells, 8 cells, 16 cells, morula, blastaea and embryos.
Results: Compared with the NC group, the percentage of fertilized egg cells in the experimental group in 2-cell stage was remarkably decreased and remarkably increased in 4-cell stage and 8-cell stage. Compared with nonpregnant female mice, the staining intensity of CD9 and CD63 and miR-21 expression were higher in pregnant female mice. MiR-21 expression in the pregnancy group was higher in extracellular vesicles than that in the nonpregnancy group. The miR-21 expression was not seen in germinal vesicles. The miR-21 expression was decreased in fertilized egg cells at 2-cell stage, and the miR-21 expression was increased with the increasing of embryonic cells. The miR-21 expression was significantly increased after embryo implantation and relatively high in the middle and late period of embryonic development.
Conclusion: Taken together, these results demonstrate that the miR-21 expression in extracellular vesicles involves in the growth of fertilized egg cells and embryonic development.
Funding: This work was supported by the National Natural Science Foundation of China [grant number 81471453]; and the National Key Research and Developmental Program of China [grant number 2017YFC1001100&2016YFC1201805].
PS58: The effect of simple artificial shrinkage prior to vitrification on clinical and neonatal outcomes
Shingo Mitsuhata, Kurashiki Medical Clinic; Yuji Endo; Yoshitaka Fujii; Momoko Hayashi; Hiroaki Motoyama
Background: This study aimed to evaluate the clinical and neonatal outcomes of blastocysts treated with two simple artificial shrinkage methods, microsuction (MS) and hyperosmotic sucrose solutions (SUC), prior to vitrification.
Methods: This was a retrospective study based on data collected between May 2014 and August 2016. A total of 220 blastocysts obtained from 149 patients were analyzed. Warmed blastocysts were divided into two groups. In the MS group, which is a technique for mechanically aspirating the fluid from the blastocoel cavity using by a microneedle. In the SUC group, which induces chemically releasing of fluid from the blastocoel due to isotonic exchange of fluid.
Results: The mean maternal age and OPU trials between the groups were similar. The survival rate of the MS blastocyst was significantly higher in comparison with the SUC (98.1% vs. 90.3%, OR 5.66, 95% CI 1.37–23.26, P<0.05). The rates of implantation and live birth were observed higher in the MS than in the SUC, with no significant differences. There were no differences in gestational age, birth weight, proportion of male babies, and rates of cesarean section and congenital abnormalities.
Conclusion: The MS treatment for blastocyst led to significantly higher survival compared with the SUC in clinical outcomes, as a results, it might be improved the final outcomes. The MS method has several possible advantages, including simplicity, reliability and feasiblility, at all laboratories. It may be available for further technique of blastocentesis.
PS59 – (Chinese language abstract – see Chinese supplement)
Wang Jing, The Zhongda Affiliated Hosopital with Southeast University
PS60: Mutant mouse Zp1 results in defective zona pellucida and shrunken blastocysts
Dajing Yi, Institute of Reproductive & Stem Cell Engineering, School of Basic Medical Science, Central South University; Chao Lv; Yan Wang; Hong-Mei Xiao; Wenxian Yu
Background: Zona pellucida (ZP) is a layer of shell-like extracellular matrix enveloping the oocyte and early embryo, and is composed of several Zona pellucida glycoproteins (ZPs) secreted by the oocyte solely or primarily. Previously, we reported a ZP1 mutant mouse strain characterized by thinned or absent ZP of ovulated eggs and attenuated developmental competence of blastocysts. Aiming to obtain some clues to the mechanism of the ZP phenotype of this mouse strain, we explored the transportation and interaction of mutant ZP1 protein and other wild type ZPs (ZP2 and ZP3).We also performed IVF in order to find some valuable morphological changes of the blastocysts originating from the eggs of mutant mice, which may reflect the attenuated developmental competence.
Methods: (1) Zp1 gene mutated C57BL/6J mice were obtained through CRISPR/Cas9 technology. (2) Immunofluorescence. (3) Construction of expression plasmids for ZP1WT-Flag, ZP1MT-Flag, ZP2-Myc and ZP3-V5. (4) Expression in heterologous cells. (5) Immunoprecipitation. (6) In vitro fertilization and culture of preimplantation embyos.
Results: (1) In the mutant ovaries, both ZP2 and ZP3 were incorporated into a zona matrix and the observed signal was comparable with that seen in the normal ovary. ZP1 protein of mutant mice was detected only in cytoplasm and could not be localized in zona pellucida, which was inconsistent with normal mice. (2) FLAG-ZP1WT co-transfected with MYC-ZP2 and V5-ZP3, it did not interact with ZP2 and ZP3. When FLAG-ZP1MT co-transfected with MYC-ZP2 and V5-ZP3, unlike FLAG-ZP1WT, FLAG-ZP1MT interacted with ZP2 and did not interact with ZP3. (3) The eggs which ZP thinned and absent could be fertilized in vitro. The fertilization rates of ZP normal group, ZP thinned group and ZP absent group were 90.53%, 88.31%, 96.49%.Mutant mouse eggs can also develop to blastocyst stage in vitro, but the blastocyst formation rate is low. The morphology of mutant mouse blastocysts was smaller than that of normal mouse eggs.
Conclusion: We described a mouse strain with truncated ZP1 completely sequestrated in the cytoplasm and probably intracellularlly polymerized with wild type ZP2, leading to ZP thinned or absent eggs which would generate shrunken blastocysts with impaired developmental competence.
Funding: This study was supported by National Key R&D Program of China (2016YFC1201805 and 2017YFC1001100), National Natural Science Foundation of China (81471453 and 81501248).
PS61: Essential requirement of mammalian PUMILIO family in embryonic development
Kaibo Lin, Nanjing Medical University; Shikun Zhang; Qinghua Shi; Eugene Yujun Xu
Background: Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to a highly conserved RNA binding protein family PUF (Pumilio/FBF), whose homologs play critical regulatory roles in embryonic development and stem cell maintenance in invertebrate, and it remained unknown if they are also required for mammalian.
Methods: To identify physiological function of PUF family in mammalian, we generated Pum1 and Pum2 null mice model and established double-knockout embryonic stem cell (ESC) lines. Next we characterized the phenotype and explored the molecular mechanism underlying defects of double-knockout embryo in mice and ESC through qRT-PCR, western blot, HE staining, immunochemistry and AP staining.
Results: Our data show that loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3′UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.
Conclusion: Loss of PUM family proteins led to embryonic lethality by E8.5.
Severe gastrulation defects in double knockout embryos.
Pum family is dispensable for establishment of pluripotent stem cells and development to blastocyst stage.
Upregulated Endoderm marker-GATA6 may promotes epiblast cells endoderm priming and gastrulation failure in double knockout embryo.
Funding: This work was supported by National Basic Research Program of China (973 program, 2013CB945201 and 2015CB943002); National Science Foundation of China (81270737); Natural Science Foundation of Jiangsu Province (BK2012838); Provincial Innovation and Entrepreneurship Grant as well as NIH grant U01 HD045871. Funding for open access charge: Provincial Shuangchuang Program.
PS62: Whether the recovery of an embryo will affect the clinical pregnancy
Chen Bo Bo, Dongguan Third People’s Hospital Reproductive Center
Background: To investigate whether the speed at which embryos recover from cleavage during freeze-thaw embryo transplantation may affect the outcome of clinical pregnancy.
Methods: Retrospective analysis of 1359 frozen cycles of two embryos from January 2014 to November 2018. The cleavage velocity and quality of the embryos were graded at the end of thawing and before transplantation (1.5~2 hours after culture), and were divided into A0 group (no embryo growth), A1 group (one embryo growth) and A2 group (two embryos growth) according to the condition of embryo recovery cleavage.According to the rate of embryo cleavage recovery, it was divided into B0 group (no blastomere growth), B1 group (1~2 blastomeres growth) and B2 group (>2 blastomeres growth). The clinical pregnancy outcomes of each group at different ages were compared and multiple regression analysis was performed.
Results: After adjusting for factors such as protocol, number of transplants, endometrium grafts, distance to the fundus of the uterus, embryo score, embryo polykaryosis, and transplant personnel, the rate of embryo cleavage recovery was positively correlated with clinical pregnancy outcome only in the population less than 30 years old (OR: 5.18, 95% CI (1.14, 23.54), P=0.0331). However, there was no statistically significant difference in the clinical pregnancy outcome of freeze-thawing embryo with cleavage recovery at different ages (P>0.05).
Conclusion: This study indicated that the rate of recovery of cleavage of resuscitated embryos in vitro culture for 1.5 to 2 hours was associated with a higher pregnancy outcome in young people, while the recovery of cleavage of resuscitated embryos had no effect on the clinical pregnancy outcome, so the extension of in vitro culture time of freeze-thawed embryos (>2 hours) might be helpful to improve the clinical pregnancy outcome.
PS63: Correlation between standard blastocyst morphology evaluation, developmental rate, euploidy and pregnancy: a single center, retrospective, observational study involving 624 embryo biopsies
Hongmao Liu, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Guangxin Yao; Shuanggang Hu
Background: With the improvement of embryo culture and cryopreservation technology, trophoblast cell (TE) biopsy and preimplantation genetic screening (PGS) are considered as promising methods for selecting euploid embryos for transfer. Understanding the role of blastocyst morphology evaluation in the PGS cycle will help to further optimize cycle management and improve clinical outcomes. This study aims to investigate the association between blastocyst morphology, developmental rate, euploidy and implantation potential.
Methods: A total of 624 embryos obtained from 194 cycles of intracytoplasmic sperm injection (ICSI) followed by PGS were included in this retrospective study. Blastocysts were evaluated according to the Gardner’s scoring system. The screening was performed using Miseq platform. We evaluated the relationship between blastocyst morphology/developmental rate and euploidy/implantation/miscarriage.
Results: Of the 624 biopsied blastocysts, 342 were euploid and 282 aneuploid. It was observed that 61.62% of D5 and 36.31% of D6 blastocysts were euploid (P<0.001), while 68.54% of top quality TE and 38.06% of poor quality TE blastocysts were euploid (P<0.001). The implantation rates were 62.82% of D5 and 50.00% of D6 euploid blastocysts (P=0.148), while 64.38% of top quality TE and 61.76% of poor quality TE euploid blastocysts (P=0.775). No statistically significant differences in the miscarriage rate were found between D5 and D6 pregnant cycles (3.80% vs. 9.09%, P=0.301), even between top quality TE and poor quality TE pregnant cycles (3.42% vs. 8.82%, P=0.328).
Conclusion: Both blastocyst morphology and developmental rate were predictive of the PGS data to improve the selection of euploid embryos. None of the blastocyst features (morphology and developmental rate) was predictive of the implantation potential of euploid embryos.
Funding: Research was funded in part by the National Natural Science Foundation of China (grant number 81571435).
Endometriosis; Endometrium and Fallopian Tube; Benign Uterine Disorders
Thursday, 11 April 2019
PS64: Correlation and significance of programmed death factor ligand PD-L1 and PI3K/AKT signaling pathway in adenomyosis
Chunzi Lv, Shandong Provincial Hospital Affiliated for Shandong University; Lukanxuan Wu; Mingjiang Li
Background: Programmed death ligand-1 (PD-L1) is the main ligand for the immune inhibitory receptor Programmed death-1 (PD-1). This ligand is mainly expressed by immune cells, and also expressed on a great deal of non-immune cells. Other studies suggest that intracellular signals of PD-1 are transduced through PI3K/AKT pathway, and the activity of most immune cells is partly influenced by the PI3K/AKT pathway. In this study, we investigated whether PD-L1 expression is controlled by PI3K/AKT pathway, and correlations between the three in adenomyosis.
Methods: Expression of PD-L1, PI3K and pAKT were researched in primary normal eutopic cells and endometrial cells from patients with adenomyosis. The effect of PI3K/AKT signal pathway on expression of PD-L1 by endometrial cells were investigated.
Results: Some association was found between the level of PD-L1 expression and activation of AKT and its upstream molecule PI3K. Expression of PD-L1 in normal endometrial epithelial cells was inhibited, while the protein expression of PI3K, pAKT were normal. However, in endometriotic eutopic epithelial cells, the expression of PD-L1, PI3K and pAKT were significantly increased.
Conclusion: The high expression of PD-L1 and PI3K may facilitate epithelial cell proliferation in adenomyosis and promote the survival of endometriotic lesions. The high expression of PD-L1 may be related to the activation of PI3K/AKT pathway.
Funding: The study was supported by the grant NO. 2017YFC1001202 from the National Key Research and Development Program of China.
PS65: The microbiota of the uterine cavity and its relationship with intrauterine adhesions following induced abortion
Guojun Zhou, West China Second University Hospital; Tianji Liao; Jing Tan; Tingting Liu; Ying Long; Bin Luo; Huili Zhu; Wei Huang
Background: The vagina has long been regarded as home for a large number of bacteria, and an altered status of vaginal bacteria may be relevant to certain gynecological diseases and obstetrical events. With the help of new techniques, researchers have demonstrated that the upper genital tract, which had been believed to be sterile, is dominated by vaginal bacterial species.
Intrauterine adhesions (IUA) influence women’s menstrual patterns and impair their fertility. Curettage and other intrauterine procedures are commonly associated with IUA. Due to the high rates of induced abortion in China, IUA tends to be a widespread problem that negatively affects Chinese women’s reproductive health.
IUA is related to trauma-like vacuum suction and subsequent inflammation inside the uterine cavity. However, it remains unknown whether inflammation existed before surgery due to the existence of intrauterine microorganisms or inflammation is simply resulted from the iatrogenic procedure. Based on strictly pre-operative examination and aseptic operation, we speculated that intrauterine microbiota is highly likely to be responsible for post-abortion IUA.
Methods: We compared the uterine bacterial microbiota by using 16S rRNA gene amplicon sequencing and real-time quantitative polymerase chain reaction. 38 Chinese women of reproductive age were recruited, and samples from the reproductive tract were collected to analyze the bacteria, especially the intrauterine part in physiological (current pregnancy or not) or pathological situations (IUA following an induced abortion).
Results: 65 samples were analyzed by amplification and sequencing of the V3–V4 regions of 16S rRNA genes. Lactobacillus was found to be dominant in most vaginal and cervical samples. In the uterine cavity, Lactobacillus no longer dominated, and bacteria, such as Prevotella, took the lead in abundance. There was no significant difference in the microbiota between examinees with and without intrauterine adhesions. However, the microbiota of examinees with no previous curettage differed from that of the others.
Conclusion: Our study concludes that the intrauterine microbiota changes interpersonally and under certain conditions. There is a trend that the abundance of Lactobacillus decreases from the vagina up to the uterus. The bacterial profiles of examinees with previous curettage are more complex than those of nulligravida, suggesting intrauterine operation and pregnancy might be the cause.
PS66: The effect of number of embryo transferred on the outcome of ectopic pregnancy after embryo transfer in the patients with salpingectomy or proximal tubal occlusion
Yuan Zhang, The First Affiliated Hospital with Nanjing Medical University; Li Shu; Xiang Ma; Jiayin Liu
Background: The incidence of ectopic pregnancy after assisted reproductive technology (ART) is significantly higher than that after natural pregnancy, especially in some rare locations including interstitial tubal pregnancy. Previous studies have shown there is an increased incidence of interstitial pregnancy in patients with a history of pelvic surgery, especially those who underwent salpingectomy or proximal tubal occlusion, but the related risk factors have not been fully elucidated. The purpose of this study was to investigate the effect of different number of embryos transferred on the outcome of ectopic pregnancy in patients who had a history of salpingectomy or proximal tubal occlusion.
Methods: A retrospective analysis was conducted on the clinical outcomes of in vitro fertilization and embryo transfer (IVF-ET) in 11773 patients with at least one surgery history of unilateral/bilateral salpingectomy or proximal tubal occlusion: 4795 subjects in single embryo transfer group and 6978 in the double embryo transfer group. There was no significant difference in infertility duration, BMI, basic FSH and AMH between the two groups. The number of embryo transfer, clinical pregnancy rate, implantation rate, ectopic pregnancy rate and interstitial pregnancy rate were analyzed.
Results: The clinical pregnancy rate of single embryo transfer group was 40.08%, which was significantly lower than that of double embryo transfer group (50.14%). The implantation rate of single embryo transfer group was 39.61%, which was significantly higher than that of double embryo transfer group (32.14%). The rate of ectopic pregnancy in single embryo transfer group was 1.04%, which was significantly lower than that in twin embryo transfer group (1.77%). The incidence of Interstitial tubal pregnancy in single embryo transfer group was 0.21%, which was significantly lower than that in double embryo transfer group (0.63%).
Conclusion: The number of embryo transfer is a risk factor for ectopic pregnancy, especially interstitial pregnancy, in patients who have undergone unilateral/bilateral salpingectomy or proximal tubal occlusion. Single embryo transfer can reduce the risk of ectopic pregnancy, especially interstitial tubal pregnancy, and improve the safety of assisted reproductive technology along with a certain reliable implantation rate and clinical pregnancy rate.
PS67: Unfavorable oocyte retrieval caused by insufficient cumulus expansion during IVF in patients with endometriosis
Yaoxue Yin, Nanjing Medical University; Zhen Hou; Yugui Cui; Jiayin Liu
Background: Endometriosis (EMS) has been identified as a chronic inflammation characterized by pelvic pain and infertility, with great debilitating effects in reproductive aged women. ART (Assist reproductive technology), especially IVF (in vitro fertilization) makes it feasible that the infertile women with EMS have their own offspring. We found previously tough operative oocyte retrieval in EMS women. This study was to explore the underlying mechanism on the unfavorable oocyte retrieval during IVF.
Methods: This retrospective study was conducted, including women undergoing IVF recorded in our database between January 2013 and December 2017. The study included women with endometriosis-associated infertility, and the control group with tubal factor or male factor alone. Meanwhile, those women aged above 40 years, with polycystic ovary syndrome (PCOS), with decline in ovarian reverse (DOR), and whose number of punctured follicles blew 6, were excluded. Totally, 5269 women were analyzed (635 in the EMS group and 4634 in the control group). The clinical outcomes were compared between the two groups. The expressions of cumulus expansion-related genes, such as HAS2/PTX3/PTGS2/TNFAIP6, in cumulus cells of 19 infertile patients with EMS and 24 in the control group were analyzed by realtime-PCR.
Results: The mean oocyte retrieval rates were 0.763 (0.00258) and 0.718 (0.00575) (P<0.0001). Mean rinsing times of follicle were 1.108 (0.0256) and 3.857 (0.0766) (P<0.0001). The endometriosis proportion of population in the no-obtained oocyte was significantly higher, with the oocyte number from 0 to 2. There were no significant differences in the mean number of oocyte retrieval (odds ratio [OR] 1.002, P=0.8753), fertilization (OR 1.058, P=0.1112), clinical pregnancy rate (OR 1.002, P=0.9752) and the live birth rate (OR P=0.1978) between the two groups. Women with EMS had significantly lower expressions of cumulus expansion-related genes (HAS2/PTX3/PTGS2/TNFAIP6) in cumulus cells.
Conclusion: In conclusion, we proposed that the lower expression levels of cumulus expansion-related genes in cumulus cells may account for the insufficient cumulus expansion, suggesting an underlying mechanism involved in the poor oocyte retrieval.
PS68: Pregnancy outcome of endometriosis patients with repeated implantation failure, treated or not
Chenyi Zhong, Clinical Center for Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University; Liusijie Gao; Yundong Mao
Background: Our previous studies found that patients with Endometriosis (EMS) who had satisfactory treatment wound get a better pregnancy outcome of IVF/ICSI. How about the pregnancy outcome in those EMS patients with repeated implantation failure (RIF)? This study was designed to investigate retrospectively whether EMS treatment improve the pregnancy outcome of IVF/ICSI in those patients with RIF.
Methods: The cycle of 1386 endometriosis patients with RIF was selected from the CCRM database of our reproductive center (excluding the oocyte donation cycles). Before or after the time of RIF diagnosis, the patients was divided into untreated group,early treatment group and late treatment group. And according to the different treatment methods or satisfaction degree of treatment,patients were divided into different groups. In the cohort study, we compared the pregnancy outcomes of each group.
Results: (1) According to treatment time, the pregnancy rates of untreated, early and late treatment group were 29.7%, 35.9%, 31.3% (F=2.86, P=0.24), respectively. The abortion rates were 15%. 15.5% and 12.5% (F=4.96, P=0.08). The live birth rates of three groups were 20.5%, 29.3% and 26.1%, respectively and it was significantly higher in early treatment group than in untreated group (F=6.62, P=0.04). The cumulative live birth rates were 25.7%, 46.1% and 45%, respectively. After treatment, the cumulative live birth rate increased significantly. Difference between three groups was statistically significant (F=22.07, P<0.001). (2) According to the different treatment methods, the clinical pregnancy rates, the abortion rates, live birth rates and the cumulative live birth rates of untreated group, operation group and drug group did not show significant difference. (3) According to the patients’ satisfaction of disease control, differences of the clinical pregnancy rates, abortion rates, live birth rates and cumulative live birth rates of untreated group, satisficed group and unsatisfied group had no statistical significance.
Conclusion: (1) The treatment is necessary for EMS patients with RIF, which can improve cumulative live birth rate. (2) The cumulative live birth rate and live birth rate can be significantly improved by the early treatment.
PS69: LincRNA02075 regulates the proliferation and apoptosis of endometrial stromal cells by the anti-proliferative protein
Chenyi Zhong, Clinical Center for Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University; Liusijie Gao; Jingqin Mao; Yundong Mao
Background: Our previous studiesy found that the anti-proliferative protein (PHB) was closely related to pathophysiology of endometriosis (EMS). If the expression of PHB was down-regulated, the proliferation of ESCs was promoted while the apoptosis of ESCs inhibited. Interestingly, the lncRNA-linc02075 is a non-coding RNA in the upstream of PHB gene. This study was designed to explore the effects of linc02075 on the proliferation and apoptosis of endometrial cells and the mechanism related to PHB, so as to discuss the detailed effect of PHB in promoting the development of endometriosis.
Methods: (1) RNA was extracted from normal human endometrial tissues, eutopic endometrial tissues and Ectopic endometrial tissues of EMS patients, then camparing the difference of expression of linc02075 among the groups by qPCR. (2) Endometrial stromal cells were cultured in an in vitro normoxic environment, then the location of linc02075 in endometrial stromal cells were detected by FISH. (3) Changing the expression of linc02075 by overexpression plasmid, then we use CCK-8 to check the proliferation and tunel to check the Apoptosis of endometrial stromal cells.
Results: (1) Compared with the normal group (NE), in the EMS patient’s eutopic endometrium (EU) the expression of linc02075 in the endometrial tissue is reduced, and The expression level in the EMs patient’s ectopic endometrial group (EC) is increased. (2) Linc02075 is expressed in both the nucleus and the cytoplasm of endometrial stromal cells. (3) By changing the expression level of linc02075, the proliferation and apoptosis of endometrial stromal cells changed. When the expression level of linc02075 is increased, the proliferation ability of the cells is decreased and the apoptosis is increased.
Conclusion: (1) Linc02075 plays a positive role in the pathophysiology of endometriosis. The expression of Linc02075 in ectopic endometrial tissue is significantly increased. (2) Linc02075 regulates the proliferation and apoptosis of endometrial stromal cells by the PHB-related mechanism. (3) Linc02075 could be developed as a therapeutic target of EMS.
PS70 – (Chinese language abstract – see Chinese supplement)
Chen Xiao, JinHua People’s Hospital; Ouyang Luan
PS71: CCR5/CCR5 ligands induced myeloid-derived suppressor cells drive the process of endometriosis
Kaihuan Bi, Reproductive Medicine Center, The First Affiliated Hospital of Anhui Medical University; Zhimin Lu; Huanhuan Jiang; Zhimin Lu; Cao Yunxia; Kangxia Wang
Background: Immunologic disorder has been reported to promote the progression of endometriosis. Recently, several studies showed that myeloid-derived suppressor cells (MDSCs) drive the progression of endometriosis. It has been known that different chemokine receptors/chemokine signalings are related to the enrichment of MDSCs. However, the role of C-C chemokine receptor (CCR) 5 and its ligands is not established. This study aims to test if CCR5 and its ligands drive MDSCs accumulation and play a role in the progression of endometriosis.
Methods: We first defined CCR5 ligands and other cytokines in peripheral blood (PB) and peritoneal fluid (PF) from endometriosis (EMT) patients by ELISA kit. Then we characterized CCR5+MDSCs in PB and PF by flow cytometry.
Results: Our data showed that EMT patients displayed a significantly higher production of plasma CCL5, IL-17 and PF CCL5 and IL-12 as compared to those from controls. Furthermore, the levels of PF CCL 5 were elevated in advanced staged patients as compared to those from early staged patients. Accumulations of CCR5+MDSCs in PF were detected in advanced EMT patients as compared to those from early staged patients and controls. And EMT patients also showed an elevation of CCR5+MDSCs in PB. By relevance analysis, we found that enrichment of CCR5+MDSCs correlated with enhanced production of CCL5 in PF from EMT patients.
Conclusion: Our study revealed that CCR5 and its ligands drive the progression of endometriosis by enhancing the accumulation of MDSCs.
PS72: The overexpression of ERK activating the NF-KB signal and promoting the pathogenesis of women with endometriosis
Wangshu Li, The Maternity Hospital of DaLian; Rui CaoChunfang Ha
Background: Endometriosis is an estrogen-dependent inflammatory disease that affects up to 10% of women of childbearing age and accounts for 50% of female infertility cases. It is closely related to the adverse results of assisted reproductive technology (ART), including reduced oocyte recovery, low implantation rate and low pregnancy rate. A better understanding of the pathogenesis of endometriosis-associated infertility is essential to improve the efficacy of infertility. Endometriosis, the growth of endometrial tissue outside the uterine cavity, is associated with chronic pelvic pain, dysmenorrhea and subfertility. The etiology and pathogenesis of endometriosis are largely unknown. We therefore examined the relevant literature to identify published data on kinase activity in endometriotic tissue and to determine whether they were activated by components of the endometriotic extracellular environment. We focused on three specific pathways involving nuclear factor (NF)kB, mitogen-activated protein kinase (MAPK).
Methods: We used Western blot and real-time PCR to detect the expression of NF-κB p65 and ERK1/2 proteins and mRNA in eutopic and ectopic endometria of patients with endometriosis. Primary culture and identifcation of endometrial stromal cells (ESCs) was performed to investigate ERK 1/2 and NF-κB p65 expression levels in ESCs after ERK-specifc small interfering RNA(siRNA) downregulation.
Results: Expression levels of ERK1/2 and NF-κB p65 proteins in eutopic and ectopic endometria of endometriosis patients were higher than in normal endometria. Treatment of ESCs with siRNA directed against ERK reduced ERK, NF-кB nuclear translocation proteins and mRNA expression.
Conclusion: Our results suggest that ERK regulates the expression of NF- κ B p65 in endometriosis by activating NF- κ B signal transduction, thus providing new insights into the invasion and metastasis mechanism of endometriosis. It may also change the pelvic microenvironment and lead to infertility.
Funding: National Natural Science Foundation of China (No. 81660251).
PS73: The good clinical prediction value of sole CD138 count in the patients suffered one fresh embryo transfer failure
Xiangxiu Fan, The Reproductive and Genetic Hospital of CITIC-Xiangya; Fei Gong; Huijun Chen; Jingnan Liao; Xiaofeng Li
Background: Chronic endometritis (CE) is a chronic infectious disease, generally asymptomatic or has vague symptoms, so it is often ignored. But recently it has been rise up for focusing more attention on the potential association with various subgroups of reproductive failure. But it is so difficult to diagnose chronic endometritis because there are no typical clinical or ultrasound findings in these. Recently, Syndecan-1(CD138), seems plasma cell relative specific, which can be used to improve the diagnosis of chronic endometritis. meanwhile, it is believed overvalued. Therefore, we mainly aimed to explore the clinical predictive value of sole cd138 count in an infertile population.
Methods: We retrospective analyze 141 infertile subjects, who had experienced an endometrial biopsy in a natural cycle preceding frozen embryo transfer. These patients whose results of a histopathological examination and transvaginal ultrasound examination is no abnormal and had transferred at least one good embryo in the following frozen embryo transfer cycle. Syndecan-1(CD138) staining was identified by immunohistochemistry staining. The quantification of CD138 cells worked out by two different approaches; this may help us to know if the different approaches of quantitating CD138+cells would influence the ability of prediction. Additionally, to find out the risk factors related to cd138+count, the clinical data of these patients were collected.
Results: Through receiver operat characteristic curve, the CD138 positive cells count for successful clinical pregnancy was determined. The areas under the ROC curve in two approaches respectively are 0.660 and 0.658; it seems that there are no obvious differences between two approaches. The clinical pregnancy rate (80.04% vs. 52.7%) and embryo implantation rate (64.9% vs. 46.8%) were significantly higher in the patients whose CD138 count was under 1. And the clinical data show us the Pelvic effusion in the sampler day might be a risk factor for the cd138 positive.
Conclusion: CD138 counting could be a valuable marker of predicting frozen embryo cycle pregnancy outcome with whom suffered one fresh embryo implantation failure. We advise these patients to have a cd138 test in advance, whose trans-vaginal ultrasound results shows Pelvic effusion in sampler day. What’s more, the two means of counting CD138 would not differ the ability of prediction.
Funding: The National Science Foundation of China (81501328).
PS74: Alpinetin ameliorates inflammatory response in LPS-induced endometritis in mice
Wang Jing, The Zhongda Affiliated Hosopital with Southeast University
Background: Alpinetin, a plant flavonoid isolated from Alpinia katsumadai Hayata, has been known to have anti-inflammatory effects.
Methods: In the present study, we focused on the protective effects of alpinetin on LPS-induced endometritis in mice. Mouse model of endometritis was induced by LPS and alpinetin was given 1 h before LPS treatment.
Results: According to our results, alpinetin had protective effects against LPS-induced endometritis via attenuating uterus histological changes and MPO activity. LPS-induced inflammatory response was inhibiting by alpinetin as confirmed by the inhibition of TNF-α, IL-1ß, and IL-6 production. Furthermore, LPS-induced NF-κB activation was significantly suppressed by alpinetin. In addition, the expression of PPAR-γ was dose-dependently increased by the treatment of alpinetin.
Conclusion: Taken together, the data of this study showed that alpinetin had protective effects against LPS-induced endometritis in mice, the beneficial effects were associated with the activation of PPAR-γ and inhibition of NF-κB signaling pathway.
PS75 – (Chinese language abstract – see Chinese supplement)
PS76: Proteome analysis of human fallopian tube reveals enrichment of mesenchymal stem cell-associated proteins
Chenyuan Wang, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine; Shan Lu; Jie Gao; Fan Zhong; Gaopi Deng
Background: Human fallopian tube (hFT) plays a vital role in selecting spermatozoa. A few proteome analyses has been performed using hFT from tubal ectopic pregnancy, however, there is no complete proteomic profiling of normal hFT as reference proteome even in the Human Proteome Draft collections.
Methods: We collected normal hFT from 12 hysterectomy samples with myoma of uterus. Proteins were analyzed by mass spectrum-based proteome profiling. Peptide annotation and enriched pathway analysis were carried out using the web-accessible DAVID tool and Ingenuity Pathway Analysis.
Results: We reported a draft proteome of hFT comprising 5416 identified proteins, which is a physiological reference to complement the Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, and vesicle-associated proteins showed high abundance in hFT. Besides, we also found extraordinary high coverage of mesenchymal stem cell-associated proteins in the hFT.
Conclusion: The hFT proteome contains many markers newly identified by mass spectrum. Ribosome, cytoskeleton, vesicle and protein-folding-associated proteins were enriched in hFT. The extraordinary high coverage of mesenchymal stem cells-associated proteins in the hFT proteome suggests that hFT might contain mesenchymal stem cells.
Funding: Fully funded by the Guangdong Key Project Specialized for Preponderance Diseases in TCM [No: 2015YZY019].
PS77: Study of hCG in improving endometrial thickness and blood flow in recurrent implantation failure patients during frozen-thawed embryo transfer cycles
Wei Wang, The Reproductive Medicine Special Hospital of the 1st Hospital of Lanzhou University, Key Laboratory for Reproductive Medicine and Embryo; Yue Yuan; Xuehong Zhang
Background: To observe the effect of hCG on the endometrial thickness and endometrial blood flow in recurrent implantation failure (RIF) patients during frozen-thawed embryo transfer (FET) cycles, then to investigate the mechanism of hCG in improving endometrial receptivity.
Methods: A prospective study was carried out for RIF patients with artificial hormone replacement program during FET cycles. The RIF patients were randomly divided into hCG group: hCG 150 IU/d was intramuscular injected from the seventh day of menstrual cycle until the sixth day after embryo transfer, and control group: intramuscular injection of equal volume physiological saline from the seventh day of menstrual cycle until the sixth day after embryo transfer.
Results: There was no significant difference in the endometrial thickness, artery branches of the endometrial blood flow, PI and RI of uterine radiate artery, PI and RI of basal area spiral artery on the day of hCG/physiological saline onset (Day-0) between the two groups (P>0.05); there was no significant difference in the quality of transferred embryo and transferred embryo number between the two groups on the day of embryo transfer (Day-ET) (P>0.05); the endometrial thickness and artery branches of the endometrial blood flow in hCG group were higher than those of control group on the Day-ET (P<0.05); there was no significant difference in the PI and RI of uterine radiate artery between the two groups on the Day-ET (P>0.05); the PI and RI of basal area spiral artery in hCG group were smaller than those of control group on the Day-ET (P<0.05). There was no pregnancy when RI>0.72 and PI>1.53. The embryo implantation rate and clinical pregnancy rate of hCG group were significantly higher than those of control group (P<0.05).
Conclusion: For RIF patients with small-dose hCG added during their artificial hormone replacement FET cycles, hCG could enrich endometrial blood flow by promoting endometrial angiogenesis, then to increase endometrial thickness, and increase embryo implantation rate and clinical pregnancy rate finally.
PS78: Expression and role of HIF1α in adenomyosis
Yang Yang, Qilu Hospital of Shandong University; Yufei Huang; Xiaohui Deng; Lan Chao
Background: To investigate the expression and role of hypoxia-inducible factor-1alpha (HIF-1α) in adenomyosis eutopic endometrial tissues.
Methods: Immunohistochemistry dyeing was used to detect localization of HIF-1α in adenomyosis eutopic endometrium and normal endometrium. Real-time PCR was used to detect the mRNA level of HIF-1α, hexokinase 2 (HK2) in adenomyosis eutopic endometrium and normal endometrium. Western blotting were used to detect the protein level of HIF-1α and HK2 in adenomyosis eutopic endometrium and normal endometrium.
Results: HIF-1α was mainly expressed in the glandular epithelial cytoplasm of normal endometrial tissue and adenomyosis endometrial tissue. The mRNA expression of HIF-1α and HK2 in adenomyosis eutopic endometrium were higher than those in normal endometrium. The protein expression of HIF-1α and HK2 in adenomyosis eutopic endometrium were higher than those in normal endometrium. There was no significant difference in the expression of HIF-1α and HK2 in the proliferative phase (P0.05).
Conclusion: HIF-1α may promotes glycolysis and plays an important role in the development of adenomyosis.
PS79: MiR-210-3p protects endometriotic cells from oxidative stress-induced cell cycle arrest by targeting BARD1
Xiang Lin, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine; Yongdong Dai; Songying Zhang
Background: Endometriosis is associated with benign but adversely developed cysts in the extrauterine environment. The oxidative imbalanced environment induces DNA damage and affects cell cycle progression of endometrial stromal cells (ESCs) and endometrial epithelial cells, but how endometriotic cells maintain proliferation in the presence of oxidative stress is not clear. Growing evidence has indicated that the ectopic hypoxic microenvironment and oxidative stress can stimulate the growth of endometriotic cells, which is mainly due to the increase of HIF-1α.
Methods: Immunohistochemistry; Alkaline comet assay; High-throughput sequencing; qRT-PCR and Western blot; FISH analysis; Luciferase assay; Flow cytometry assay; Endometriotic mouse model analysis; linear regression analysis and univariate linear regression analyses.
Results: Endometriotic lesions show increased expression of HIF-1α and an oxidative stress induced DNA damage marker-8-OHdG; In vitro hypoxia culture induces DNA damage in ESCs and Ishikawa cells; Hypoxic culture stimulates miR-210-3p expression in ESCs and Ishikawa cells; MiR-210-3p is overexpressed in endometriotic lesions; MiR-210-3p knockdown results in G2/M cell cycle arrest under hypoxia; MiR-210-3p knockdown under hypoxia causes cell cycle arrest signal activation; MiR-210-3p decreases BARD1 expression by directly targeting its 3’-UTR and BARD1 is an indicator of endometriosis severity; Complementing BARD1 expression in hypoxia recovers cell cycle arrest activation; MiR-210-3p inhibitor alleviates endometriosis progression in vivo.
Conclusion: Oxidative stress (OS) and hypoxia are both involved in endometriosis but exhibit inconsistent functions. Excessive OS in endometriotic lesions triggers the DNA damage response and results in cell cycle arrest, while hypoxia is essential for establishment of ectopic lesions. Our study illuminates a novel mechanism that resolved OS-generated DNA damage and hypoxia-stimulated cell cycle progression in endometriosis through the activity of a hypoxia-responsive miRNA—miR-210-3p. We propose upregulated miR-210-3p in endometriosis directly targets BARD1, leading to physiological and biochemical function impairment of the BRCA1 complex, which results in an inactivation of cell cycle arrest in response to DNA damage and finally trigger cell cycle progress and endometriosis development.
PS80: A cohort study of the effects of different interventions on IVF/ICSI outcomes in patients with endometriosis
Chunxiang Yang, Reproductive Medicine Center, Department of Obstetrics and Gynecology, Northern Jiangsu People’s Hospital, Yangzhou University; Yuan Zhang; Chunxiang Wu; Jie Huang; Li Shu; Zhen Hou; Jinyong Liu; Xiang Ma; Wei Ding; Jiayin Liu; Yundong Mao
Background: Observe the effects of different interventions on IVF/ICSI outcomes in women with endometriosis related infertility, and explore ways to effectively improve in vitro fertilization and embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) pregnancy outcomes in patients with endometriosis.
Methods: This was a retrospective cohort study, a total of 488 women who were treated with IVF/ICSI were enrolled from January 2008 to December 2017. Patients were divided into four groups, the untreated group (group A, n=102), the GnRH-a treatment group (group B, n=97), the surgery-only group (group C, n=152), and the surgery +GnRH-a treatment group (group D, n=137). The pregnancy outcomes of IVF/ICSI under different interventions were compared. In addition, the last three groups were stratified according to the level of disease control satisfaction, and compared the differences in IVF/ICSI outcomes among different levels of control satisfaction.
Results: The group D has higher clinical pregnancy rate (44.8% vs. 17.2%, 22.8%, 31.9%, P<0.05) and cumulative live birth rate (85.4% vs. 44.1%, 71.1%, 73.7%, P<0.05) than the other three groups (group A, B, C). The group D has lower abortion rate (22.2% vs. 45.2%, 38.0%, 24.3%, P<0.05) and higher live birth rate (27.0% vs. 9.2%, 13.0%, 23.5%, P<0.05) than the other three groups (group A, B, C). In group C, the clinical pregnancy rate (37.3% vs. 27.7%, P<0.05) and cumulative live birth rate (82.6% vs. 66.3%, P<0.05) were higher in satisfied group than those in the unsatisfied group. Within group D, the clinical pregnancy rate (50.7% vs. 39.0%, P<0.05) and cumulative live birth rate (92.4% vs. 78.4%, P<0.05) of the satisfied group were higher than those of the unsatisfied group. The difference was statistically significant.
Conclusion: GnRH-a and surgical treatment can improve IVF/ICSI pregnancy outcome in patients with endometriosis, especially surgery combined with GnRH-a treatment, the outcome of IVF/ICSI in patients with endometriosis will be significantly improved. Patients with endometriosis had significantly improved IVF/ICSI outcomes when their condition was controlled satisfactorily.
PS81: Aberrant overexpression of RNA editing enzyme ADAR1 promotes endometriosis progression
Zhe Li, The Third Affiliated Hospital of Zhengzhou University; Lijun Sun; Lulu Wang
Background: Endometriosis is a benign lesion in histopathology, while it has the biological characteristics of malignant tumors. Overexpression of ADAR1 can increase tumor-related characteristics in cellular and mouse model studies, such as cell proliferation, migration and invasion, and the knockdown of ADAR1 reduces these characteristics. Endometriosis has similar biological characteristics to tumors. Therefore, we wonder whether ADAR1 may have some influence on the occurrence and development of endometriosis. Herein, we investigated the relationship between ADAR1 and endometriosis by detecting the expression of ADAR1 in endometriosis and normal endometrium cells and comparing the effect of ADAR1 on proliferation, apoptosis, invasion and migration of endometriosis cells.
Methods: Eutopic endometrium was respectively collected from women with endometriosis as study group by laparoscopy and the women without endometriosis group as control group by hysteroscopy. The expression level of ADAR1 mRNA was measured by real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot and immunohistochemistry were performed to assess the ADAR1 protein expression levels. Then, we investigated the effect of ADAR1 on the biological function of eutopic endometrial cells in endometriosis by examining cell viability, proliferation, invasion and migration.
Results: RT-qPCR analysis showed that the ADAR1 mRNA expression was upregulated in eutopic endometrium of endometriosis compared to that in the endometrium of patients without endometriosis. Western blot and immunohistochemistry indicated that ADAR1 protein expression in patients with endometriosis was significantly higher than that in the control group (P<0.05). Moreover, in vitro assay demonstrated that ADAR1 promoted endometrial cell viability, proliferation, migration and invasion.
Conclusion: In summary, the expression of ADAR1 gene in eutopic endometrium of endometriosis is over-expressed compared with that in normal endometrium of the control group. And ADAR1 is a promotor for the progression of endometriosis. This paper presents preliminary evidence that ADAR1 may be involved in the pathogenesis of endometriosis.
PS82: Niche cellular components of endometrial stem cells
Rachel Chan, University of Hong Kong; Ernest Ng; William Yeung
Background: In every menstrual cycle, the human endometrium is shed and the whole endometrium is regenerated in the following cycle. The endometrial stem cells are believed to be responsible for the tissue regeneration. They reside in the basalis layer of the endometrium. We are interested in a population of endometrial mesenchymal stem cell-like cells (eMSCs). These cells co-express two surface markers CD140b and CD146. They form colonies, undergo self-renewal and can differentiate into four mesenchymal (adipogenic, myogenic, osteogenic, or chondrogenic) lineages. We have identified eMSC-like cells in ovarian endometrioma. The surrounding microenvironment (niche) regulates activities of somatic stem cells. We test the hypothesis that the myometrial cells and the endometrial cells are niche cells that regulate the biological activities of eMSCs.
Methods: Full thickness endometrial tissue comprising myometrium were obtained from ovulating women. Endometrial cells on Day 2 of the menstrual cycles were obtained by endometrial aspirates. Coculture of eMSCs with or without myometrial cells and endometrial cells in culture insert were performed. The number of eMSCs colonies formed and the expression of eMSCs markers were determined.
Results: Significantly more eMSCs are found in the basalis layer than the functionalis layer. The eMSCs in the two layers have similar clonogenic and self-renewal activities. Coculture with myometrial cells and endometrial cells on Day 2 of the menstrual cycles enhanced the clonogenicity, self-renewal and total cell output of eMSCs. The positive effects did not require direct niche cells-eMSCs contact. The results suggested that secretory product(s) from the myometrial cells and the endometrial cells acted on the eMSCs for the beneficial effects.
Conclusion: Myometrial cells and endometrial cells are niches cells of eMSCs and regulate their activities by paracrine factor(s). Efforts are being made in understanding the molecular mechanism regulating these activities of eMSCs. In future, we may be able to treat women with thin endometrium by manipulation of the niche environment.
Funding: The study is supported by the GRF grants 17125815 and 17158316 to EHYN, Sanming Project of Shenzhen and Shenzhen Knowledge Innovation Plan for Basic Research 20180264.
PS83: Sonic hedgehog signaling induces intrauterine adhesion through inhibiting endometrial stromal autophagy
Cheng Wei, Zhejiang University; Xiaona Lin; Yibin Pan; Songying Zhang
Background: Intrauterine adhesions (IUA) are the second leading cause of female infertility, mainly caused by trauma or infection. Therefore, it is important to explore the mechanism of IUA development. Previous research found that activation of the sonic hedgehog (SHH) pathway plays a key role in the pathogenesis of IUA. In this abstract, we focus on whether SHH pathway affects fibrosis by regulating autophagy.
Methods: IUA and normal endometrium in proliferative phase were collected. The key molecules of SHH pathway (SHH, PTCH1, GLI2, etc) and autophagy-related molecules (LC3B, Beclin1, etc) were compared between the two groups by RT-qPCR and Western bloting. Secondly, to investigate how Shh pathway regulates autophagy, Shh inhibitor (Cyclopamine/GANT61) dose and time experiments are performed on immortalized human endometrial stromal cell line (T-HESC), with LC3B expression and tandem mCherry-GFP fluorescence microscopy performed to reflect autophagy status, and with collagen I and a-SMA expression examined to reflect fibrotic status. Thirdly, to assess whether the regulation of Shh pathway on endometrial fibrosis are autophagy-dependent, autophagy drugs (3-MA/chloroquine) combined with GANT61, are used to study the role of Shh-autophagy on fibrosis.
Results: Among clinical samples, SHH/GLI2 protein and PTCH1 mRNA are increased, autophagy-related proteins are decreased in IUA group, suggesting that in IUA Shh pathway is activated, while autophagy are impeded. Secondly, in T-HESC, Shh inhibitor (Cyclopamine/GANT61) can increase LC3B-II in a gradient-dependent/time-dependent manner, accompanied by a decrease of fibrotic markers (collagen I/aSMA). Upon LC3B-II turnover assay and tandem mCherry-GFP fluorescence microscopy, it is found that GANT61 can promote autophagosome production. In autophagy inhibitor reversal experiments, autophagy drugs (3-MA/chloroquine) can reverse the effect of GANT61 on fibrotic markers.
Conclusion: Activation of SHH pathway impedes autophagy of endometrial stromal cells, which plays an important role in the process of endometrial fibrosis.
PS84: Interlukin-1 regulates oxytocin receptor in endometrial stromal cells and decidual cells via NF-κB pathway
Yeqing Fu, Reproductive Medicine Center of Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology; Cong Sui; Xiyuan Dong; Junning Yao; Shulin Yang; Hanwang Zhang
Background: Oxytocin (OT) and its corresponding receptor (OTR) are known to be crucial in the process of parturition and lactation. In recent years, more biological processes that OT/OTR system participates has been revealed, including cell proliferation, differentiation, angiogenesis and inflammatory response. A recent study found elevated expression of oxytocin receptor on day 7-9 of pregnancy in mouse endometrium, especially at the implantation sites. Thus, we hypothesized this elevation might be induced by signals from embryos and/or trophoblasts, such as IL-1 family, which has been believed to be associated with successful implantation.
Methods: Endometrial stromal cells or decidual cells were treated with IL-1β with different conerntation (0, 0.01, 0.1, 1, 10 ng/mL) and time (2, 4, 6, 8 hour). Then p65 siRNA was administrated to inhibit the NF-κB pathway. OTR expression in each group was meaured after the treatments, respectively. IL-1β was also injected into uterine or peritoneal cavity or of mice to investigate whether OTR was regualted in vivo.
Results: The results showed that administration of IL-1β elevated the expression of OTR mRNA and protein in both endometrial stomal cells and decidual cells, in a dose and time dependent manner. However, this elevation was weakened by p65 siRNA, which blocked the expression of p65. The intrauterine and intraperitoneal injection of IL-1β both upregulated the expression of OTR in the endometrium of mice.
Conclusion: This study showed that OTR expression in endometrium was upregulated by IL-1β via NF-kB signaling pathway.
Funding: This study was supported by grant from the National Natural Science Foundation of China(No. 81471459).
PS85: Analysis of the pregnancy outcomes in 55 women with a didelphus uterus after in vitro fertilization-embryo transfer
Qingqing Wu, Reproductive & Genetic Hospital of Citic-xiangya; Xihong Li; Yan Ouyang; Pei Cai; Qingwen He
Background: Didelphus uterus is a congenital malformation resulting from the abnormal development of Mullerian ducts during embryogenesis. According to the literature, the incidence of didelphus uterus is about 1/10000. The two independent uterus have fertility function, but because of severe uterine malformation, miscarriage can occur during pregnancy and even infertility can be caused.
Methods: The clinical data of 55 infertility patients who were diagnosed with a didelphus uterus and got clinical pregnancy via IVF treatment from January 2003 to December 2016 were retrospectively analyzed. According to ultrasound examination, the 55 patients were divided into singleton-pregnancy group (n=44) and twin-pregnancy group (n=11). The pregnancy outcomes between these two groups were compared.
Results: There was no significant difference in the mean maternal age, infertility duration and infertility factors between the singleton-pregancy group and the twin-pregnancy group (P>0.05). When compared to the singleton-pregnancy group, twin-pregnancy had a significantly higher perinatal mortality [40% (8/20) vs. 0 (0/35), P<0.001], and highr rates of preterm delivery [72.7% (8/11) vs. 18.2% (8/44), P=0.001], and low birth weight[83.3% (10/12) vs. 14.3% (5/35), P<0.001].
In addition, twin pregnancy can also significantly lower the gestational weeks at delivery (30.7±6.5 vs. 38.1±2.2, P=0.006) and the birth weight (1965.0±419.4 vs. 3023.7±606.9,P<0.001). Simultaneously, the rates of miscarriage [9.1% (1/11)vs. 15.9% (7/44), P=1.000] and live birth [54.5% (6/11) vs. 79.5% (35/44), P=0.124] in twin pregnancy group were lower than those in the singleton-pregnancy group, but the difference was not statistically significant.
Conclusion: Singleton-pregnancy could obtain better pregnancy outcomes than twin-pregnancy in women with didelphus uterus anomaly after in vitro fertilization-embryo transfer. Therefore, reducing the incidence of twin pregnancy in women with a didelphus uterus is clinically necessary.
Funding: The Science and technology project of Health and Family Planning Commission of Hunan Province (No. C20180898) and the Citic-Xiangya Research Fund (No. KYXM-201703).
PS86: Comparative study of twin pregnancy outcomes between women with a unicornuate uterus and a didelphus uterus after in vitro fertilization-embryo transfer
Pei Cai, Institute of Reproductive and Stem Cell Engineering, Central South University, China; Yan Ouyang; Xihong Li; Qingqing Wu; Fei Gong; Qingwen He
Background: The unicornuate uterus and didelphus uterus result from the abnormal development of Müllerian ducts during embryogenesis. The prevalence of them were estimated to be 0.5% (0.3-0.8%) and 0.3% (0.2-0.5%) in women with infertility, respectively. Our previous study demonstrated that twin pregnancy in women with a unicornuate uterus had worse pregnancy outcomes compared to those with a normal uterus after in vitro fertilization-embryo transfer (IVF-ET). However, the study regarding outcomes of women with a didelphus uterus carrying a twin pregnancy is rarely reported. The difference of twin pregnancy outcomes between women with a didelphus uterus and a unicornuate uterus after IVF-ET is also unclear.
Methods: 54 cases of twin pregnancy in women with a unicornuate uterus from and 16 cases of twin pregnancy in women with a didelphus uterus from January 2003 to December 2017 after IVF-ET at our reproductive centre were retrospectively analyzed. The diagnosis of uterine malformations were based on American Fertility Society classification system. Women with the monochorionic twins and spontaneous or selective reduction in one fetal of twins were excluded. The pregnancy and obstetric outcomes between these two groups were compared.
Results: Compared with the unicornuate uterus group, didelphus uterus had higher rates of very preterm birth [42.9 vs. 17.0%; P=0.044; OR 3.66; 95% confidence interval (CI) 0.99-13.46] and low birth weight (89.5 vs. 58.2%; P=0.011; OR 6.10; 95% CI 1.32-28.21); the live birth weight (1944±387 vs. 2284±476 g; P=0.005) was significantly lower in the didelphus uterus group. Additionally, miscarriage rate (12.5 vs. 13.0%; P>0.05; OR 0.96; 95% CI 0.18-5.15) was similar between the two groups; preterm delivery rate (75.0 vs. 57.4%; P >0.05; OR 2.23; 95% CI 0.64-7.80) and perinatal mortality (32.1 vs. 16.0%; P>0.05; OR 2.50; 95% CI 0.95-6.56) were higher and live birth rate (62.5 vs. 75.9%; P >0.05; OR 0.53; 95% CI 0.16-1.74) and the gestational age at delivery (31.3±5.7 vs. 34.3±4.0 wk; P>0.05) were lower in the didelphus uterus group, but those difference were not significant between the two groups.
Conclusion: For twin pregnancy, women with a didelphus uterus had significantly worse obstetric outcomes than those with a unicornuate uterus.
Funding: The Science and technology project of Health and Family Planning Commission of Hunan Province (No. C20180898) and the Citic-Xiangya Research Fund (No. KYXM-201703).
PS87: Twin pregnancy outcomes of women with a Caesarean section scar after in vitro fertilization-embryo transfer
Yuyao Mao, Reproductive & Genetic Hospital of Citic-xiangya; Xihong Li; Yan Ouyang; Jingzi Xiao
Background: The Caesarean delivery rate has increased markedly in the past few decades throughout the world. Women with a scarred uterus, always with greater age, are more likely to seek pregnancies via in vitro fertilization-embryo transfer (IVF-ET). However, IVF procedure increases the probability of twin pregnancy and the Caesarean scar has been reported to be associated with many complications. Whether reduction is necessary for twin pregnancy in women with a Caesarean section scar becomes a problem. The objective of our study was to investigate the pregnancy outcomes of twin pregnancies in women with a Caesarean scar after IVF-ET.
Methods: The study group comprised 96 patients with a Caesarean scar from January 2015 to December 2016, and 167 patients with a normal uterus during the same time period were selected as the control group. The pregnancy outcomes of these two groups were compared. Only patients who were diagnosed as intrauterine dichorionic twin pregnancy by early transvaginal sonography in our hospital were included.
Results: Compared with the control group, the study group had higher rates of premature delivery (46.9% vs. 41.3%, P=0.381) and live birth (96.9% vs. 92.8%, P=0.172), lower rates of early pregnancy loss (2.1% vs. 2.4%, P=1.0), late miscarriage (1.0% vs. 4.2%, P=0.265), term delivery (50.0% vs. 52.1%, P=0.743), low birth weight (36.2% vs. 41.0%, P=0.288) and low perinatal mortality (0.5% vs. 1.6%, P=0.418), but the differences were not statistically significant. The gestational age at delivery (36.6±1.6 vs. 36.5±2.34 wk, P=0.726) and the live birthweight (2569.7±450.9 vs. 2516.8±497.7 g, P=0.237) were similar between the two groups. While a Caesarean scar was associated with a significant increased risk of Caesarean section (98.9% vs. 90.4%, P=0.008).
Conclusion: The pregnancy outcomes of twin pregnancies in women with a Caesarean scar were similar to those of a normal uterus after IVF-ET, but a significantly higher cesarean section rate was also observed in a scarred uterus.
Funding: The science and technology major project of the ministry of science and technology of Hunan Province, China (2017SK1031-4).
PS88: Effect of different endometrial factors on pregnancy outcome of embryo transplantation
Jiang Lijie, Peking University International Hospital; Li Tian
Background: Many studies have shown that hysteroscopy can significantly improve the embryo transfer pregnancy outcomes. However, it is not clear that hysteroscopy has the same effects in different endometrial diseases. The purpose of this study is to compare the outcomes of embryo transplantation after hysteroscopy for different endometrial diseases.
Methods: 130 patients with different endometrial diseases who underwent hysteroscopy before embryo transplantation in Peking University International Hospital from 2015.9 to 2018.11, these patients are divided into pregnancy group and non-pregnancy group. The age, infertility years, preoperative B-ultrasound diagnosis (intrauterine space occupying, endometrium thinning, endometrium heterogeneity, normal intrauterine cavity) and postoperative pathological diagnosis (intrauterine adhesion, endometrial polyp, endometritis, normal intrauterine cavity) of the patients in the two groups were counted respectively, and the differences in the above aspects between the two groups were compared. T test was used for the age and infertility years of the two groups, preoperative b-ultrasound diagnosis and postoperative pathological diagnosis were performed by chi-square test.
Results: (1) There is no difference between two groups in the age (32.92 vs. 32.96, P=0.486), and gestational age (2.11 vs. 2.32, P=0.623); (2) There is no difference in pregnancy rate of different diseases diagnosed by preoperative b-ultrasound (P=0.134); (3) Different postoperative diagnosis leads to different rate of pregnancy (P=0.026). Moreover, further study shows that there is no statistical difference in the pregnancy rate of uterine adhesion, endometritis and normal uterine cavity, but the pregnancy rate of endometrial polyps is higher than that of non-pregnancy (69.7% vs. 30.3%).
Conclusion: Endometrial polyps after hysteroscopy may lead to infertility easier.
PS89: Proviral insertion in murine lymphomas 2 (PIM2) oncogene phosphorylates Î²-catenin and promotes endometriosis progression
Tingting Yang, Reproductive Hospital of Weifang Medical University; Zhenhai Yu; Chun-E Ren
Background: Endometriosis is a common gynecologic disease, the exact molecular mechanisms remain poorly elucidated. In tumors, PIM kinases 2 mediate the biological function through phosphorylating a wide range of target proteins. However, only a few studies have shown promising effects of PIM2 in endometriosis. While Wnt/β-catenin signaling is recently found to be aberrantly expressed in endometriosis. This study sought to evaluate whether PIM2 promotes the development of endometriosis through Wnt/β-catenin signaling.
Methods: The expression of PIM2 was investigated using immunohistochemistry in a panel of normal endometrium (n=20), eutopic endometrium (n=20) and ectopic endometrium (n=40). Methylation status of the EFEMP1 promoter. We also exam the expression of EMT related marker such as E-cadherin, Vimtain, β-Catenin and FMT related a-SMA, collagen I. Up- or down-regulation of PIM2 were achieved by transfection with pCMV6/GFP/Neo-PIM2 or PIM2 inhibitor 4A. Effects of PIM2 on tumor proliferation, invasion and migration were evaluated by MTT, plate colony formation, Transwell and wound healing assay. Through DNA constructs and Immunoprecipitation and GST-pulldown assays to prove PIM2 directory combine to β-Catenin and the site of phosphorylation of β-Catenin. The endometriosis mouse model was used to investigate function of PIM2 in vivo.
Results: The expression of PIM2 in ectopic endometrium was higher than normal and eutopic endometrium. PIM2 was negative related with E-cadherin and positive with Vim and β-catenin. In vitro and vivo, PIM2 promoted the development of endomtriosis and tissue fibrosis. PIM2 phosphorylated β-catenin at the site of Ser552 and promoted the activity of Wnt signaling.
Conclusion: Our findings propose that PIM2 can infulance the progress of EMT and FMT in endometriosis. The function is through phosphorylation of the key protein β-catenin of Wnt signaling.
Funding: The study was supported by research grants from Innovation Fund of National Natural Science Foundation of China (Grant no. 81602440, 81602301, and 81471048).
PS90: Management of Â endometrialÂ polypsÂ or endometrial polyp—like hyperplasia incidentally diagnosed during IVF
Wanjun Jiang, Institute of Reproductive and Stem Cell Engineering, Basic Medicine College, Central South University; Fei Gong; Hui Chen
Background: An endometrial polyp is a frequently encountered abnormality of the uterine cavity that may interfere with normal embryo implantation. The aim of the current study was to investigate the clinical efficacy of hysteroscopic removal of polyps or endometrial polyp-like hyperplasia during controlled ovarian hyperstimulation.
Methods: A retrospective, matched, cohort study at the Reproductive and Genetic Hospital of CITIC-XIANGYA. We enrolled the women in whom endometrial polyps or endometrial polyp--like hyperplasia were incidentally diagnosed by transvaginal ultrasound, office hysteroscopy and histological examination during IVF (group 1). The exclusion criteria were: (1) age ≥40 years, (2) pathology conditions such as hydrosalpinx, uterine adenomyosis, uterine fibroids (submucosal fibroids, non-mucosal fibroids >3 cm or compressing the endometrium) and uterine malformations (unicornuate uterus, bicornuate uterus, double uterus, T-shaped uterus and uterine mediastinum), intrauterine adhesion, (3) no embryo transfer. The women who had removal of endometrial polyps or endometrial polyp--like hyperplasia before IVF 1-2 months (group 2) were 1:1 matched for the age, body mass index, rank of fresh embryo transfer, endometrial thickness at embryo transfer and the type and number of embryos transferred. The primary outcome was ongoing pregnancy rate (OPR); the secondary outcomes were implantation rate (IR), clinical pregnancy rate (CPR), early miscarriage rate (EMR) and ectopic pregnancy rate (EPR).
Results: Total of 194 women were included for final analysis. The OPR (67.0% vs. 70.1%), IR (51.6% vs. 52.7%), CPR (73.2% vs. 74.2%), EMR (8.5% vs. 4.2%) and EPR (1.1% vs. 1.1%) were similar between group 1 and group 2 (P>0.05).
Conclusion: For women with endometrial polyps or endometrial polyp-like hyperplasia incidentally diagnosed during IVF, pregnancy outcomes are not compromised after hysteroscopic management followed by fresh embryo transfer.
PS91: Endometrioma facilitated surrounding ovarian tissue fibrosis and attenuated oocyte quality
Libing Shi, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine; Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province; Yinli Zhang; Feng Zhou; Songying Zhang
Background: To elucidate whether the endometriotic loci of endometriomas specifically synthesized transforming growth factor beta 1 (TGF-β1) and how it affects surrounding ovarian tissue and oocyte quality.
Methods: Comparing mRNA and protein expression of fibrosis-related factors between the cystic walls of endometriomas and mature teratomas, detection of TGF-β1 mRNA synthesis in endometriomas, and testing TGF-β1 fibrotic effect in vitro, to understand the role of TGF-β1/Smad signaling in fibrosis formation in the tissue surrounding endometriomas. Investigating oocyte nucleus maturation and cytoplasm maturation on endometriomas rat model through comparing tublin and JC-1 expressing respectively.
Results: Compared with the mature teratomas, the cystic walls from endometriomas demonstrated severe adhesion to ovarian tissue and obvious fibrosis, which was proven by the increased mRNA expression of fibrotic markers and highly TGF-β1 protein expression. Additionally, TGF-β1 mRNA was confirmed to be specifically synthesized in the endometriotic cells. And cultured EnSCs showed obvious fibrosis after exposed to TGF-β1. Interestingly, components of the TGF-β1/Smad pathway and their phosphorylated forms were expressed in the same location as TGF-β1, TGFβR1, and fibrotic factors expressed in the endometriotic cystic wall. The nucleus maturation and cytoplasm maturation of endometriomas rats’ oocytes are decreased.
Conclusion: Endometriotic cells of endometriomas synthesize TGF-β1 leading to fibrosis and adhesion to ovarian tissues by activating TGF-β1/Smad signaling. And the oocyte quality around endometriomas is attenuated.
Funding: This work was supported by grants from the Natural Science Foundation of China (81471505, 81402131 and 81601236).
Thursday, 11 April 2019
PS92: Assisted reproductive technology service provision and utilization in China
Fu Bai, National Center for Women and Children’s Health, China CDC; Dongying Wang
Background: This study aimed to describe the development trend of ART (Assisted Reproductive Technology) service provision (clinic numbers, regional distributions and type of services provided) and ART service utilization in China from 2012 to 2016.
Methods: This study conducted base on the open data of National Health Commission of the People’s Republic of China. Two cross-sectional official reporting data of ART clinics and ART services were described. Clinic numbers, regional distributions and type of services and total number of ART cycles referred to 2012 and 2016 were analyzed and reported respectively.
Results: The-past-five-year has witnessed great progresses in ART development in China. According to the national reports, the numbers of clinics increased greatly from 356 in 2012 to 451 in 2016. As a vast imbalance of both territory and economic development exists, the clinic numbers, geographical distributions and services type provided showed great uneven allocation accordingly. The ART clinics in eastern regions occupied 53.9% in 2012, whereas this percentage slightly increase to 54.5% in 2016, while in the west, the percentage is 16.7% in 2012, just increased a little bit to 17.5% in 2016. With the development of this technology, almost all types of ART services can be provided in China. In 2016, the number of clinics which can provide intrauterine insemination using husband (IUI-H) only is 121, intrauterine insemination using donor (IUI-D) is 60, In Vitro Fertilization and Embryo Transfer (IVF-ET)/Intracytoplasmic sperm injection (ICSI) is 327 and preimplantation genetic diagnosis (PGD) is 40. Furthermore the total number of ART clinics was significantly correlated with regional GDP (r=0.868) and Population (r=0.797). In 2012, total initiated cycles were 507190 (103730 artificial insemination cycles included), with 158287 live births. While in 2016, total initiated cycles were 1068216 (161376 artificial insemination cycles included), with 311309 live births.
Conclusion: From 2012 to 2016 in China, the numbers of ART clinics, services provision and clinic distribution all manifested significantly changes. ART service availability in China have been remarkably improved, while ART service provision and utilization imbalance still exist.
PS93 – (Chinese language abstract – see Chinese supplement)
PS94: Infertility and its treatment-an insight into its affect on oral health related quality of life, a cross sectional study in Delhi, India
Swati Jain, National Health Mission, Maulana Azad Institute of Dental Sciences, New Delhi; Kirti Vikrant Mohanty
Background: Infertility is emerging as a modern age pandemic disease. The Psychosocial impact associated with infertility remains major concern throughout the globe. Primarily it remains a woman’s social burden. Oral health also gets affected by the treatment modalities of infertility and is an integral component of general health. The present study aims to assess the influence of infertility and its treatment on Oral health related quality of life (OHRQOL) and oral health status.
Methods: A total of 119 patients attending the OPD of infertility clinic of a Multispecialty hospital in Delhi, India from January to September 2018 were selected through simple random sampling for the study. Oral Health Impact Profile-14 (OHIP) questionnaire was used to assess OHRQOL. Clinical Oral health status was also assessed. Statistical analysis was done using SPSS v21.0 software package.
Results: The final sample consisted of 95 patients (mean age 28.2±11.2 y). The mean duration of infertility treatment was 13.8±3.2 months. All participants were dentate with only 5.2% subjects having total number of natural teeth less than 20. The most prevalent items for OHIP-14 (mean 8.87; SD 10.95) were uncomfortable to eat foods, and diet has been unsatisfactory. Chewing problems were significantly related to OHIP-14 (P<0.001) scales. Higher OHIP-14 scores were significantly associated with dental treatment needs (P=0.029) and poor self-rated oral status (P=0.001). Poor OHRQOL scores were significantly associated with increasing duration of infertility treatment and with patients referred for IVF treatment.
Conclusion: The oral problems were not a major concern for this group of patients, which could indicate the adaptation to impaired oral health or a change in health priorities. However, the poor oral health status was perceived more by patients undergoing infertility treatment for longer duration and worsened with increasing gestation age which might be primarily contributed to the effects of hormonal fluctuations and neglect of oral hygiene. Further it reflects underlying psychological impact of infertility on oral health as well. Hence it is necessary to reinforce knowledge about significance of oral health in patients undergoing treatment for infertility by means of appropriate preventive and educational programs.
Thursday, 11 April 2019
PS95: Autologous peripheral blood mononuclear cell instillation to improve pregnancy outcomes after recurrent implantation failure: a systematic review and meta-analysis
Yang Wu, The Reproductive Medicine Special Hospital of The 1st Hospital of Lanzhou University; Lifei Li; Xuehong Zhang; Kehu Yang
Background: Recurrent implantation failure (RIF) is a common cause of disappointment and a big challenge after assisted reproduction technology treatments. Autologous peripheral blood mononuclear cell (PBMC) instillation is a new adjunctive therapy for improving outcomes among patients with RIF. Our objective with this study was to evaluate the existing literature to explore whether PBMC instillation could improve pregnancy outcomes among patients with RIF.
Methods: We searched PubMed, EMBASE, the Cochrane library, China Biomedicine (CBM), China National knowledge Infrastructure (CNKI), China Wan Fang data, and the VIP Database for Chinese Technical Periodicals (VIP) to identify relevant literature that reported the effectiveness of autologous PBMC instillation in women with RIF. We included subgroup and sensitivity analyses using Stata 12.0.
Results: Four randomized controlled trials (RCTs) and three non-randomized controlled trials (non-RCTs) were included. The results of the four RCTs showed that PBMC improved outcomes in all patients compared with placebo or no-treatment (clinical pregnancy rate (CPR): odds ratio (OR)=2.35, 95% confidence interval (CI)=1.53-3.60, I2=0%; implantation rate (IR): OR=2.41, 95%CI=1.54-3.76, I2=0%; live birth rate (LBR): OR=2.43, 95%CI=1.32-4.49, I2=0%). However, the results of the three non-RCTs indicated that there was no statistically significant difference in the outcomes and that the heterogeneity was higher (I2>0%). The subgroup analysis further suggested that there was a statistically significant increase in CPR, IR and LBR in the three or more implantation failure subgroups (CPR: OR=2.83, 95%CI=1.29-6.22, I2=0%; IR: OR=3.74, 95%CI=1.71-8.19, I2=0%; LBR: OR=3.03, 95%CI=1.15-7.98, I2=0%). No significant differences were observed in the miscarriage rate or endometrial thickness on embryo transfer (ET) day.
Conclusion: All the patients with RIF seems to be able to get benefit from Autologous PBMCs instillation. Among patients with three or more implantation failures, this treatment significantly improved IR, LBR, and CPR compared to controls. It is a safe adjunctive therapy to in vitro fertilization (IVF) treatment.
PS96: MiR-148a-3p may contribute to flawed decidualization in recurrent implantation failure by modulating HOXC8
Yan Junhao, Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University; Qian Zhang; Yan Junhao; Qian Zhang; Tianxiang Ni; Yujie Dang; Lingling Ding; Jingjing Jiang; Jing Li; Mingdi Xia; Na Yu; Jinlong Ma; Chen Zi-Jiang
Background: Recurrent implantation failure (RIF) has become a predicament in human reproductive treatment whereby couples fail to achieve pregnancy despite multiple embryo transfers. At present, its precise pathogenesis remains enigmatic. Suboptimal decidualization is increasingly recognized to intertwine with embryo implantation defects. Compelling evidence indicated an essential role for microRNAs (miRNAs) in various physiologic and pathologic processes, whereas little is known about endometrial miRNA signature and its functional mechanism in RIF pathogenesis.
Methods: The miRCURY LNA™ microRNA Array was utilized to screen different miRNA expressions in receptive endometrial tissues collected from 8 RIF patients and 10 female controls, and reverse transcription and quantitative real-time PCR (RT-qPCR) was used to quantify selected miRNAs and predicted miRNA target genes. Immortalized human endometrial stromal cells (HESCs) were cultured for proliferation and differentiation assays after enhancing miR-148a-3p expression or inhibiting putative target gene HOXC8 expression. RT-qPCR, western blot and luciferase reporter assays were performed to demonstrate the relationship between miR-148a-3p and HOXC8 gene.
Results: We identified endometrial miRNA and mRNA expression profiles during the implantation window for RIF and found that miR-148a-3p was significantly up-regulated in RIF endometrial tissues. Forced expression of miR-148a-3p notably attenuated HESCs in vitro decidualization. Mechanistic studies revealed that miR-148a-3p directly bound to the HOXC8 3’untranslated region (3’UTR) and suppressed HOXC8 expressions in mRNA and protein levels. Further investigations demonstrated that inhibition of HOXC8 in HESCs induced similar effects on decidual process as those induced by miR-148a-3p overexpression.
Conclusion: Our findings suggested that elevated miR-148a-3p might account for suboptimal decidualization in RIF by negatively regulating HOXC8, raising the possibility that miR-148a-3p might be a novel therapeutic target in RIF.
PS97: Cuscuta chinensis improved endometrial receptivity
Li Sun, Shengjing Hospital of China Medical University; Jichun Tan
Background: Cuscuta chinensis has been shown to increase beta-endorphin levels in the hypothalamus, increase pituitary LH levels, and improve ovarian function in OHSS models and ovariectomized rats. These all proved that Cuscuta chinensis has a regulatory effect on the hypothalamic-pituitary-ovarian axis. However, the effect of Cuscuta chinensis on the endometrial transformation, intimal growth and embryo implantation synchronization is unknown. Our experiment was to investigate the effect of crude extract of Cuscuta chinensis on endometrial receptivity.
Methods: First, the crude extract of Cuscuta chinensis was added to the culture medium of human endometrial stromal cells in a gradient concentration to determine its effect on cell proliferation, apoptosis and cycle. Secondly, the effects of endometrial receptivity-related indicators and proteins related to PI3K/Akt and p38/MAPK proliferation pathways were verified. Finally, the rats were given a gradient concentration of crude extract of Cuscuta chinensis and a pregnancy test was performed to observe its effect on embryo implantation.
Results: Crude extract of Cuscuta chinensis could promote the proliferation of human endometrial stromal cells, inhibit its apoptosis, and increase the percentage of cells in G1 phase. In addition, the expression of ERα, ERβ, PR, FOXO1, Integrin αVβ3, LIF, IL6, IL1β was increased after 72 hours of addition of crude extract of Cuscuta chinensis at 25 ug/mL and 50 ug/mL. At the same time, the expression of PI3K/Akt and p38/MAPK proliferation pathway-associated proteins was promoted. Animal experiments showed that the crude extract of Cuscuta chinensiss significantly increased the embryo implantation rate of embryo implantation impaired rats.
Conclusion: The crude extract of Cuscuta chinensis might affect the expression of PI3K/Akt and p38/MAPK proliferation pathway-related proteins, thereby promoting the proliferation of endometrial cells, inhibiting apoptosis, and finally up-regulating the endometrial receptivity related indicators and improving embryo implantation rate. It could be seen that crude extract of Cuscuta chinensis could improve endometrial receptivity and was expected to become a new method of alternative treatment in assisted reproductive technology.
PS98: Dnmt3b is essential for the endometrial decidualization in mice during peri-implantation period
Mengyue Chen, Chongqing Medical University; Jing Long; Yingxiong Wang; Mengyue Chen; Jing Long; Rufei Gao
Background: Implantation, a critical and complicated step for the establishment and maintenance of pregnancy, requires molecular events and a dialogue between the endometrium and the implanting embryo (Guzeloglu-Kayisli et al 2009). DNA methylation is an epigenetic mechanism which regulates gene expression through recruiting proteins or inhibiting the binding of transcription factors to DNA in the mammalian genome (Moore et al 2013). Dnmt3b is responsible for the establishment of methylation during implantation and plays a role in establishing methylation at germ line genes (Walton et al 2011). The methylation of key genes regulated by Dnmt3b are still unknown. Which function altered through Dnmt3b during embryo implantation is waiting for further study.
Methods: Dnmt3b–/– PRCre mice, Artificial decidualization, Real-time PCR, Western Blot, Immunohistochemistry, methylation sequencing, Breeding experiment, Statistics.
Results: (1) cKO group was significantly infertile than control group. (2) cKO mice might possess a more receptive uterus. But the expression of decidualization factors were significantly declined in cKO group. (3) Embryos were absorbed in more than half of cKO mice but none in control group on day 7 of pregnancy. The lower expression of decidualization factors in cKO mice indicated the damaged decidualization function. (4) The damaged decidualization function was also presented in Dnmt3b conditional knockout mouse using artificial induced decidual model. (5) Methylation sequencing was performed on the genomes of control and cKO mice. It’s reported that the many decidualization factors were presented different expression. And the details need our further analysis and discussion.
Conclusion: We established Dnmt3b conditional knockout mouse model successfully, and found the damaged decidualization function on D5 and D7 of pregnancy and absorbed embryos on day 7 of pregnancy. Furthermore, the damaged decidualization function was also presented in Dnmt3b conditional knockout mouse using artificial induced decidual model. To explore the possible factors, methylation sequencing was performed on the genomes of control and cKO mice. It’s reported that the many decidualization factors (MMP2, MMP9, BMP2, HOXA10) were presented different expression. And the details need our further analysis and discussion.
Funding: National natural science foundation projects, 31571190.
PS99: The effect of vitamin D level in follicular fluid on the cycle characteristics and pregnancy outcome of in-vitro fertilization/intracytoplasmic sperm injection-embryo transplantation
Yanling Wan, Medical College of Qingdao University; Cuifang Hao
Background: Vitamin D is a fat-soluble hormone with widespread actions acts via the vitamin D receptors, which are located not only in bone but also in the reproductive system. Our aim was to address the effect of vitamin D level in follicular fluid on the cycle characteristics and pregnancy outcome of IVF/ICSI-ET among women with or without polycystic ovary syndrome(PCOS).
Methods: Prospective study. 63 infertility women underwent IVF/ICSI from January 2018 to April 2018 were included. According to their 25-hydroxyvitamin D(25OHD) level in follicular fluid, the patients were divided into low level group (<13.8 ng/mL, n=27) and high level group (≥13.8 ng/mL, n=36). The effect of different follicular fluid 25OHD levels on cycle characteristics and clinical pregnancy rate of IVF/ICSI were analyzed. Furthermore, the patients were divided into PCOS and non-PCOS group, the cycle characteristics and clinical pregnancy rate were compared.
Results: (1) Serum 25OHD level: There was a significant linear correlation between the levels of 25OHD in serum and follicular fluid on the day of ovum pick up (PCOS group, r=0.881, P<0.001; non-PCOS group, r=0.826, P<0.001). There was no significant difference in the trends of serum 25OHD level repeated measured on the day prior to starting IVF/ICSI, ovum pick up day and the fifth day after ET between PCOS and non-PCOS groups (P>0.05). However, of both groups, the lowest levels of serum 25OHD were on the day of ovum pick up (P<0.05). (2) Comparison of cycle characteristics and IVF/ICSI outcome: In women with PCOS, the rates of MII, embryo implantation and clinical pregnancy in 25OHD high level group were significantly higher than low level group (P<0.05). In women without PCOS,the cycle characteristics and IVF/ICSI outcome were of no significant differences between two 25OHD level groups (P>0.05).
Conclusion: The serum vitamin D levels on different days were variational in women underwent IVF/ICSI. Follicular fluid vitamin D may play a role in the enhancement of the rates of embryo implantation and clinical pregnency in women with PCOS.
Funding: National Science Foundation of China [grant number 81671416].
PS100: Adverse effect of high-level estrogen on placental tight junction
Xinru Xia, Nanjing Medical University; Jie Liu; Yuan Zhang; Yugui Cui; Jiayin Liu
Background: Studies have shown that super-physiological levels of estrogen during IVF are associated with adverse pregnancy outcomes. We have found CLDN8 significantly changed in mRNA levels between the IVF and the control groups. We hypothesize that the IVF cycle super-physiological estrogen environment may affect the invasive ability of trophoblast cells by affecting the structure and function of placenta tight junctions, causing placental-related pregnancy complications.
Methods: BeWo and JEG-3 cells were treated with 0, 10-7 and 10-6M E2 for 12 hours. Transmission electron microscope, Transepithelial resistance and paracellular permeability were measured to evaluate the integrity of tight junction structures and function among choriocarcinoma cells. Tight junction proteins: CLDN3, CLDN4, CLDN5, CLDN8, ZO-1 and OCLN, were determined by immunofluorescence and western blotting to evaluate the expression of tight junction proteins. ERα agonist PPT or ERβ agonist DPN or ER inhibitor ICI 182780 was added to demonstrate whether E2 acts through ER.
Results: Under transmission electron microscopy (TEM), it can be seen that the tight junction ultrastructure is more severely destroyed with the increasing concentration of E2. It changes from continuous linear to intermittent, and even disappears. Transepithelial resistance decreased in the cells after treatment of 10-6M E2 for 12 hours (P<0.05). At the same time, paracellular permeability increased in the cells after treatment of 10-6M E2 for 12 hours (P<0.05). Expression of CLDN3 and OCLN was decreased in the cells treated with 10-6M E2 (P<0.05). When 10-6M PPT or 10-6M DPN was added, as with 10-6M E2, TEER decreased, CPP increased, and CLDN3 and OCLN expression decreased. Adding ICI 182780 reversed the effect of 10-6M E2.
Conclusion: The super-physiological level of estrogen can disrupt tight junction in structure and function, suggesting that the super-physiological level of estrogen in IVF cycles may affect the normal function of placenta through affecting the normal structure of tight junction in trophoblasts. This may lead to placenta-related pregnancy complications.
PS101: Thick endometrium (over 15 mm) on the day of HCG administration does not influence pregnancy outcomes in IVF-ICSI cycles: retrospective cohort study
Shanshan Gao, Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University; The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, China; Jingjing Jiang; Yingying Qin; Rong Tang
Background: Previous studies have suggested that endometrium below 7 mm will lead to poor pregnancy rate. However, study investigating the role of thicker endometrium is limited, and whether a thick endometrium (exceeding 15 mm) will impair endometrial receptivity is still controversial. The aim of this study is to observe the influence of thick endometrium (≥15 mm) on ongoing pregnancy rate, ectopic pregnancy rate and early abortion (<13 wk) rate.
Methods: A retrospective analysis of IVF-ET and ICSI-ET cycles was conducted from January 2016 to December 2017 in Reproductive Hospital Affiliated to Shandong University, Jinan, China. Patients with downregulation protocol or antagonist protocol were involved, while patients with thin endometrium (<8 mm) were excluded. Hysteroscopy was performed before ovarian stimulation to exam and pretreat endometrial diseases. Embryo transfer was conducted on either day 3 or day 5 after oocyte retrieval. Patients were divided into two groups based on the endometrial thickness (group A: 8–14.5 mm; group B: ≥15 mm) on the day of HCG administration. Ongoing pregnancy rate (OPR), early miscarriage rate (EMR), and ectopic pregnancy rate (EPR) were analyzed. Binary logistic regression analysis was carried out to determine the association between endometrial thickness and pregnancy outcomes.
Results: The range of endometrial thickness was from 8mm to 22mm. A total of 13988 transfer cycles were analyzed, among which 460 cycles were recorded with a thick endometrium (≥15mm, Group B). Significant differences were found in OPRs (46.07% and 55.22%, P<0.05), and EPRs (1.74% and 0.70%, P<0.05) between group A and B. No difference was found in the EMRs between the two groups (11.56% and 10.80%, P>0.05). After adjusting regression analysis for age, number of embryos transferred, protocol for controlled ovarian stimulation, embryo transfer timing, type of ART treatment, primary or secondary infertility and progesterone level on HCG administration day, results showed that endometrial thickness remained predictive for OPR (adjusted OR=2.391 95%, CI 1.970-2.902, P<0.001) and EPR (adjusted OR=0.129 95%, CI 0.044-0.380, P<0.001).
Conclusion: Our findings suggest that thick endometrium (≥15 mm) have a favorable effect on pregnancy outcomes, i.e. ongoing pregnancy rate increased while ectopic pregnancy rate reduced. Therefore, canceling fresh embryo transfer because of a thick endometrium is not recommended.
PS102: Lower conception probability observed in menstrual cycles with higher physiological stress as measured by Wrist Worn technology
Brianna Goodale, Ava Women AG; Mohaned Shilaih; Catrin Argyle; Maureen Cronin; Brigitte Leeners
Background: Recent literature has demonstrated the negative effects of stress on the probability of conception as well as pregnancy outcomes. Heart rate variability (HRV) parameters are known to correlate with both perceived and physiological stress. Until recently measuring HRV was only possible in clinics. We sought to test the utility of wearable technology in tracking HRV and its relationship to conception. Pilot data from this study was published in an abstract for the 2018 American Society for Reproductive Medicine Scientific Congress. We build upon our initial findings here, reporting outcome variables from a larger sample size and additional HRV measures.
Methods: Healthy women aged 18-42, with self-reported regular cycles (Mean cycle duration=28.27, SD=2.86), were recruited for a prospective observational study. Participants (n=380) wore the Ava bracelet nightly during sleep for up to a year or until the end of their pregnancy. HRV parameters were calculated from R-R intervals recorded by the bracelet’s optical sensor. Each cycle, ovulation was estimated using a LH-urine test, and users took a HCG-urine test to check for pregnancy. Perceived stress and other potential confounders were recorded through a daily questionnaire. We assessed the effect of perceived and physiological stress on time to pregnancy via mixed effects and Cox proportional hazard models with random intercept and slopes.
Results: We regressed the time-to-a positive pregnancy test on participants’ per cycle mean daily stress level, mean daily mood, mean HRV ratio, mean daily alcohol consumption, and total sexual encounters. The total number of times a woman had sex per cycle was positively correlated with the time to pregnancy, hazard rate=1.07, P=0.012. The resulting shared frailty revealed a significant effect of HRV ratio on conception likelihood, such that women with higher average HRV ratio in the luteal phase were less likely to become pregnant, hazard ratio=0.59, P=0.031. When both measures of stress were included in the final model, only physiological stress was significantly associated with lower chances of a positive pregnancy test.
Conclusion: Only physiological stress negatively affected the probability of conception. Wearable technology serves as a useful tool for studying peri-implantation and early pregnancy factors that are otherwise challenging to collect.
Funding: Research was funded in part by the Swiss Commission for Technology and Innovation and Ava AG.
PS103: Predictive value of serum kisspeptin levels on 14 and 21 days after frozen-thawed embryo transfer
Kai-Lun Hu, Peking University Third Hospital; Rong Li
Background: Recently, two independent studies suggested that the serum or plasma kisspeptin levels could be used as a potential biomarker to diagnosis pregnancy viability in the first (6-10 wk gestational age) and/or second trimester (5.9–22.1 wk gestational age). However, its utility as a serum biomarker in the very early first trimester is currently unknown. Furthermore, the utility of the serum kisspeptin levels in patients undergoing IVF-ET has not been investigated. To explore whether the serum kisspeptin levels on 14 and 21 days after frozen-thawed embryo transfer (ET) could predict the early pregnant outcome of patients. Patients are divided into non-pregnant (NP) group, biochemical pregnancy (BP) group, single pregnancy (SP) group, Miscarriage group, and Twin group.
Methods: Patients: 133 patients undergoing in vitro fertilization (IVF) and frozen-thawed ET.
Main Outcome Measure(s): The concentration of serum kisspeptin and hCG on 14 and 21 days after frozen-thawed ET.
Results: Serum kisspeptin levels on 21 days after frozen-thawed ET are significantly higher than 14 days after ET in the SP group, the Miscarriage group, and the Twin group, but not in the BP group (P<0.0001, P<0.0001, P<0.0001, P>0.05, respectively). For 14 days after frozen-thawed ET, compared to the Twin group, other four groups show significantly higher levels of serum kisspeptin levels (NP group, BP group, Miscarriage group, SP group, P<0.01, P<0.05, P<0.05, P<0.05, respectively). For 21 days after frozen-thawed ET, compared to the BP group, the Miscarriage group, the SP group, and the Twin group has a significantly higher serum kisspeptin levels (P<0.001, P<0.01, P<0.001, respectively). Serum kisspeptin levels on 14 days after ET are negatively correlated with embryo implantation rate (P<0.05). Serum kisspeptin levels on day 21 after ET have a poor predictive value of miscarriage compared to serum HCG levels (AUC=0.533, AUC=0.777, respectively).
Conclusion: Serum kisspeptin levels on 14 days after frozen-thawed ET are negatively correlated with embryo implantation rate. Serum kisspeptin levels on day 21 after frozen-thawed ET have a poor predictive value of miscarriage.
Funding: This work was supported, in part, by the National Key R&D Program of China (2016YFC1000201, 2016YFC1000601), the National Natural Science Funds for general program (31501201, 81471427, 81771650, 81571400, 81771580).
PS104: Intrauterine perfusion with granulocyte colony-stimulating factor improves clinical outcomes of patients with thin endometrium during frozen embryo transfer cycle
Li Yue; Zou Libo; Wang Rong
Background: To explore the variations of endometrium and clinical outcomes after intrauterine perfusion with granulocyte colony-stimulating factor (G-CSF) in patients with thin endometrium during frozen embryo transfer (FET) cycle.
Methods: One hundred patients with thin endometrium during FET cycle were enrolled and randomly divided into two groups: 50 cases as the research group and 50 as control group. All of patients received the same HRT program. The research group was given G-CSF intrauterine perfusion on day 5 and day 10 during HRT. The endometrial thickness (EMT) of each group was observed by transvaginal B ultrasound on the progesterone conversion day and clinical results were compared. Expression of leukemia inhibitory foctor (LIF) mRNA from endometrium of transfer-cancelled patients of the two groups was detected and compared.
Results: Compared with the control group, the research group had lower cycle ablation rate, thicker endometrium and higher implantation rate (P<0.05). The abortion rates of the two groups have no statistical significance (P>0.05). Expression of LIF in the research group is higher than control group (P<0.05).
Conclusion: Intrauterine perfusion with G-CSF can improve the endometrium thickness during HRT of FET and improve higher implantation rate.
PS105: Dysfunction of DNA damage-inducible transcript 4 in the decidua is relevant to the pathogenesis of preeclampsia
Yang Jieqiong, Renji Hospital; Cong Zhang; Chen Zi-Jiang
Background: Preeclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation and af- fects 3–5% of all human pregnancies worldwide. However, the pathogenesis of PE still remains poorly understood. A deficiency in decidualization is considered a contributing factor to the development of PE. The DNA damage inducible transcript 4 (DDIT4) gene encodes a protein whose main function is inhibiting mammalian target of rapamycin (mTOR) under stress, and several studies have demonstrated that its expression promotes tumor cell apoptosis.
Methods: We investigated the expression of DDIT4 in deciduae and their relationship with PE. A total of 38 decidua samples were collected, of which 22 were from normal pregnant (NP) and 16 from severe PE. DDIT4 expression in decidua and the levels of DDIT4 in cultured human endometrial stromal cells (hESCs) and primary hESCs during decidualization were determined. To further investigate the role of DDIT4 in human decidualization, RNA interference was used to silence DDIT4 in hESCs and primary hESCs.
Results: The results indicated that DDIT4 was markedly decreased in the decidua of severe PE compared with those from uncomplicated pregnancies. The expression of DDIT4 in human endometrial stromal cell (hESC) line and primary hESCs was up-regulated during decidualization. Knockdown DDIT4 in hESCs and primary hESCs caused a significant reduction in the transcription of decidualization markers, insulin-like growth factor binding protein 1 and prolactin. In addition, silencing DDIT4 caused up-regulated p-mTOR and p-p70s6k and reduced apoptosis, whereas rapamycin, an inhibitor of mTOR, reversed the result of apoptosis. Moreover, the expression of cleaved-caspase 3 in severe PE was significantly lower than that of uncomplicated pregnancies, which was unfavorable for trophoblast invasion.
Conclusion: Our data suggest that DDIT4 is critical for normal decidualization and the apoptosis of decidual cells. DDIT4 deficiency is likely involved in the development of PE.
Funding: This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31471399 and 31671199), the National Key R&D Program of China (2017YFC1001403), and the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20152515) to CZ, by a grant from the Major Program of the National Natural Science Foundation of China (81490743) to Z-JC and by the Shanghai Commission of Science and Technology (17DZ2271100).
PS106: The effect of progesterone on the oviductal physiology in mammals
Cheng Li, Shandong University; Jian Zhang; Hefeng Huang
Background: Our previous study indicated that progesterone could lead to ectopic pregnancy following contraception failure. However, our understanding of the effect of levonorgestrel and progesterone on the oviductal physiology is limited.
Methods: The present study focused on the in vitro effects of progesterone on oviductal receptivity.
Results: Our results show that progesterone cannot affect the embryo implantation, without changes in receptivity markers including IGFBP1, MUC1, ACVR1B, TGFb, and STAT3. Furthermore, JAr spheroid-fallopian tubal epithelial cell attachment assay showed no differences after we treated the OE-E6/E7 with different dose of P4, the percentage of attached JAr spheroid did not show any significant difference among groups.
Conclusion: In summary, our data shows that super-physiological level of progesterone can not change tubal receptivity.
Funding: This study was supported by a National Natural Science Foundation of China [grant number 81671482 and 31401226].
PS107: Simultaneous intrauterine pregnancy and abdominal pregnancy following in vitro fertilization and literatures were reviewed
Min Yan, Yili Prefecture Maternal and Child Health Hospital; Zhen Hou; Lingjie Hu; Xiaoqin Guo; Jing Liu; Xialapati Abuduwaili; Wulitai Shadeerhuojia
Background: To analyze the diagnosis and treatment of intrauterine pregnancy and abdominal pregnancy following in vitro fertilization.
Methods: A case of intrauterine pregnancy and abdominal pregnancy after freeze-thawing embryo transfer in our center was studied.
Results: Patients with emergency laparoscopy, intraoperative pelvic large hematoma. Doctors found embryonic tissue, a side oviduct was absent and the other side oviduct was resected because of hemorrhage. Postoperative bleeding costed more blood transfusion. postoperative pathology revealed that embryonic tissue in the abdominal cavity and (the left side of the oviduct) chronic salpingitis.
Conclusion: With the application of assisted reproductive technology, the rate of heterotopic pregnancy and the rate of peritoneal pregnancy are significantly increased, so clinicians should be more alert to the occurrence of such dangerous pregnancy in the process of diagnosis and treatment.
Funding: Support from Project of Jiangsu Province helping Xinjiang for Yili Center of Reproductive Medicine; Project of key intelligence of women and children’s health care of Jiangsu Province (FRC201748).
PS108: The clinical outcomes of different stage embryos in frozen-thawed embryo transfer for patients with recurrent implantation failure
Lihua Yang, Reproductive Medicine of Jinhua People’s Hospital
Background: As the World’s first In Vitro Fertilization baby was born in 1978, the number of global Assisted Reproductive Technologies cycles increases year by year. Now, approximately 1.68% of all infants born in China every year are received by ART. Although we have made great achievements in this field, we still face many frustrating problems in clinic, such as recurrent implantation failure (RIF), which is a difficult problem in ART.In recent years, blastocyst culture have been introduced as an alternative for improving the chance for IVF. The aim of this study was to evaluate the clinical outcomes of different stage embryos in frozen-thawed embryo transfer for patients with RIF.
Methods: Medical records of patients who underwent twice or more failed attempts of frozen-thawed embryo transfer (FET) at cleavage stage and still have frozen cleavage stage embryos at Jinhua People’s Hospital from 2015 to 2017 were reviewed. Patients having immunological and intrauterine diseases were excluded. Included patients were divided into two groups according to undergoing early cleavage stage FET or blastocyst transfer (BT) by sequential culture after vitrified frozen cleavage embryo recovery. In group A (blastocyst stage, n=132), blastocysts were cultured to D5 or D6 after freeze-thawing of D3 cleavage stage embryos. If the blastocysts formed, the transfer cycles existed. In group B (cleavage stage embryo, n=164), after freeze-thawing of the D3 cleavage embryos, the transfer cycles existed if embryos survived. The number of embryos transferred, clinical pregnancy rate, implantation rate, multiple pregnancy rate, miscarriage rate, ectopic pregnancy rate and live birth rate were compared between two groups.
Results: Blastocyst formation rate of D3 cleavage stage embryo after frozen-thawed was 55.27%. Cancellation rate in group A was 6.06%, while no cases of cancellation were seen in group B. There was a higher pregnancy rate in group A with 46.77% versus 36.59% in group B, even though their difference did not reach statistically significant level. Implantation rate in group A was higher than group B (P<0.05). There were no differences in multiple pregnancy rate, miscarriage rate, ectopic pregnancy rate and live birth rate between the two groups (P>0.05).
Conclusion: Blastocyst transfer can get better clinical results in patients with RIF, but it does have the risk of transfer cancellation.
Funding: Funded by the Program for Jinhua S&T bureau, Zhejiang, China.
PS109: Long-acting GnRH agonist is associated with increased live birth rate for patients with early pregnancy loss in IVF/ICSI cycles
Dan Pan, Northwest Women’s and Children’s Hospital; Juanzi Shi; Hanying Zhou; Na Li
Background: The aim was to compare the efficacy of long-acting and shortacting gonadotropin-releasing hormone (GnRH) agonists on live birth rate for patients with early pregnancy loss in vitro fertilization.
Methods: In this retrospective study, long-term pituitary downregulation, achieved with long- and short-acting GnRH agonists (GnRHa), was performed for patients who had early pregnancy loss in vitro fertilization (n=577).
Results: Pregnancy results were compared between the long and short-acting GnRH group. There was no difference in female age,number of retrieved oocytes, endometrial thickness on HCG day, fertilization rates between the two groups. The clinical pregnancy rates (75.45% vs. 65.27%, P=0.001) and live-birth rates (LBR) (65.27% vs. 50.00%, P=0.001) appeared higher in the study group.
Conclusion: Live birth rate is significantly higher following long-acting GnRH agonist compared with short-acting GnRH agonist for patients who had early pregnancy loss in IVF/ICSI cycles.
PS110: GnRH-a increase endometrial receptivity through microRNA-125b-5p suppresses leukemia inhibitory factor expression in frozen-thawed embryo transfer cycles
Junxia An, The Reproductive Medicine Special Hospital of the 1st hospital of Lanzhou University, Lanzhou; Lifei Li; Xuehong Zhang
Background: There are currently various protocols for frozen-thawed embryo transfer (FET). However, which is the optimal mean of preparing the endometrium remains controversial.
Methods: In this randomized controlled study, we evaluated 305 patients during 2 years (February 2012–April 2015). The patients were randomly allocated to the following treatment groups: group A, comprising 59 patients who received E2+P4; group B, comprising 53 patients who received 3 cycles of GnRHa+E2+P4; and group C, comprising 57 patients who received 4 cycles of GnRHa+E2+P4; group D, comprising 53 patients who received 5 cycles of GnRHa+E2+P4; and group E, comprising 52 patients who received 6 cycles of GnRHa+E2+P4;. The inclusion criteria were regular menstrual cycles (length 24–35 days) and age 21–45 years.
Results: The major outcome of the study-clinical pregnancy rate (CPR) per embryo transfer cycle — was statistically lower in group A than group B and C (49.15% vs. 62.26% vs. 56.14%, P<0.05). Similar results were found for the CPRs with fetal heartbeat per embryo transfer cycle [42.37%] in group A vs. [58.49%] in group B vs. 14/42 [45.61%] in group C. The opposite trend was found when we limited our analysis to miscarriage rate per embryo transfer cycle between group A and B (37.29% vs. 24.52% P<0.05). There were no significant differences among group A, group D and group E regard to the clinical pregnancy rate and clinical pregnancy rate with FHB per embryo transfer cycle (P>0.5). Similarly, the expression levels of endometrial integrin avb3, LIF, VEGF, OPN, Hoxa10 and HOXA11 mRNA in group B and C were sig¬nificantly increased, and had no differences with group D and E on the day of embryo implantion with treatment. By contraries, the expression of miR-125b-5p were significantly lower in group B and C, compared with the group A, D and E. Similarly, And there was no obvious differences among group A, D and E.
Conclusion: The clilical pregnancy rates were better in the hormone therapy with 3 or 4 cycles of GnRHa groups, hormone therapy FET with 3 or 4 cycles of GnRHa for pituitary suppression did result in significantly improved CPR when compared with hormone therapy FET without GnRHa or of 5 or 6 cycles of GnRHa. Some molecules such as integrin avb3, LIF, VEGF, OPN, Hoxa10, Hoxa11 and miR-125b-5p might participate in this process.
PS111: Research of intrauterine administration of HCG-activated autologous PBMCs for repeated implantation failure and the related mechanism
Zhang Huakun, Shenzhen Maternity & Child Healthcare Hospital; Yao Jilong; Liu Qingzhi; Li Xuemei
Background: To investigate the effect of intrauterine administration of HCG-activated autologous PBMCs on pregnancy outcome and changes of intrauterine cytokines in patients with recurrent recurrent implantation failure (RIF) and the related mechanism.
Methods: Sixty-five RIF patients who received frozen-thawed embryo transfer (FET) were enrolled in this study. Twenty eight patients received autologous PBMCs intrauterine perfusion therapy and uterine fluid extraction before the frozen-thawed embryo transfer as the research group and the other 37 as the control group. PBMCs was isolated from autologous peripheral blood before ovulation and co-cultured with HCG for intrauterine perfusion 3 days before transplantation. Routine FET was performed in both groups and pregnancy outcomes were followed up. Detection of cytokines related to uterine secretions before and after intrauterine administration with Bio-plex chip analysis system.
Results: There were no significant differences in age, infertility years, basic sex hormone levels, the number of previous transplant cycles, the mean intimal thickness and the number of embryos transferred between the two groups (P>0.05). The clinical pregnancy rate (42.86% vs. 16.22%) and implantation rate (25.86% vs 9.21%) were compared between the research group and the control group respectively, which were significantly higher in the research group than the control. The expression of Eotaxin in uterine secretions increased after intrauterine administration (P<0.05), while the expression of INF-γ decreased (P<0.05).
Conclusion: Intrauterine administration of HCG cocultured PBMCs is a safe and effective treatment for RIF patients which can improve the clinical pregnancy rate of FET cycles in these patients and ameliorate the expression of local cytokines in uterine cavity to promote embryo implantation.
Funding: Research was funded in part by the Shenzhen Science and Technology Project (Project No.:JCYJ20160427191500406), Shenzhen, China.
PS112: Activation of uterine Smad3 pathway is crucial for embryo implantation
Juan Li, Reproductive Medicine Center of Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology; Xiyuan Dong; Peiwen Yang; Shulin Yang; Lan Wang; Hanwang Zhang; Cong Sui
Background: Embryo implantation is a complicated physiological process tightly regulated by multiple biological molecules including growth factors. Transforming growth factor-betas (TGF-βs) and their most specific signal transduction factors, Smads, are expressed in endometrium during the window of implantation. Recent researches indicated that Smad dependent TGF-β signaling may play an important role in the process of embryo implantation.
Methods: In this study, the expression of TGF-β1, TGF-β receptor type I (TβRI), Smad3 and p-Smad3 in the endometrium of mice during implantation window were measured. A specific Smad3 phosphorylation inhibitor was administrated into the uterine cavity of mice on day 3 of pregnancy. Then the number of implanted embryos was counted.
Results: The results showed that the mRNA expression of TGF-β1, TβRI and Smad3 were elevated on Day 4, Day 5 and Day 6 of pseudo pregnancy, comparing with that in the unmated female mice. However, the elevation on Day 6 was lowered, comparing with that on Day 4 or Day 5. The expression of both Smad3 and p-Smad3 protein reached the highest level on Day 4, and then declined gradually on Day 5 and Day 6. The p-Smad3/Smad3 ratio, which represented the phosphorylation level of Smad3 protein, was also increased on Day 4, Day 5 and Day 6 of pseudo pregnancy.
The expression of p-Smad3 was measured 4 hours after Sis3 administration and a complete inhibition of Smad3 phosphorylation was observed by using western blot. The number of implanted embryo in the Sis3 treated group was dramatically decreased, comparing with that in the DMSO treated group (2.22±1.30 vs. 5.44±1.24, P<0.001) and the sham group (2.22±1.30 vs. 7.11±1.36, P<0.001).
Conclusion: This study revealed the expression features of key factors in TGF-β/Smad3 pathway during the implantation window in the endometrium of mice and found that TGF-β/Smad3 pathway was enhanced and activated during this period, suggesting the important role of TGF-β/Smad3 pathway during embryo implantation. The results of animal experiments validated that the activation of this pathway in endometrium is critical for embryo implantation.
Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 81471459 and No. 81701450).
PS113: Integrated analysis of the transcriptome and proteome provides new insights into the molecular regulation on endometrial receptivity
Shuanggang Hu, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Yuan Wang; Guangxin Yao; Yun Sun
Background: Competent endometrial receptivity is a prerequisite for successful embryo implantation. Identification of novel key molecules involved in endometrial receptivity is essential to better interpret human implantation and improve pregnancy rates in assisted reproduction treatment. In the past decade, using gel-based proteomics, only a limited number of differentially expressed proteins (DEPs) between prereceptive and receptive endometrium have been identified.
Methods: Endometrial proteomes were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics. Integrated analysis of proteome and transcriptome was perfomed and pearson correlation coefficients were applied to measure the correlation between mRNA and protein expression levels. Gene ontology, pathway and protein-protein-interaction (PPI) network analysis were performed on concordant DEPs. Quantitative RT-PCR and Western blot were used to detect the expression of ACSL4.
Results: In this study, we identified 173 DEPs between LH+2 and LH+7 human endometrium using iTRAQ proteomics technology. Furthermore, we first performed the integrated analysis of proteome and transcriptome and found that the expressions of mRNA and protein only showed limited positive correlations for differentially expressed genes (DEGs) and DEPs. We identified 63 novel concordant DEPs with differential expressions at both mRNA and protein levels between prereceptive and receptive endometrium. By gene ontology, pathway and protein-protein-interaction (PPI) network analysis on concordant DEPs, five hub proteins- ACSL4, ACSL5, COL1A1, PTGS1 and PLA2G4F, were determined. The protein expression of ACSL4 was significantly up-regulated in receptive endometrium compared to prereceptive endometrium and significantly down-regulated in adenomyosis patients.
Conclusion: This study provides the first gel-independent quantitative proteomes of the LH+2 and LH+7 human endometrium using iTRAQ technology. A complete picture of gene activity in human endometrium is firstly achieved by combined survey of proteome and transcriptome. The identified concordant DEPs and hub proteins may be valuable for elucidating the underlying mechanisms governing endometrial receptivity.
Funding: Research was funded in part by the National Natural Science Foundation of China (81571435, 81571499 and 81771648).
PS114: Dynamic changes of circRNA expression in human endometrium between days LH+2 and LH+7 potentially contribute to endometrial receptivity
Shuanggang Hu, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Yuan Wang; Guangxin Yao; Yun Sun
Background: Compatible endometrial receptivity is essential for successful embryo implantation. While the effects and regulatory mechanisms of protein-encoding genes and miRNAs have been extensively addressed, the involvement of circular RNAs (circRNAs) on endometrial receptivity have not yet been clarified. The aim of this study is to identify and compare the expression changes in circRNAs between days LH+2 and LH+7 in human endometrium.
Methods: CircRNA expression profiles in endometrial biopsies from six women (3 for LH+2, 3 for LH+7) were measured using Arraystar Human circRNA Array. Bioinformatic analyses were performed to identify the differentially expressed circRNAs. Furthermore, RNase R treatment, RT-PCR, sanger sequencing and qRT-PCR were used to confirm the circular characteristic and expression changes of circRNAs.
Results: Using circRNA microarray, 1 circRNA (hsa_circRNA_101280) was found to be up-regulated and 3 circRNAs (hsa_circRNA_102293, hsa_circRNA_104789, and hsa_circRNA_104791) were down-regulated on day LH+7 endometrium compared with day LH+2 endometrium (fold-change >5 and P<0.01). These 4 differentially expressed circRNAs were successfully verified by qRT-PCR. Hsa-circRNA-101280 is a stable exonic circRNA derived from the Slain1 gene locus, is predominantly expressed in endometrial epithelial cells, and is significantly up-regulated during the establishment of endometrial receptivity. Hsa-circRNA-101280 was predicted to interact with 5 miRNAs-hsa-miR-491-5p, hsa-miR-141-5p, hsa-miR-200b-3p, hsa-miR-200c-3p, and hsa-miR-429. The circRNA/miRNA/target gene network analysis indicated that hsa-circRNA-101280 might be involved in transcriptional regulation and macromolecular metabolism. The expression of hsa-circRNA-101280—but not its linear cognate gene, Slain1—was significantly decreased in the endometrium of adenomyosis patients.
Conclusion: This study revealed the circRNA expression profile in human endometrium, and we identified differentially expressed circRNAs between LH+2 and LH+7 endometrium. The present study indicated that hsa-circRNA-101280 might be associated with the defective endometrial receptivity caused by adenomyosis, and thus provides novel insight into the role of circRNAs in endometrial receptivity.
Funding: Research was funded in part by the National Natural Science Foundation of China (grant numbers 81771648, 81571435, 81571499).
PS115: A novel molecule in human cyclic endometrium: lncRNA TUNAR is involved in embryo implantation by modulating HOXA10 expression
Yuan Wang, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shuanggang Hu; Yun Sun
Background: Embryo implantation rate remains an inefficient process in in vitro fertilization and embryo transfer (IVF-ET) cycles. Although much research on the molecular mechanisms of embryo implantation has been carried, the role long noncoding RNA (lncRNA) plays in the establishment of endometrial receptivity remains unclear. We aim to investigate the expression levels of TCL1 upstream neural differentiation-associated RNA (TUNAR) in human cyclic endometrium and clarify the role of TUNAR in the development of endometrial receptivity.
Methods: We detected the level of TUNAR in endometrial biopsies collected at the late proliferative phase, LH+2 and LH+7, as well as from patients with or without adenomyosis, by real-time PCR. Homeobox A10 (HOXA10) expression during the menstrual cycle was measured by real-time PCR and western blot. Correlation analysis was conducted to explore the relationship between TUNAR and HOXA10. We also investigated the effect of TUNAR on HOXA10 expression in primary cultured endometrial stromal cells (ESCs) through siTUNAR and pZW1-snoVector-TUNAR transfection.
Results: The expression of TUNAR, found to be highly expressed in the nucleus of ESCs in the endometrium, was down-regulated at LH+7 and was increased in patients with adenomyosis. A positive relationship between the expression of TUNAR and HOXA10 in endometrium was discovered. Furthermore, knockdown of TUNAR in primary human ESCs inhibited HOXA10 expression at mRNA and protein level, while overexpression of TUNAR exhibited the opposite effect.
Conclusion: LncRNA TUNAR was proven to be expressed in human cyclic endometrium for the first time. It might be involved in embryo implantation by regulating the level of HOXA10. Our study helps us to better understand the molecular regulation of endometrial receptivity.
PS116: Diagnostic value of transvaginal sonography in heterotopic interstitial pregnancy after in vitro fertilization - embryo transfer
Jingzi Xiao, Reproductive & Genetic Hospital of Citic-xiangya; Xihong Li; Yan Ouyang; Yuyao Mao
Background: Heterotopic pregnancy (HP) is a type of pathological pregnancy defined by the coexistence of intrauterine and extrauterine gestations. HP is rare in the general population, with an incidence of 1/3889 to 1/30 000, the incidence of HP is considerably higher in women undergoing in vitro fertilization - embryo transfer (IVF-ET) procedures, at approximately 1/100 pregnancies. Heterotopic interstitial pregnancy (HIP) is a rare type of HP, it has serious consequence once it breaks, thus, the early diagnosis and early treatment seem extremely important to these pregnant women. Transvaginal sonography (TVS) is the first diagnostic method for HP, the objective of this study is to assess the value of TVS in the diagnosis of HIP after IVF-ET.
Methods: This was a retrospective study, women who underwent IVF-ET and TVS between January 2005 and December 2016 were included. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the TVS in the diagnosis of HIP were analyzed. All HP diagnoses were confirmed by surgery and pathological analysis.
Results: Overall, 70230 consecutive women who underwent IVF had TVS during this study, 722 women (1.03%) were diagnosed on TVS as having an HP, and 156 women (21.61%) of the HP were diagnosed with an HIP. While 2 cases were missed. 12 women who received expectant treatment were excluded from the analysis. The overall sensitivity and specificity of TVS in the diagnosis of HIP were 98.61%, 100.00%, respectively, with PPV and NPV of 100.00% and 99.46%. Among the 142 HIP cases who received surgical treatment, the live birth rate of the intrauterine pregnancy (IUP) was 71.13% (101). However, 25.35% of these cases (36) suffered an early IUP loss and 3.52% (5) miscarriages occurred after the surgical procedures.
Conclusion: Early TVS performed by an experienced sonographer has a high sensitivity for making the correct diagnosis of HIP after IVF-ET, which makes the early treatment of the interstitial pregnancy and the high live birth rate of IUP to be possible.
Funding: The Science and Technology project of Health and Family Palnning Comminssion of Hunan Province (No.C20180289).
PS117: ADAMTS-7 improves the growth and invasiveness of trophoblasts via focal adhesion kinase signaling in early pregnancy
Yu-Han Meng; Chun-E Ren; Jie Qiao
Background: ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be associated with cell migration and invasion. However, its function on trophoblasts remains unknown. In this study, we are aimed to investigat the role of ADAMTS-7 on trophoblasts in human first trimester gestation.
Methods: The expression of ADAMTS-7 in trophoblasts and HTR8/SVneo cells is examined by immunohistochemistry and quantitative real-time PCR. BrdU incorporation and Annexin V/PI staining are utilized to measure the effect of ADAMTS-7 on the proliferation and apoptosis of HTR8/SVneo cells, respectively. In addition, we detect the role of ADAMTS-7 on the invasion ability of HTR8/SVneo cells using matrigel invasion assays. The activation of FAK and integrinβ1 induced by ADAMTS-7 were determined by western blot.
Results: ADAMTS-7 and its substrate cartilage oligomeric matrix protein (COMP) were highly expressed in both primary human trophoblasts and human trophoblast cell lines. TGF-β1 induced a continuous and significant decrease of ADAMTS-7. Inversely, IL-1β up-regulated the ADAMTS-7 level in a dosage dependent manner. In addition, knockdown of ADAMTS-7 inhibited the growth and invasion of HTR8/SVneo cells. To the contrary, ADAMTS-7 overexpression promoted the growth and invasion of HTR8/SVneo cells. ADAMTS-7 knockdown led to a decreased level of FAK Tyr-397 phosphorylation.
Conclusion: Our results suggest that ADAMTS-7 may regulate trophoblasts invasion through focal adhesion kinase (FAK) signaling.
Funding: This work was supported by the National Natural Science Foundation of China (81501275, 81370759, 81300482), the National Key Technology R&D Program in the Twelve Five-Year Plan (2012BAI32B01).
PS118: Effect of vitamin D intervention on re-IVF fresh embryo transplantation
Guohong Song; Junli Zhao
Background: To investigate the effects of vitamin D intervention on patients undergoing in vitro fertilization (IVF) fresh embryo transfer and the pregnancy outcomes of patients with different serum vitamin D levels after receiving the same dose of vitamin D supplementation.
Methods: A total of 43 infertile patients who were scheduled to undergo IVF fresh embryo transfer after the failure of IVF fresh embryo transfer from December 2016 to September 2018 in the Reproductive Medicine Center of Ningxia Medical University General Hospital were selected. Serum 25 (OH) D was detected, and vitamin D deficiency was given to vitamin D2 10 mg Q2 weeks intramuscular injection 3 times +calcium carbonate D3 500 mg QD oral treatment for six weeks and then re-entered IVF cycle Embryo transplantation, comparing the clinical indicators of fresh embryo transplantation before and after vitamin D treatment. According to the level of serum 25 (OH) D deficiency, the patients were divided into group A: 50 nmol/L< 25 (OH) D <75 nmol/L, that is, vitamin D deficiency group (3 cases). Group B: 25 nmol/L<25 (OH) D <50 nmol/L, that is, vitamin D deficiency group (30 cases); Group C: 25 (OH) D <25 nmol/L, that is, severe vitamin D deficiency group (10 cases), compared the pregnancy outcomes of the three groups of patients with vitamin D supplementation and the age, BMI, thyroid function, AMH and vitamin D levels of this three groups.
Results: (1) The difference of Gn days, Gn dosage, embryo age, transplantation endometrial morphology and thickness, and E2, P and E2/P on the transplant day before and after treatment with vitamin D in the long-term IVF fresh embryo transfer. There was no statistical significance (P>0.05), and the number of eggs obtained before and after treatment was (7.02±3.776) and (8.67±4.868), respectively, and the number of MII eggs was (5.63±3.437) before and after treatment (7.33±4.087) and 2PN numbers were (2.70±2.435) and (4.21±3.385) before and after treatment, and the differences were statistically significant (P <0.05). (2) Patients with different serum 25(OH)D levels were treated with the same dose of vitamin D. The clinical pregnancy rate of group A was 33.33%, the clinical pregnancy rate of group B was 13.33%, and the clinical pregnancy rate of group C was 40.00%. There was no significant difference in age, BMI, thyroid function, AMH and serum 25(OH)D levels in infertile patients (P>0.05).
Conclusion: (1) Vitamin D may affect the outcome of IVF fresh embryo transfer by affecting the ovarian function and egg quality of infertile patients. (2) Patients with different serum vitamin D levels received the same dose of vitamin D supplementation, the severe vitamin D deficiency has the highest clinical pregnancy rate, so it is speculated that vitamin D treatment for patients with severe vitamin D deficiency can help improve the clinical pregnancy rate. (3) There was no correlation between serum vitamin D levels and age, BMI, thyroid function and AMH in infertile patients.
Male and Female Fertility Preservation
Thursday, 11 April 2019
PS119: CDC42 in oocytes plays an indispensable role in the activation of primordial follicles by regulating phosphoinositide 3-kinase signalling in mice
Hao Yan, China Agricultural University; Jiawei Zhang; Yan Zhang; Chao Wang; Yingying Qin; Guoliang Xia; Hua Zhang
Background: The non-renewal primordial follicles serve as the basic unit of female mammalian reproduction and the only natural source of fertilizable ova. Naturally, only a limited number of primordial follicles enter the growing follicle pool each wave through a process called primordial follicle activation, while the rest maintains dormant state. The balance between dormancy and activation of primordial follicles maintains a proper reproductive lifespan in organisms. Disorders in primordial follicle activation might lead to various ovarian diseases such as primary ovarian insufficiency (POI). In the past decade, several signaling pathways in both oocytes and preGCs have been revealed to play a functional role in controlling primordial follicle activation, such as phosphoinositide 3-kinase (PI3K) signaling in oocytes and mTORC1 signaling in preGCs. However, our understanding of the molecular networks regulating the activation progress is still incomplete.
Methods: Ovary in vitro culture system, Lentiviral infection, RNAi, IP, kidney capsules transplantation and biochemical techniques were performed in this study.
Results: Our results show that CDC42 expression increases in oocytes during the activation of primordial follicles. Knockdown of Cdc42 expression significantly suppresses primordial follicle activation in cultured mouse ovaries. Conversely, the follicle activation ratio is remarkably increased by overexpression of CDC42 in ovaries. We further demonstrate that CDC42 governs the activation process by binding to PI3-kinase catalytic subunit beta (p110β) and regulating the expression levels of PTEN in oocytes. Finally, we extend our study to potential clinical applications and show that a short-term in vitro treatment with CDC42 activators could significantly increase the activation rates of primordial follicles in both neonatal and adult mouse ovaries.
Conclusion: Our results reveal that CDC42 controls the activation of primordial follicles in the mammalian ovary and that increasing the activity of CDC42 with specific activators might improve the efficiency of in vitro activation approaches, opening avenues for infertility treatments.
Funding: Research was funded by the National Key Research & Developmental Program of China (2017YFC1001100), National Natural Science Foundation of China (31371448 and 31571542), National Basic Research Program of China (973 Programs: 2013CB945501; 2014CB943202).
PS120: Oocyte expressing E-cadherin acts as a key factor maintaining the ovarian reserve in mice
Hao Yan, China Agricultural University; Tuo Zhang; Wenying Zheng; Yi Yang; Hua Zhang; Guoliang Xia; Chao Wang
Background: In mammals, female fecundity is determined by the size of the primordial follicle (PF) pool, which is established during the perinatal period. As a non-renewable resource, the preservation of dormant PFs is crucial for sustaining female reproduction throughout life. Although recent studies has successfully reconstituted PFs in vitro from mouse pluripotent stem cells and generated fully potent mature oocytes. However, the efficiency of this process is low. The underlying mechanisms of PF formation and maintaining may provide clue for elucidating diseases such as POI and improving the efficiency of the in vitro reconstitution of PFs from stem cells.
Methods: Ovary in vitro culture system, Lentiviral ovary infection, TUNEL staining and conventional biochemical techniques were applied in this study.
Results: In this study, we demonstrated that E-cadherin plays a crucial role in the maintenance of PFs in mice. E-cadherin is specifically localized to the cytomembrane of oocytes in PFs. Knock-down of E-cadherin resulted in significant PF loss due to oocyte apoptosis. In addition, the expression pattern of NOBOX is similar to that of E-cadherin. Knock-down of E-cadherin resulted in a decreased NOBOX level, whereas overexpression of Nobox partially rescued the follicle loss. Furthermore, E-cadherin governed NOBOX expression by regulating the shuttle protein, β-catenin, which acts as a transcriptional co-activator. Notably, oocyte membrane-expressed E-cadherin was also responsible for maintaining the PF structure by facilitating cell-cell adhesive contacts with surrounding pre-granulosa cells expressing N-cadherin.
Conclusion: E-cad in oocytes of PFs plays an indispensable role in the maintenance of the PF pool by facilitating follicular structural stability and regulating NOBOX expression. These findings shed light on the physiology of sustaining female reproduction.
Funding: This work was supported by the National Basic Research Program of China (973 Program: 2014CB943202; 2013CB945501), National Key Research and Developmental Program of China (2017YFC1001100), Institution of Higher Education Projects of Building First-class Discipline Construction in Ningxia Region (Biology, NXYLXK2017B05), National Natural Science Foundation of China (31371448; 31571540), Beijing Natural Science Foundation (5182015; 7182090).
PS121: SIRT3: An emerging participant in Delaying Ovarian Aging
Li Yamin, Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology/Center of Reproductive Medicine; Wang Xiaofei; Ping Su
Background: Sirtuin 3 (SIRT3) is one member of the mammalian sirtuin family (SIRT1~7). It locates on mitochondria and has been detected in various tissues such as ovary, heart, liver, kidney etc., and plays an important regulatory role in cell proliferation, differentiation, senescence, apoptosis and metabolism by deacetylating mitochondrial proteins. Additionally, a series of changes in mitochondria occur during ovarian aging, including decreased numbers, structural abnormalities, mtDNA deletion mutations, decreased oxidative phosphorylation, and increased ROS. The article reviews the biological functions of SIRT3, researches on mitochondria and ovarian aging, and further will be benefit for exploring its value in delaying ovarian aging.
Methods: PubMed and Web of Science were performed with relevant keywords (Sirtuin3, SIRT3, function, ovarian aging, senescence and disease). The reviewed articles were browsed and the same articles were removed. All references were restricted to those written in English.
Results: In the ovary, SIRT3 is localized to follicles and corpus luteum. Some researchers showed that its transcriptional level and translational level are closely related to ovarian function, but specific mechanisms remain unclear. However, other researchers have discovered that SIRT3 participates in central nervous system disease, kidney disease, hepatocellular disease, cardiovasular disease, and cancer, etc., via regulating mitochondrial function, such as oxidative stress regulation, ATP production, and DNA repairing. The mechnisms include: SIRT3 inhibits the production of reactive oxygen species (ROS) and increasing the elimination of ROS by deacetylating manganese superoxide dismutase (MnSOD), isocitrate dehydrogenase 2 (IDH2), etc., to participate in alleviating oxidative stress. And SIRT3 can regulate the production of basal ATP through deacetylating electron transport chain related enzyme, such as L-glutamic dehydrogenase (GDH). Besides, 8-oxoguanine-DNA glycosylase 1 (OGG1) is one of the newly discovered target proteins of SIRT3, which can scavenge 8-dihydroguanine (8-oxoG) in the genome to repair damaged DNA. Briefly, SIRT3 participates in many biological processes via regulating mitochondrial function and may contribute to delay ovarian aging via above mechanisms.
Conclusion: SIRT3 plays an important role in improving mitochondrial function and may participate in delaying ovarian aging process, which is expected to become a new target for prolonging ovarian life.
PS122: Growth retardation and glucose metabolism disorder in offspring mice produced from ovarian tissue cryopreservation and transplantation
Hongyan Wang, Ningxia Human Sperm Bank; Xue Zhou; Lianghong Ma; Qingyuan Sun; Yanyan Xu
Background: Ovarian tissue cryopreservation (OTC) and orthotopic implantation are used more and more widely. Ovarian tissue cryopreservation (OTC) provides an effective option for preserving fertility in patients with cancer suffering from fertility loss due to cancer treatment. Despite the successful application of OTC and orthotopic implantation, but the potential risks are not well understood. Previous studies indicated that the technique was associated with potential risks, and the health of the offspring required evaluation.
This study provides an important basis for evaluating the side effects of the technique on the growth and development of the offspring. It also provides a basis for women to choose OTC and orthotopic transplantation to preserve fertility. Of course, the potential risks of OTC and orthotopic transplantation need to be studied more forcefully in the future.
Methods: This study aimed to investigate the effects of OTC and orthotopic transplantation process on the growth and glucose metabolism of offspring mice. We compared the offspring mice conceived after OTC and orthotopic transplantation with the naturally bred mice in terms of growth and glucose metabolism, and further investigated the mechanisms regulating the expression of Grb10 in mouse liver and related signaling alterations.
Results: Compared to normal mice, the body weight of offspring was significantly lower in the cryopreservation transplantation group, while the fasting blood glucose level and serum insulin level of the offspring mice were higher. The expression of Grb10 significantly increased in the offspring of the transplantation group. The DNA methylation status on 4 of 11 sites of the Grb10 promoter region was significantly decreased in the transplantation group. Additionally, miR-145b was predicted to regulate Grb10 expression, and the correlation between miR-145b and Grb10 was confirmed by luciferase reporter assay. Moreover, up-regulation of Grb10 impaired insulin signaling in the transplantation group.
Conclusion: Therefore, ovarian tissue vitrification and orthotopic transplantation may result in growth retardation and symptoms of glucose metabolism disorder in offspring mice. Of course, the potential risks of OTC and orthotopic transplantation need to be studied more forcefully in the future.
PS123: LSD1 Contributes to germ cell immortality by regulating the transcription of autophagy adaptor Sqstm1/p62
Meina He, China Agricultural University; Tuo Zhang; Guoliang Xia; Chao Wang
Background: Physiologically, the size of the primordial follicles (PFs) pool within the ovaries determines the reproductive lifespan of female mammals. Interestingly, the establishment of the pool is accompanied by a programmed remarkable germ cells loss around the time of birth and the reason is unclear. Although recent studies have revealed that apoptosis and autophagy are involved in the process of germ cells loss, the underlying mechanisms need substantially study.
Methods: In this study, we used ovary in vitro culture system to specifically inhibit and overexpress genes in ovary to investigate the dynamics of change and as well as funtions of epigenetic regulators in oocyte development and the initially established primordial follicle pool, to demonstrate the mechnisms of oocyte massive loss and POI or even female infertility.
Results: We found autophagy was involved in germ cell massive loss in physiological conditions. We further evidenced the level of Lysine-specific demethylase 1 (LSD1) in the ovary reduces time dependently while germ cells initiating programmed death perinatally. Specific disruption of LSD1 protein level resulted in significantly decreased of germ cells number in cultured ovaries. While overexpress Lsd1 in ovaries protects germ cells against deaths. Finally, we identified that LSD1 repress the transcription level of autophagy adaptor p62 to regulate the level of autophagy in oocyte.
Conclusion: Our results reveled that autophagy is involved in germ cell massive loss in physiological conditions. And the epigenetic modification role of LSD1 in modulating the key factor of autophagy protects germ cells from premature loss while the PF pool is establishing in mice.
Funding: This study was supported by grants from the National Key Research & Developmental Program of China (2017YFC1001100); the National Basic Research Program of China (2014CB943202; 2013CB945501).
PS124: Resveratrol improves the survival and oocyte quality of cryopreserve-thawed ovarian tissue after subcutaneous transplantation in mice
Dalin Wang, Nanjing Medical University First Affiliated Hospital; Dongying Gan; Gege Han; Yanqiu Hu
Background: To assess if resveratrol could improve the survival and oocyte quality of cryopreserve-thawed ovarian tissue after auto-transplantation in mice.
Methods: A total of 120 6-8 week-old ICR mice were randomly distributed into fresh control, vitrified warmed control and experimental groups. The experimental groups were as follows: saline, carboxymethyl cellulose nitrate (CMCN) and resveratrol. Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules.In the experimental groups, the mice were administered CMCN (5%, orally) and resveratrol (20 mg/kg, orally) after auto-transplantation for one week, whereas the saline control mice received saline. The ovaries and blood samples were collected 3, 7 and 21 days after auto-transplantation for histological analysis, tereoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, serum FSH and E2 level, and related gen (MDA, SOD, NF-κB, IL-6 and SIRT1) express by qRT-PCR. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovaries day 21.
Results: The group that received resveratrol showed a significantly improved inprimordial follicle (D3: P<0.05, D7: P<0.05 and D21: P<0.05), antral follicle (D21: P<0.05), apoptotic follicle (D7: P<0.05), serum FSH and E2 level (D21: P<0.05), MDA (D3: P<0.05), SOD (D3: P<0.05, D7: P<0.05), NF-κB (D3: P<0.05), IL-6 (D7: P<0.05) after transplantation when compared with the saline groups. However, no difference was noted in the fertilization and cleavage rates.
Conclusion: Treatment with resveratrol can prevent ovarian follicle damage and restore ovarian function promptly through anti-inflammatory and anti-oxidative mechanisms.
PS125: Ovarian stimulation or in vitro maturation for fertility preservation in female cancer patients
Ying-chun Guo, Reproductive Medicine Research Center, The Sixth Affiliated Hospital, Sun Yat-sen University; Ting-ting Li; Pan-yu Chen; Cong Fang
Background: To determine the effect of different ovarian stimulation protocols or in vitro maturation for fertility preservation in female cancer patients.
Methods: A retrospective study was conducted in 30 females who underwent fertility preservation procedures in our center. Thirty two cycles were included, 12 of which administrated mini/antagonist protocol, 13 of which used PPOS protocol and 7 of which performed urgency ovulation.
Results: Patients with breast cancer (19/30), lymphoma (6/30), intestinal cancer (2/30), ovarian tumor (2/30) and nasopharyngeal carcinoma (1/30) were included in this study, all of which searched for fertility preservation before chemotherapy. Female patients were included in this study with an average age of 28.4 and an average AMH value of 20.59 ng/mL. Patients spent on average for 7.48 days to retrieve oocytes since their attendance day, while for patients in the urgency ovulation group, only 2.57 days on average were spent (P<0.05). Total amount of Gn was on average 1309.38 U per patient and no significant difference was found between groups. For patients with breast cancer, letrozole were administrated and the average estrogen level on trigger day reached 541.40 pg/mL, which were comparable between ovarian stimulation protocols. The maturation rate of oocytes from ovarian stimulation cycles was 89.96% (206/229), which of that in emergency in-vitro maturation cycles was 38.71% (24/62). For cycles with embryos frozen, the rate of 2PN (76.47%, 39/51) and available embryo (94.44%, 34/36) in the PPOS group were higher than those in the other groups (P<0.05).
Conclusion: The development capability of oocytes from young cancer patients before adjuvant chemotherapy are comparable with those of other infertility patients. Peak estradiol levels of breast cancer patients were controlled by the administration of letrozole. In vitro maturation of oocytes performed at random time of the menstrual cycle may result in a lower maturation rate, which is associated with the time limit of the follow-up cancer treatment. Improvement in the invitro cultivation system is needed for cancer patients with limited time for fertility preservation. In conclusion, clinicians should consider a more holistic approach for female cancer patients, which focuses not only on the characteristic of the primary cancer but also on the phase of the menstrual cycle at their attendance day.
PS126: Lysobisphosphatidic acid activates primordial follicles through regulating PI3K signalling pathways
Weijie Yang, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine; Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province; Yerong Ma; Jiamin Jin; Yinli Zhang; Songying Zhang
Background: The reproductive lifespan of female mammals is determined by the primordial follicle pool before birth, and ovarian function declines with aging and loss of primordial follicles. However, there are still numbers of dormant primordial follicles in perimenopause women’s ovary, and they can be activated by some stimulators to get eggs and have babies. Lysophosphatidic acid is a serum-derived small phospholipid, which has various biological actions, especially developmental effects. Here we established an in vitro model to detect whether lysobisphosphatidic acid could activate primordial follicles and its potential mechanism.
Methods: To evaluate the function of lysobisphosphatidic acid on primordial follicles, we cultured three-day-old newborn mouse ovaries in vitro. Tissues were collected for immunoblotting analysis after cultured for 24 hours and immunohistochemistry staining of Foxo3a was used to identify the activation of primordial follicles. After seven days of culture, we did RT-qPCR to test the change of oocyte development related genes and analysed the proportion of different development stage of follicles through histological sections.
Results: We found more primordial follicles were activated after treatment of lysobisphosphatidic acid than control group with translocation of Foxo3a and the upregulation of PI3K-AKT-mTOR signaling. RT-qPCR revealed that expression of oocyte development gene elevated significantly. And histological sections showed that lysobisphosphatidic acid treated ovary had better follicle development.
Conclusion: We demonstrated successfully that lysobisphosphatidic acid could activate the primordial follicles in vitro, which suggested a possible new method in fertility improvement and treatment of primary ovary insufficiency in future.
Funding: The effect of lysophosphatidic acid (LPA) on the in vitro maturation and early embryo development potential of oocytes, 2016KY085, Zhejiang Medical and Health Science and Technology Program Research Fund Project.
The effect and mechanism of LPA on the developmental potential of oocytes cultured in vitro, 16020320648, Special Fundation for Clinical Research of Chinese Medical Association-Research and Development Project of Young Scientists in Reproductive Medicine.
PS127: An adapted carrier for the cryopreservation of human testicular sperm
Min Wang, Reproductive Medical Center, the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University; Zhengmu Wu; Yuting Hu; Yong Wang; Yajing Tan; Yuqian Xiang; Li Wang; Jin, Hefeng Huang
Background: Does the adapted carrier Cryoplus improve the quality of frozen-thawed sperm compared with the effects of conventional containers, and what is the effect of the novel carrier on clinical outcomes?
Methods: This study included 30 testicular sperm samples for detecting the quality of testicular sperm cryopreserved with the adapted carriers. Semen samples from 27 cases of oligozoospermia were used to investigate whether the adapted carrier improved freeze-thaw sperm quality compared with the effects of 0.25-ml straws and 2-ml cryogenic vials. The retrospective study included another 104 azoospermic patients to investigate the clinical outcomes of testicular sperm cryopreserved with the adapted carrier. This study was conducted on men with mostly obstructive azoospermia (OA).
Results: The adapted carrier required a lower sperm cooling time and fewer sperm thawing procedures compared with conventional containers. The adapted carrier improved post-thawing sperm motility compared with 2-ml cryogenic vials but not compared with 0.25-ml straws. No differences were evident regarding post-thawing sperm DNA fragmentation (SDF) among the three carriers. Testicular sperm cryopreservation with the adapted carrier decreased post-thawing sperm motility and increased SDF. No difference was evident between ICSI cycles using fresh or frozen testicular sperm in terms of fertilization or the good-quality embryo rates. Additionally, no difference was evident between FET cycles using fresh or frozen testicular sperm in terms of the clinical pregnancy, implantation, spontaneous abortion rates, live birth rates or birth weight.
Conclusion: The adapted carrier improved the post thawing sperm motility compared with the effects of 2-ml cryogenic vials. The ICSI-FET outcomes indicate that testicular sperm cryopreserved using the adapted carrier is not inferior to fresh testicular sperm. The use of the adapted carrier for freezing human testicular sperm especially from OA is simple and effective.
Funding: This work was supported by the Shanghai Medical Guide Project from the Shanghai Science and Technology Committee (Grant Number: 16411969600), the Shanghai Health Bureau Fund (Grant Number: 20124Y036), and the Interdisciplinary Program of Shanghai Jiao Tong University (Grant Number: YG2015QN30).
PS128: Achievement of in vitro activation of primordial follicle in mice and human by SIRT1 activator, Resveratrol
Tuo Zhang, China Agricultural University; Meina He; Xinhua Du; Guoliang Xia; Chao Wang
Background: To maintain female reproduction, only a few primordial follicles (PFs) are activated to enter the growing follicle pool during each wave, whereas the majority of PFs are quiescent for later use. Although specific proteins such as mTOR in granulosa cells or AKT and Pten/PI3K in oocytes have been proven to be important for the in vitro activation (IVA) of PFs, and these proteins have even been successfully used to rescue the fertility of patients with either premature ovarian insufficiency (POI) or cancer survivors, the low efficiency and the uncertain safety of the drugs used raises concern.
Methods: To study the function of SIRT1 during primordial follicle recruitment, an in vitro ovary culture system was employed in this study, Based on this model, either RSV, SRT1720 (a specific activator of SIRT1) or CMV-driven Sirt1-overexpression and knockdown vector was introduced into cultured ovaries.
Results: Here, SIRT1, an NAD+-dependent deacetylase that was prominently expressed in the nucleus of pregranulosa cells and oocytes and increased progressively during PF activation, was shown to contribute to the activation of PF independent of its deacetylase activity in mice. Notably, IVA of PFs by up-regulation of SIRT1 in the cultured ovaries with its activator Resveratrol (Res) extracted from grape seeds was cheap, efficient and relatively safe. The underlying mechanism demonstrated that SIRT1 activation increased both Akt1 and mTOR expression by acting more as a transcription co-factor to directly bind to the respective gene promoters than as a deacetylase. The effectiveness of IVA of PFs in human ovarian tissues with Res was also demonstrated.
Conclusion: Activation of SIRT1 with Res induces IVA of PFs in mice and human via classical signaling pathways, and this approach could be an effective candidate strategy to develop more efficient procedures for future clinical use.
Funding: The National Key Research & Developmental Program of China grant number 2017YFC1001100 to G.X.; the National Basic Research Program of China grant numbers 2014CB943202 to C.W.; 2013CB945501 to G.X., 2014CB138503 to B.Z.
PS129: Expression of microRNA-105-3p in peripheral blood of patients with polycystic ovary syndrome and its effects on the growth and apoptosis of human ovarian granulosa cells
Wang Fang; Zou Libo
Background: To detect the expression of miR-105-3p in peripheral blood of patients with PCOS, and observed the its effects on the growth and apoptosis of human ovarian granulosa cells (KGN) in vitro.
Methods: Peripheral blood samples from 30 PCOS patients who came to our hospital’s reproductive center were collected as the experimental group. Peripheral blood of 30 healthy women of the same age was used as the control group. Total RNA was extracted from peripheral blood by Trizol method, cDNA was synthesized by reverse transcription method, and expression of miR-105-3p was analyzed by real-time PCR. KGN cells were routinely cultured in vitro, and transfected with miR-105-3p mimics and mimics negative control, and the expression level of miR-105-3p was detected. The effects of miR-105-3p on the proliferation and apoptosis of KGN cells were observed by MTT colorimetric and enzyme-linked immunosorbent assays.
Results: Compared with the control group, the experimental group had higher body mass index, luteotropic hormone, estradiol, prolactin, total testosterone, free testosterone, ovarian volume and ovarian follicular number, while follicle stimulating hormone and number of menses per year were lower (P<0.05). The expression levels of miR-105-3p in the experimental group and control group were 0.29±0.15 and 1.0±0.31, respectively, and the difference was statistically significant (P<0.05). After 48 hours of KGN cell transfection with miR-105-3p mimics and mimics negative control, the expression of miR-105-3p was 16.39±3.56 and 1.0±0.17, the difference was statistically significant (P<0.05). In vitro transfection of miR-105-3p mimics to KGN cells significantly inhibited cell growth compared to the mimics negative control treated cells (P<0.05). After 48 hours of KGN cell transfection with mimics negative control, the concentrations of Caspase-3, Bax and Bcl-2 were 1.26±0.35, 4.18±0.77 and 5.94±0.93 pg/mL, respectively, whereas the concentrations in miR-105-3p mimics group were 4.17±0.62, 9.03±1.38 and 2.70±0.64 pg/mL, respectively, and the differences were statistically significant (P<0.05).
Conclusion: The expression of miR-105-3p was down-regulated in the peripheral blood of PCOS patients. Overexpression of miR-105-3p in vitro significantly inhibited the growth of KGN cells and promoted apoptosis, suggesting that miR-105-3p is closely related to the pathological process of PCOS.
PS130: Peroxiredoxin4, a protein which shows protective effect on ovarian function
Yan Meng, The First Affiliated Hospital with Nanjing Medical University; Zhengjie Yan; Xiuru Liang
Background: Peroxiredoxin 4 (Prdx4) is a member of Prdx family. As revealed by our previous research work, the expression of Prdx4 was in close contact with ovary-aging. Furthermore, we also discovered that it was located in endoplasmic resticulum (ER) of granular cell and involved in regulating the endoplasmic reticulum stress (ER stress) in granulosa cell. This research work aimed at exploring the protective effect and the molecular mechanism of Prdx4 on ovarian function.
Methods: We established mice model with gene prdx4 knock out by the CRISPR/Cas9 technology. All mice aged 8 months were divided into two groups: Prdx4-KO and wild type group. For the purpose of evaluating the protective effect of Prdx4 on ovarian function, we detected the representative estrous cyclicity of these mice during 12 consecutive days. Secondly, all mice were sacrificed and then blood was collected by removing eyeball. And then we calculated ovary-to-body weight ratio and detected the expression of senescence-associated protein P16 with the help of immunohistochemistry. Thirdly, the number of different types of follicles were counted by hematoxylin and eosin (H&E) staining for the assessment of ovarian reserve. Eventually, the levels of oxidative stress related factors 4-HNE, 8-OHdG, NTY, and three ER stress pathways related markers were examined by immunohistochemistry and real-time PCR. All the statistical analyses were performed using SPSS v.16. software. All the statistical comparisons between the two groups were analysed by t test.
Results: In comparison with the control group, the prdx4-KO group presented significantly smaller ovary weight/body weight ratio (P<0.05), more irregular estrous cycle, higher expression of senescence-associated protein P16, and lower proportion of primordial follicles (P<0.05). In addition, the prdx4-KO group expressed higher oxidative stress related factors. By screening three pathways of ER stress, we found that ATF4 changed obviously. The level of ATF4 and CHOP were up-regulated in the ovary of prdx4-KO group.
Conclusion: Prdx4 shows protective effect on ovarian function. As for the molecular mechanism of Prdx4 on ovarian function, we hypothesized that Prdx4 may inhibit ER stress through PERK-eIF2α-ATF4-GADDl53/CHOP pathway in granulosa cells.
PS131: In vitro activation of primordial follicles application in ovarian freezing and thawing
Zhang Jing, State Key Laboratory of Reproductive Medicine; Li Jing
Background: As chemotherapy often causes ovarian damage and raises risks of ovarian failture and infertility, ovarian fertility preservation becomes an urgent problem. The target of ovary cortex cryopreservation is primordial follicles, which are the storage of oocyte in the ovary. It has the unique advantage which can help not only preserve the fertility of patients, but resume the female’s endocrine function also up to a point. However, efficiency of ovary cortex cryopreservation is not as high as we are concerned. During the freezing and thawing process, lots of primordial follicles will be loss. The only method which is designed to theprimordial follicle is in vitro activation of primordial follicle (IVA). Binding the technique of in vitro activation primordial follicle and ovary cryopreservation and thawing can decrease the damage of primordial follicles after thawing of ovary, and can advance the activation and development of the primordial follicle as well. This novel combination therapy with the two-step system of in vitro cultivation of ovary, will serve as a more efficient and safe system of ovary cortex cryopreservation and thawing. It also can enable us to develop a product of kit to do this preservation process easily and quickly.
Methods: Paired ovaries from PD3 ICR female mice were excised and washed 3 times in media. The paired ovaries are frozen normally, but one of them is thawed normally and the other is thawed combining with IVA. The frozen-thawed ovaries are transplanted to 6 weeks old female mice kidney capsule, then we get transplanted ovaries after 14 days. The ovaries are fixed in 4% paraformaldehyde and stained with hematoxylin for morphological observation.
Results: Comparing the normal frozen-thawed group, we found that the IVA treated frozen-thawed ovaries we significantly larger. Moreover, ovarian histology showed that the IVA treated frozen-thawed ovaries had more follicles at preovulatory stages than normal frozen-thawed ovaries (10.3% vs. 15.1%).
Conclusion: Our results indicate that frozen-thawed mice ovaries combining with IVA could be a effective way to stimulated more silence follicles to grow.
Funding: Research was funded in part by the Nanjing Medical University Science and Technology Development Fund, 2016NJMUZD018, Nanjing, China.
PS132: EIF4G1 is a novel candidate gene associated with severe asthenozoospermia
Yan-Wei Sha; Wen-sheng Liu; Xianjing Huang; Lu Ding
Background: Infertility is a major public health issue affecting an approximate 7%–12% of couples all over the world. Asthenozoospermia (AZS), also known as asthenospermia, is characterized by reduced motility of ejaculated spermatozoa and detected in more than 40% of infertile patients. With less than 1% of motile spermatozoa, severe asthenozoospermia is an urgent challenge for reproductive medicine. The mechanism of severe asthenozoospermia is complex, including metabolic deficiencies , tail anatomic abnormalities, genital tract infections, varicocele, unhealthy lifestyle, antisperm antibodies (ASA) and necrozoospermia.
Methods: Whole genome sequencing and Sanger sequencing, Papanicolaou staining, Transmission Electron Microscopy, Western blot analysis, Immunostaining.
Results: The sperm from patient showed defects in the mitochondrial sheath and flagella. Biallelic mutations c.2521C>T: p.P841S and c.2957C>G: p.A986G in EIF4G1 gene were identified with whole exome sequencing (WES) and sanger sequencing. Furthermore, eIF4G1 protein was extremely low and the protein of COXIV (a mitochondrial marker) and ATP6 (ATP synthesis enzyme) were also decreased in mutant sperm. This infertility was overcome by Intracytoplasmic sperm injections (ICSI) as his wife was pregnant successfully.
Conclusion: Our findings suggest that EIF4G1 may be a new candidate gene relevant to severe asthenozoospermia.
Funding: The National Natural Science Foundation of China (Grant No. 31171375, No. 81871200); the Science Technology Guidance Project of Fujian Province [Grant No. 2017D018]; the Linqiaozhi Funding Supporting Youth Project of Xiamen Maternity and Child Care Hospital [Grant No. FYLQZ2015004].
PS133: Application of hUCMSC for premature ovary failure treatment
Yue Zhao, Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China; Xiangyan Ruan; Alfred Mueck
Background: Premature ovary failure (POF), featured by the cessation of ovarian function before 40 years of age, has become one of the most common causes of infertility in women. But there is no a completely effective treatment regime approved as the best treatment yet. However, Human umbilical cord mesenchymal stem cell (hUCMSC) transplantation has become a promising therapy to restore ovarian function. Our study investigates that hUCMSC can restore the ovarian function of mice with POF induced by chemotherapy and gives new insights into the potential clinical application of hUCMSC for POF treatment.
Methods: Female CD-1 mice at 7-8 weeks age were treated with a single dose of busulfan (in DMSO, 12 mg/kg subcutaneously) and cyclophosphamide (120 mg/kg, intraperitoneally), and the mice of control group were treated with DMSO only. One week after the treatment, hUCMSC were transplanted into POF mice via tail vein or in situ. For tail vein transplantation, mice were transplanted with UCMSC (1×106 cells) by tail vein injection. For in situ transplantation, mice were transplanted directly through bilateral ovaries with UCMSC (1×106 cells). At 14, 21 and 28 days after the UCMSC transplantation, respective 6 mice from each group were euthanized, and blood was collected to measure E2, FSH, and AMH. Ovaries sample were collected for HE staining (ovarian follicle counting and morphological analysis), WB, qPCR and apoptosis assay.
Results: Transplantation of hUCMSC into POF mice increased the level of serum E2 and decreased the level of FSH significantly, and improved the number of follicles. hUCMSC transplantation also reduced the ovarian cells’ apoptosis in POF mice. Both in site and tail vein transplantation of hUCMSC were effective in POF mice.
Conclusion: hUCMSC transplantation for the treatment of POF mice shows positive effects on rescuing ovarian function, and the main action included reduction of apoptosis, increase of number of follicles, restoration of hormone level etc., whereas the exact mechanism is still need further and deeper investigation to illustrate. However, application of hUCMSC represents a promising therapy for POF treatment.
Funding: Research was funded in part by Capital Medical University Research and Development Fund [PYZ2018161]; Beijng Municipal Administration of Hospitals’ Youth Programme [QML2018140]; Beijing Obstetrics and Gynecology Hospital, Captital Medical University [FCYY201811].
PS134: Smad2 is involved in growth of human luteinized granulosa cells through regulation of CYP19A expression
Huiming Ma, Ningxia Medical University; Dongmei Chen; Junli Zhao; Qiaoni Hou; Shuai Zhao
Background: Estrogen plays a very important role in male or female fertility. The synthesis of estrogen is catalyzed by aromatase. Aromatase, encoded by the CYP19A1 gene, is the key enzyme for estrogen biosynthesis. The aim of this study was to examine the effects of Smad2 inhibition on CYP19A1 (the P450 aromatase gene) in human luteinized granulosa cells.
Methods: Human luteinized granulosa cells were seperated from follicular fluid through puncture in IVF-ET. The present study examined the CYP19A activation through lentiviral vector based Smad2 gene overexpression, human granulosa cells were treated using either lentiviral vector based CRISPR/Cas9 nickase mediated knockout gene editing or with the absence of 200 nmol/L the small-molecule inhibitor SM16 for 24 h to evaluate the effect of Smad2 inhibition on CYP19A activation, results in the expression of CYP19A and the regulation of cell apoptosis, as measured by real-time PCR and western blotting and flow cytometer.
Results: Our results show that an increase of CYP19A expression and an decrease of cell apoptosis were observed upon Smad2 overexpression (P<0.05). Interestingly, SM16 treatment and knockdown of Smad2 attenuated the decreased phosphorylation of Smad2 and suppressed CYP19A expression in granulosa cells compared to controls (P<0.05). Furthermore,these results were further confirmed that inhibition of Smad2 reduced the CYP19A mRNA and protein expression (P<0.05).
Conclusion: The present studies suggest that inhibition of Smad2 stimulates the expression of CYP19A protein in human granulosa cells through inhibition of SMAD2 phosphorylation. It suggest that Smad2 signaling could play a pivotal role in growth of human luteinized granulosa cells.
Funding: This study was supported by the National Natural Science Foundation of China (Grant no. 81660813).
Male and Female Infertility
Thursday, 11 April 2019
PS135: Novel variants in WEE2 cause fertilization failure and female infertility
Zhao Shuai, Center for Reproductive Medicine, Shandong University, Jinan, China; Shigang Zhao; Zhao Shuai; Chen Tailai; Yu Mengru; Bian Yuehong; Cao Yongzhi
Background: Successful fertilization is defined by the presence of the second polar body and two pronuclei in the fertilized oocytes. Total fertilization failure (TFF) is a frustrating event that all available oocytes fail to fertilized after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) treatment. Except for some cases caused by possible gamete defects, which can be overcame in the subsequent ICSI treatment, there are women in rare cases enduring recurrent pronucleus formation failure. The etiology of repeated cycles of non-pronucleus zygote after fertilization remains unknown. WEE2 (WEE1 homolog 2, also known as WEE1B) has been known to function in the formation of pronuclei (PN) during fertilization.
Methods: Case-control genetic study.
Results: We identified five subjects that were subjected to homozygous or compound-heterozygous variants of WEE2 [case 1 (from a consanguineous family) with homozygous frameshift variant: c.293_294insCTGAGACACCAGCCCAACC (p.Pro98Pro fs×2); case 2 with homozygous missense variant: c.1576 T>G (p.Tyr526Asp); the other three cases with compound-heterozygous variants, case 3: c.991C>A (p.His331Asn) and c.1304_1307delCCAA (p.Thr435Met fs×31); case 4: c.341_342 del AA (p.Lys114Asn fs×20) and c.864G>C (p.Gln288His); case 5: c.1A>G (p.0?) and c.1261G>A (p.Gly421Arg)]. Except c.1576 T>G (from case 2) and c.864G>C (from case 4), which have been previously reported as rare single nucleotide polymorphisms (SNPs), other 6 variants were novel and were predicted to be deleterious by software. The parental genotypes of case 1 and case 2 indicated that the detected homozygous variants were inherited in an autosomal recessive mode. All the detected variants were absent from the control cohort.
Conclusion: Novel variants found in WEE2, which is autosomal-recessive inherited, may cause recurrent pronucleus formation failure and female infertility.
Funding: National Key Research and Development Program of China (2017YFC1001500), the National Natural Science Foundation of China (81622021, 81601256), the National Natural Science Foundation of Shandong Province (JQ201816)and the China Postdoctoral Science Foundation (2016M600542, 2018M630453).
PS136: Lysyl oxidase blockade ameliorates anovulation in polycystic ovary syndrome
Chuyue Zhang, Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University; Jin Ma; Wangsheng Wang; Yun Sun; Gang Sun
Background: Aberrant ovarian ECM remodeling and inflammation may contribute to the development of polycystic ovary syndrome (PCOS). It remains unknown whether proinflammatory factor is a contributing factor for the over expression of lysyl oxidase (LOX), an enzyme responsible for the cross-linking of collagens, in PCOS ovaries and whether inhibition of LOX can improve PCOS conditions.
Methods: The mRNA abundance of LOX and IL-1β in granulosa cells from patients was measured with quantitative real-time polymerase chain reaction. The LOX activity and IL-1β level in follicular fluid from patients were measured by LOX activity assay and enzyme-linked immunosorbent assay kits respectively. The effect of IL-1β on LOX expression were examined in the primary cultured human granulosa cells. The role of LOX in ovulation was investigated in a dehydroepiandrosterone (DHEA)-induce PCOS rat model with administration of LOX inhibitor β-aminopropionitrile (BAPN). The numbers of retrieved total oocytes and MII oocytes were recorded upon superovulation stimulation.
Results: Increased abundance of LOX and IL-1β was observed in the granulosa cells and follicular fluid in PCOS patients. IL-1β increased LOX expression in a concentration-dependent manner. Inhibition of LOX with BAPN ameliorated irregular estrous cyclicity, polycystic ovary morphology and anovulation in PCOS rats, but appeared to be ineffective in the improvement of oocyte quality.
Conclusion: We have demonstrated in this study that over-expression of LOX is a contributing factor to acyclicity, polycystic ovary morphology and anovulation in PCOS patients, and low grade chronic inflammation of the ovary accounts, at least in part, for the over-expression of LOX. Inhibition of LOX activity may provide a potential effective therapeutic approach to the treatment of PCOS.
PS137: A comparison of medical and surgical management for first trimester pregnancy failure after in vitro fertilization treatment: a randomized clinical trial
Yan Lei, Reproductive Hospital Affiliated Shandong University; Zhang Liping; Yan Lei; Chen Zi-Jiang
Background: To ensure whether a clinical difference exists in the efficacy and future pregnancy rate of the subsequent embryo transfer between medical management and surgical management of first-trimester miscarriage after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment.
Methods: A randomized controlled trial comparing medical versus surgical treatment of first-trimester miscarriage after IVF/ICSI. The women with spontaneous miscarriage after IVF/ICSI of less than 13 weeks’ gestation who were treated with medical management (oral misoprostol preceded by mifepristone 24-48 hours earlier) and surgical uterine curettage were included, from January 2016 to January 2017, in the Reproductive Hospital affiliated to Shandong University. Then, we evaluate the efficacy of these two methods, endometrial thickness and the Human Chorionic Gonadotropin (HCG) levels at 15 days after treatments, the percentage of endometrial thickness difference before the subsequent embryo transfer and the related pregnancy outcomes.
Results: The efficacy of these two methods was significantly different. Compared to the surgical group, the number of women who had an unexpected surgical curettage was significantly higher in the medical group (15.4% vs. 0.8%, P<0.01). As to the percentage of differences (PD) of endometrial thickness before embryo transfer, significant difference existed between the two groups (P=0.036). No differences existed in the pregnancy rates of the subsequent IVF cycle, the HCG levels and endometrial thickness at 15 days after the treatments.
Conclusion: Medical method and surgical evacuation are both effective treatments for IVF patients with first-trimester miscarriage; however, significantly more unexpected surgical curettage occurred after medical management than after surgical management. The medical treatment was more preservative for endometrium.
Funding: This research was supported by the Key Research and Development Program of Shandong Province (2015GSF118124) and National Research and Development Plan (2016YFC1000600).
PS138: Effects of electroacupuncture on the PI3K/Akt/FOXO3a expression in granulosa cells of infertility women with kidney deficiency syndrome
Haicui Wu, Affiliated Hospital of Shandong University of Traditional Chinese Medicine; Jianwei Zhang; Zhengao Sun; Yan Qiao
Background: The success of assisted reproductive technology (ART) procedures, although offering a treatment option and bringing new hope to the infertility patients, may be restrained by poor oocyte quality among other challenges. Previous studies showed that acupuncture at an early stage of oocyte recruitment could improve the quality of oocytes and promote the embryo development. This study further investigated the effect of Electroacupuncture (EA) on outcomes of IVF in infertility women and explored its mechanism by observing the PI3K, Akt and FOXO3a mRNA expression in the granulosa cells.
Methods: Sixty-six infertility patients with Kidney deficiency syndrome, who were to undergo IVF-ET, were randomly assigned to either an EA group or a control group according to a random table. Besides gonadotropin therapy in both groups, the EA and control groups received electroacupuncture or placebo needles for 3 menstrual cycles before IVF, respectively. The scores of the Kidney deficiency syndrome were assessed. Other outcome measures included the number of retrieved oocytes, the fertilization rate, the rate of high-quality embryos and clinical pregnancy rate. The follicular fluid was collected on the day when the ovum was picked up, and PI3K, Akt and FOXO3a mRNA expression in the granulosa cells were measured by RT-PCR.
Results: The syndrome score in the EA group decreased significantly from 16.53±1.75 to 8.67±1.61, while it changed insignificantly in the control group, with a significant difference in the lowering score between the two groups (P<0.05). The high-quality embryo rate and clinical pregnancy rate were both superior in the EA group to the control group (69.15% vs. high-quality 60.27%, 66.67% vs. 42.42%, P<0.05). The fertilization rate was not significant when compared to the control group. As compared with the control group, the expression levels of PI3K and Akt mRNA in the EA group were significantly increased, while the expression of FOXO3a was declined.
Conclusion: For the infertile patients with Kidney deficiency syndrome undergoing IVF, EA for tonifying the Kidney as an adjunct treatment could alleviate clinical symptoms and improve high-quality embryo rate, the mechanism may be through regulating PI3K, Akt and FOXO3a expression in the granulosa cells to improve the oocytes developmental microenvironment and inhibit granulosa cell apoptosis which may explain the improvement in clinical pregnancy rate.
Oral Presentation Session 1
Friday, 12 April 2019
OR01: Follicle-stimulating hormone receptor polymorphism and seminal anti-MÃ¼llerian hormone in fertile and infertile men
Ashraf Hassan, Mansoura University; Adel Zalata; Hoda Nada; Ashock Agarwal; A. Bragais; Taymour Mostafa
Background: Follicle-stimulating hormone (FSH) is fundamental for Sertoli cell function stimulating spermatogenesis and follicular growth by a specific receptor (FSHR). This work aimed to investigate the occurrence of Asn and Ser FSHR gene variants and its relationship with seminal anti-Müllerian hormone (AMH) among normozoospermic and infertile oligoasthenozoospermic (OAT) males.
Methods: Eighty-two Caucasian males grouped into normozoospermic healthy controls (n=30) and infertile OAT males (n=52). FSHR gene variants were determined by DNA from anti-coagulated blood and underwent polymerase chain reaction (PCR) amplification and electrophoresis in detecting amplification products. AMH in seminal plasma was determined by ELISA.
Results: The results showed that the frequency of FSHR gene variants among fertile men was 46.7% Asn/Asn (N680S), 33.3% Asn/Ser, and 20% Ser/Ser, whereas among OAT men were 34.6%, 38.5% and 26.9% respectively with nonsignificant differences. Seminal AMH was significantly higher in fertile than infertile OAT men. There was significant increase in seminal AMH with Asn/Asn variant of FSHR gene than those with Asn/Ser or Ser/Ser.
Conclusion: It is concluded that FSH gene variants showed no difference in distribution between fertile or infertile OAT men. However, when correlated with seminal AMH values, there was an increase in Asn/Asn in men with high seminal AMH.
OR02: Cholesterol and desmosterol in two sperm populations separated on Sil-Select gradient
Ashraf Hassan, Mansoura University; Adel Zalata; A. Christophe; Frank Comhaire; Taymour Mostafa
Background: Sperm lipids are important for sperm viability, maturity and function. This study aimed to identify cholesterol and desmosterol composition of human spermatozoa of two sperm populations separated on Sil-Select gradient.
Methods: Forty-eight males were divided into four groups namely healthy men (n=13), asthenozoospermia (n=11), asthenoteratozoospermia (n=10) and oligoasthenoteratozoospermia (n=14). Sperm cholesterol and desmosterol were estimated in two human sperm population separated by centrifugation in a discontinuous Sil-Select gradient.
Results: The results showed that cholesterol and desmosterol were the major sterols in human spermatozoa. Spermatozoa recovered from upper/lower layer interface (fraction I) had low fertilization potential, while those from the base (fraction II) had high fertilization potential. Median values of cholesterol and desmosterol in fraction I were 2.55 micromol and 0.77 micromol/10(9) spermatozoa and in fraction II were 1.16 micromol and 0.27 micromol/10(9) spermatozoa. Cholesterol/desmosterol ratio was significantly higher in fraction II than I (4.8 vs. 3.2, P<0.01). Cholesterol, desmosterol, total phospholipids and sterols/phospholipids were negatively correlated with sperm concentrations, sperm motility, linear velocity, normal sperm morphology and acrosome reaction percentage whereas cholesterol/desmosterol ratio was positively correlated with these parameters.
Conclusion: It is concluded that the difference in sterol composition of sperm subpopulations separated on Sil-Select gradient suggests that composition of sterols is related to sperm functions.
OR03: NP95 is essential for spermatogenesis and suppresses retrotransposon and cooperates with PRMT5 and piRNA pathway in male germ cell development
Juan Dong, Tongji Medical College, Huazhong University of Science and Technology; Shuiqiao Yuan; Yujiao Wen
Background: Maintaining germline genome integrity is fundamental in living organisms, and transposable element (TE) silencing is essential for protecting the mammalian germline. In mammalian evolution, TEs are responsible for genetic diversifications. Nevertheless, in the short term, TE-induced events can threaten the genomic integrity and contribute to oncogenesis, developmental pathologies and infertility. To protect the germline from TE-induced events, several repressive pathways are responsible for silencing retrotransposons. DNA methylation plays a key role in silencing retrotransposons and function together with histone modifications. Differently, in mouse embryonic stem cells (ESCs), repressive histone marks (H3K9 methylation) are the main mechanisms in repressing retrotransposon transcription with some assistance from DNA methylation. The crosstalk between these repressive epigenetic pathways is crucial in spermatogenesis. NP95 is emerged as a key regulator between DNA methylation maintenance and histone modifications, however, how NP95 regulates the recognition of these two repressive epigenetic marks, and whether NP95 cooperates with PRMT5 for suppressing retrotransposon in postnatal male germ cells remain largely unknown.
Methods: In this study, we first identified a dynamical nuclear-cytoplasmic translocated expression pattern of NP95 in different subtype of adult male germ cells by immunofluorescence, and then discovered a critical role for Np95 in retrotransposon suppression during spermatogenesis by conditional inactivation of Np95 strategy and Co-IP methods in mice.
Results: Depletion of NP95 in postnatal germ cells resulted in complete male sterility, impaired spermatogenesis characterized by meiosis blockage at pachytene stage and massive germ cells enter apoptosis. At a molecular level, conditional loss of NP95 leaded to upregulation of retrotransposons, activation of a DNA damages and alteration of chromatin landscape remodeling in male germ cells.
Conclusion: Our data shed light on novel evidences that NP95 cooperate with PRMT5 and piRNA pathway in male germ cell development, dysfunction of NP95 leads to histone arginine methylation changes and pachytente piRNA profile abnormality. Our observations reveal previously unknown mechanistic features of NP95 functions in retrotransposon silencing for supporting spermatogenesis and male fertility.
OR04: Self-Prostatic massage as an alternative to surgical sperm recovery in spinal cord injured man with anejaculation
Satija Roshi, Dr Sunderlal Memorial Hospital; Gordon Uma
Background: We present the rare case report where with the help of self-prostatic massage sufficient sperm were recovered from the ejaculate of a man with spinal cord injury to undertake ICSI with successful outcome, avoiding surgical sperm recovery.
Methods: The spinal cord injury patient was seen in the clinic for surgical sperm recovery for anejaculation. Following discussion with the patient, it was realized that daily self-bowel evacuation was needed to be undertaken by the gentleman and a small amount of ejaculate was noted from the penis on each occasion. On laboratory assessment, sperm were noted in small quantities which were not suitable for freezing. Although a formal urological prostatic massage was undertaken, this was unsuccessful in recovering sperm, but the man himself was successful in obtaining sufficient ejaculate by self–prostatic massage. Laboratory provided an open appointment to bring in samples until suitable sperm for freeze were obtained.
Results: Thirteen semen samples were analyzed over a period of 6 weeks. Volumes ranged from 0.1 ml to 1.5 ml. Most samples showed many immotile sperm, but occasional motile sperm. A total of 4 ampules were frozen. They conceived in the second ICSI cycle and have delivered a baby
Conclusion: It may only be suitable for men who can undertake self-bowel evacuation thereby indirectly undertaking self-prostatic massage. Prostatic massage appears to be an easy, noninvasive and risk-free method to obtain spermatozoa from selected spinal cord injury patients avoiding surgical sperm recovery.
OR05: The clinical outcomes after laser assisted selection of immotile testicular spermatozoa for ICSI
Huanhua Chen, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region; Jinhui Shu; Hong Zhou; Ruoyun Lin
Background: A viable spermatozoon is a prerequisite for initiating fertilization in Intracytoplasmic sperm injection (ICSI) cycle. Motility is the primary sign used for determining the viability of a spermatozoon. However, the testicular spermatozoa is often encountered as completely immobile sperm. As is well known, completely immotile sperm does not mean dead sperm. How to select of immotile but viable testicular spermatozoa on the day of ICSI is a major challenge for embryologists.
Methods: A total of 148 couples were enrolled to the study from June 1, 2014 to June 30, 2018. These couples were divided into two groups according to whether their husbands’ testicular spermatozoa present motility after in vitro culture of 2~6 hours. The motility spermatozoa group were 113 couples, and laser assisted selection immotile spermatozoa group were 35 couples. The fertilization rate, cleavage rate, high-quality D3 embryo rate, biochemical pregnancy rate, clinical pregnancy rate, embryo implantation rate, abortion rate, live birth rate and continued pregnancy rate were compared between the two groups.
Results: There were no significant differences between laser assisted selection immotile spermatozoa group and motility spermatozoa group in terms of women mean age, mean number of oocytes, percentage of M II oocytes, normal fertilization rate, cleavage rate and percentage of high-quality D3 embryos (P>0.05). A of 27 fresh embryo transfer cycles in the laser assisted selection immotile spermatozoa group and 86 fresh embryo transfer cycles in the motility spermatozoa group were performed. There was no significant difference in the mean number of transfer embryos between the two groups (P>0.05). The biochemical pregnancy rate (70.37% vs. 48.84%), clinical pregnancy rate (62.96% vs. 45.35%), embryo implantation rate (45.00% vs. 35.48%) and live birth rate (64.70% vs. 61.54%) in the laser assisted selection immotile spermatozoa group were slightly higher than those in the motility spermatozoa group, but there was no statistical difference (P>0.05). There was no significant difference between the two groups in the abortion rate (11.76% vs. 17.94%) and the pregnancy continuation rate (23.53% vs. 23.08%) (P>0.05).
Conclusion: Laser assisted selection of viable but completely immobile testicular sperm does not increase incidence of adverse clinical outcomes and is an reliable tool for selection of viable but completely immobile testicular sperm in ICSI.
Oral Presentation Session 2
Male and Female Infertility
Friday, 12 April 2019
OR06: Evaluating the prevalence, management, and the psycho-social impact of premature ovarian insufficiency on patients attending an academic hospital reproductive endocrine clinic
Mmaselemo Tsuari, Tygerberg Academic Hospital, Cape Town, South Africa; Thabo Matsaseng
Background: Primary Objective: To measure the prevalence of premature ovarian insufficiency (POI); Secondary Objective: To determine the etiology, review the management process and approach and to assess the impact of the diagnosis on patients with POI; Design: Cross-sectional study; Setting: Reproductive Endocrine Clinic in an academic hospital; Patients: Patients living with a diagnosis of POI.
Methods: Materials and Methods: Patients interviews, review of medical records including bone scans, clinical evaluation of patients as they present at the clinic. The study was undertaken over 12 months from August 2016 to July 2017. Primary Outcome Measure: The prevalence of POI in our Reproductive Endocrine Clinic; Secondary Outcome Measures: The management of POI, and impact of the diagnosis on the women.
Results: There were 47 patients living with a diagnosis of POI and the total number of all patients seen during this period at the above clinic was 561. The prevalence of POI in our setting is 8,4% (Confidence interval 6.28%-11.06%).
The etiology of POI in 32% of the patients was idiopathic, 19% genetic syndromic disorder, all of which were Turner Syndrome, 10% were non-syndromic genetic disorders. A familial disorder was found according to family history and accounted for 2% of the causes.
Auto-immune causes accounted for 6%, infective causes 6%, cancer and chemotherapy 6%, obstetric complications 6%, iatrogenic 4% and endometriosis 2%.
A dual energy X-ray absorptiometry (DEXA) scan was done of which 32% had osteopenia, 11% had osteoporosis 21% patients a DEXA scan was requested but not done. Patients with normal DEXA scans were re-assessed 5 years later and patients with osteopenia and osteoporosis were re-assessed every 2 years while on treatment. Eighty-one percent (81%) of patients with osteoporosis were treated with bisphosphonates.
There were 55% of patients with depression and they were treated with various antidepressant.
Conclusion: The prevalence of POI in our clinic is reported to be 8.4%, which is much higher than the prevalence quoted in literature of 1%. This large difference may be due to a bias caused by a smaller sample size relative to the total number of patients seen. All patients received the standard treatment protocol and various specific treatment modalities.
Funding: Budget requirements: There was no budget allocated to the study.
OR07: Pregnancy and birth outcomes of single versus multiple embryo transfer in gestational surrogacy arrangements: a systematic review
Jutharat Attawet, University of Technology Sydney; Alex Wang; Cindy Farquhar; Elizabeth Sullivan
Background: The likelihood of livebirth following assisted reproductive technology (ART) is increased when more than one embryo is transferred. However, with the transfer of more than one embryo there is also an increased risk of multiple pregnancy and subsequent adverse maternal and perinatal outcomes. This is particularly important for gestational carriers as they may potentially have long term complications following a surrogacy pregnancy. As a result in Australia, New Zealand and Canada, single embryo transfer (SET) is strongly recommended for gestational surrogacy. Nonetheless, there is limited evidence that systematically evaluates pregnancy outcomes between single and multiple embryo transfers (multiple ET) for gestational carriers.
Methods: This study is a systematic review. Electronic databases were searched from CINAHL, Medline, Embase, Scopus and ProQuest for studies from 1980 to 2017. Cross-references and national ART reports were also manual searched. Articles without restriction of English language and study types were accessed. The key words for searching were surrogacy, gestational carriers, embryo transfer, SET and multiple ET. The primary outcomes were live delivery and multiple delivery per gestational carrier, and compared between SET and multiple ET. Mantel-Haenzel risk ratios (RRs) with 95% confidence intervals (CIs), using the numbers of outcome events in SET and multiple ET of each study were calculated suing RevMan5.3.
Results: The search returned 97 articles. Of which five met the inclusion criteria where articles focused on SET and MET carrier cycles along with primary outcome measures. Of the five studies there were 898 carriers aged from 21 to 52 years. There were 885 gestational carrier cycles with embryo transferred, 48% (432) of SET and 52% (463) of MET. The live delivery rate was not significantly different between SET and MET with RR=0.88 (95% CI: 0.61-1.30, n=4, I2=67%). There were 105 sets of twins and one set of triplets following MET. There was one set of twins following SET. The multiple delivery rate per gestational carrier was significantly lower following SET than MET (RR=0.04, 95% CI: 0.01-0.26, n=3, I2=37%). The clinical pregnancy rate was not significantly different between SET and MET (RR=0.89, 95% CI: 0.66-1.19, n=5, I2=62%).
Conclusion: SET should be strongly recommended among gestational carriers to prevent multiple pregnancy and subsequent adverse outcomes for both carrier and baby.
OR08: Fertility knowledge of women in China
Lesley Foster, SPD Swiss Precision Diagnostics GmbH; Sarah Johnson; Giulia Zanzi; Celine Mollard
Background: Education on reproductive health at school usually focuses on prevention of pregnancy and sexually transmitted infections. Therefore, when women start trying for a baby, they may lack important knowledge to help them conceive. This study sought to assess fertility knowledge amongst Chinese women.
Methods: On behalf of SPD, Ipsos Suisse SA surveyed a sample of 1001 Chinese women aged 20-45 years old who were able to have children and who chose to take part in our survey from Ipsos Panel who were predominately in economic Tier 1, 2 and 3. Interviews were conducted online (computer assisted web interview) between 27th of July to 7th of August 2018.
Results: When choosing the most accurate description of a menstrual cycle from a list of 5 options, only 37% of our participants selected the correct answer (first day of bleeding to the day before the next bleeding start), but a significantly higher proportion (63%) were able to select the correct definition of ovulation (when an egg is released from an ovary). Only 19% of participants correctly knew there were 3-6 fertile days when women are able to get pregnant in a menstrual cycle, and more than 6 in 10 (63%) believed there were 7-14 fertile days when women are able to get pregnant. However, awareness of which part of the menstrual cycle was fertile (when being able to get pregnant) was higher, with 46% correctly saying “the 2-3 weeks before the next period starts”, and 31% saying “always in the middle of the cycle”.
Conclusion: Knowledge of fertility varied for Chinese women in our survey. Of key concern is the large proportion of women who believed their fertile window was 7-14 days. This misconception could lead to mistiming of intercourse, which could mean failing to achieve pregnancy as quickly as desired.
Funding: Research was funded by SPD Swiss Precision Diagnostics GmbH, Geneva, Switzerland.
OR09: Clinical feathers and outcomes of nonobstructive azoospermia patients with different etiology undergoing ICSI-MicroTESE treatment
Xiangfeng Chen, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Yi Ma; Ping Ping
Background: The purpose of the study was to investigate clinical, laboratory, histological features, microdissection testicular sperm extraction (microTESE) and intracytoplasmic sperm injection(ICSI) treatment outcomes of nonobstructive azoospermia (NOA) patients of various etiologies, and to compare ICSI outcomes using fresh and frozen thawed sperms from MicroTESE.
Methods: A retrospective analysis was made in 595 NOA patients undergoing microTESE from January, 2013 to December, 2017. The men were classified into six groups based on etiology. Patients’ age, history, hormone profile, testis volume, testicular histology, sperm retrieval, fertile and pregnancy outcomes of ICSI were included for analysis.
Results: A total of 595 NOA patients were included in this study, with 446 (75.0%) were idiopathic NOA, 66 (11.1%) were Klinefelter syndrome (KS), 34 (5.7%) with micro-deletion of the AZFc, 33 (5.5%) cases had the history of cryptorchidism, 13(2.2%) had a history of mumps orchitis, and 3(0.5%) cases underwent chemotherapy. The overall sperm retrieval rate (SRR) was 40.3% (240/595), SRR of the cryptorchidism (84.8%, 28/33) and mumps orchitis (84.6%, 11/13) groups were much higher than that of other groups, the SRR of idiopathic group was the lowest (31.8%, 142/446). 198 ICSI cycles utilizing MicroTESE sperm were retrospectively analyzed, including 155 fresh MicroTESE ICSI cycles and 43 frozen-thawed MicroTESE ICSI cycles. Fertilization rate, cleavage rate, and clinical pregnancy rate of fresh sperm group were slightly higher than those in frozen thawed MicroTESE sperm group, but high qualified embryo rate of fresh sperm group was lower than frozen thawed group. The differences were of no statistical meaning.
Conclusion: Etiology may be an effective prognostic factor for SRR in NOA patients. NOA of definite etiology, such as cryptorchidism, has high SRR, while Idiopathic NOA, the most common type of NOA, has the lowest SRR. Using of frozen thawed sperm from MicroTESE had similar pregnant outcome to that of fresh sperm, so cryopreservation of testicular sperm seems to be more suitable and of great benefit in these cases and good results can also be expected when oocyte retrieval and ICSI are not performed at the same time.
Funding: The research was supported by the National Natural Science Foundation of China (No. 81671511).
OR10: Real-world study of recombinant human follicle stimulating hormone/luteinizing hormone (r-hFSH/r-hLH) for controlled ovarian stimulation in assisted reproductive technology treatment in routine practice in Russia
Yuliya Koloda, Russian Medical Academy of Continuous Professional Education, Moscow, Russia; Vladislav Korsak; Aydar Abubakirov; Elena Kalinina; Alesya Lvova; Emilia Richter; Joan Schertz; Thomas D’Hooghe; Oleg Rozenson
Background: Supplementation of follicle-stimulating hormone (r-hFSH) with luteinising hormone (r-hLH) for controlled ovarian stimulation (COS) as part of assisted-reproductive technologies (ART) can improve outcomes in patients with predictors of suboptimal/poor response (POR). We investigated the patient profile and administration criteria of patients who received the 2:1 fixed-ratio combination of r-hFSH/r-hLH (Pergoveris®; Merck KGaA, Darmstadt, Germany) for COS in ART centres in Russia.
Methods: Five hundred women were enrolled in this prospective, observational study at 20 centers in Russia from April 2016 to January 2017. They received r-hFSH/r-hLH throughout COS or r-hFSH for 5–7 days then r-hFSH/r-hLH from day 6–8. ART procedures were according to individual treatment practices and adjusted for patient response. Primary endpoints were patient demographics, baseline characteristics, medical history and criteria used for r-hFSH/r-hLH administration (Russian label or ESHRE Bologna criteria). Other endpoints were clinical pregnancy rate (CPR) and adverse event (AE) monitoring.
Results: Most patients (n=330 [66%]) were ≥35 years-old and 110 (22%) were ≥40 years-old; 231 (46%) had previously undergone IVF/ICSI. Patients were categorized as POR according to the Russian label (n=374) or the ESHRE Bologna criteria (n=269); patients could also meet neither (n=80) or both criteria (n=223). Mean duration of COS was 8.6 and 8.2 days, mean drug doses (IU) were 338 and 365 (r-hFSH) and 123 and 124 (r-hLH), and mean oocytes retrieved were 6.5 and 5.0, in the Russian label and ESHRE criteria groups, respectively. CPRs were 27.3% and 31.2% (per cycle) and 31.7% and 36.7% (per transfer) in the Russian label and ESHRE groups, respectively. 22 (4.4%) patients reported AEs. One AE (ectopic pregnancy) was severe but not fatal.
Conclusion: Most patients who received r-hFSH/r-hLH for COS met either or both POR criteria; however, higher CPRs were observed than those previously published for PORs (range 10.8–16.4%), supporting the use of r-hFSH/r-hLH in this population.
Funding: This research was funded in full by Merck KGaA, Darmstadt, Germany. This abstract was presented at the XXVIII annual conference of the Russian Association of Human Reproduction.
OR11: Meta-analysis of recombinant-human follicle-stimulating hormone (r-hFSH) +recombinant-human luteinizing hormone (r-HLH) vs. r-hFSH alone for controlled ovarian stimulation in assisted reproductive technologies treatment
Sandra Guedes, Merck KGaA, Darmstadt, Germany; Julie Hubbard; Robert Wan; Thomas D’Hooghe; Veronica Alam; Joan Schertz
Background: The aim of this systematic review and meta-analysis was to evaluate r-hFSH/r-hLH vs. r-hFSH alone for controlled ovarian stimulation (COS) as part of assisted reproductive technologies treatment.
Methods: MEDLINE, Embase and other relevant sources were searched for studies of women undergoing COS for multifollicular development with r-hFSH/r-hLH or r-hFSH followed by embryo transfer. Endpoints analysed were live birth rate (LBR), biochemical, clinical and ongoing pregnancy rates (PR), incidence of pregnancy loss and ovarian hyperstimulation syndrome, and assessment of outcomes in 4 subgroups: poor ovarian responders (POR), advanced maternal age (≥35 y), gonadotropin-releasing hormone (GnRH) agonist and GnRH antagonist. Outcomes were compared using a random effects model separately for randomized and non-randomized studies and reported as relative risk (RR) and 95% CI.
Results: A total of 649 publications were identified, of which 78 were eligible for inclusion in the analysis (54 randomized [9203 patients (pts)] and 24 non-randomized [14143 pts] studies). No difference in LBR was observed between treatments in the randomized studies (21 studies [3782 pts]; pooled RR 1.04 [0.90, 1.20]) or the non-randomized studies (14 studies [12874 pts]; pooled RR 1.05 [0.90, 1.23]). Little or no difference was observed for clinical PRs (RR 1.08 [1.00, 1.17]) with r-hFSH/r-hLH vs. r-hFSH alone in randomized studies (N=44). No difference was observed for any of the other endpoints in the randomized studies. Biochemical PR (RR 1.14 [1.01, 1.28]) and ongoing PR (RR 1.14 [1.05, 1.24]) were higher with r-hFSH/r-hLH vs. r-hFSH alone in the non-randomized studies. In the subgroup of POR there was a higher clinical PR in randomized studies with r-hFSH/r-hLH vs. r-hFSH alone (16 studies [2666 pts]; RR 1.22 [1.07, 1.40]).
Conclusion: This meta-analysis showed that supplementation of r-hFSH treatment with r-hLH did not increase LBR but analyses of subgroups showed a potential beneficial effect on clinical PR in POR. Further studies into the impact of r-hLH supplementation on LBR in patients with POR should be undertaken, using up-to-date, globally accepted definitions of reproductive outcomes and also definitions for POR, as proposed in the ESHRE Bologna criteria and the Poseidon classification for POR/suboptimal response.
Funding: Funded by Merck KGaA, Darmstadt, Germany.
Oral Presentation Session 3
Ethics, Human Rights and the Law
Friday, 12 April 2019
OR12: Human dignity: ART and the future of human nature
Marisa Almeida Araujo, Lusiada University - Norte (Porto); Augusto Meireis
Background: ART tecnhology, specialy CRISPR technology, brings us the debate of a modern (re)interpretation of the Human nature.
At a time when we are living an era which, as Kaufmann refers to as “postmodernism” of transition and rupture, the law will be apprehended as a reality, in the “astonishment” being, therefore, a human construction.
It seems undeniable that ART tecnhology is confronting men to a new debate, of human nature irself, were human dignity, is the boundary.
Methods: Considering, as a method of study, the analysis of the Nuffield Council on Bioethics (2016), regarding the human reproduction, and its latest version, of 2018, the “Genome Editing and Human Reproduction: Social and Ethical Issues” that, inflaming the debate, seeming to accept, albeit cautiously, that the issue may be ethically acceptable in some circumstances, namely the guaranty of the welfare of the person.
The timeliness and importance of the subject requires us to focus our analysis considering the ethical and legal issues raised by the topic, considering the results and recommendations of the reports and bioethics authors in this domain.
Results: New biological values arises, in great strength, ethical and legal problems, that will be necessary to consider to an optimisation of the principles and interests in conflict and the choice.
This valid perception of legitimacy imposes the debate, bringing out the theme to the public domain in an outcrop of democratic participation and exercise of (bio)citizenship.
The biological citizenship on the active participation of citizens, individually and as a whole in a social perspective, as a way of democratic participation in a new biological dimension. The power of today in the generation of tomorrow and the determinism of its life projects, creating an instrumental connection between “creator and creature”, has its border line in Human dignity, is the line between creating a man and creating a thing.
We are at the absolute threshold of human creation, irreversible and unrepeatable, and constitute an appropriation of the identity, mainly genetic, and the historicity that integrates it, of another.
Conclusion: ART tecnhology opens the discussion between were to establish the frontier that Human Dignity imposes, in a Kantian vision, in which man becomes not an end in itself but an object at the service of others.
Funding: This work is financed by national funds by FCT - Foundation for Science and Technology, under the Project UID/DIR/04053/2016.
OR13: Challenges in global governance of reproductive technologies: legal and ethical issues
Sayani Mitra, University of Liverpool; Lucy Frith
Background: Infertility is a growing social and medical problem and the use of ARTs is rising, particularly in low and middle-income countries (LMIC). There are substantial differences in how ARTs are regulated and who has access to ARTs in different countries and people are increasingly travelling abroad to use ARTs. How to regulate these technologies in such a globalised context urgently needs to be addressed.
Methods: This paper will discuss the development of new approaches for the effective regulation and oversight of the rapidly expanding global provision of ARTs. How to regulate these technologies in such a globalised context urgently needs to be addressed. We will present an overview of the key debates. It will explore these policy challenges by adopting an interdisciplinary approach, informed by ethico-socio-legal and global health perspectives.
Results: The paper will examine how concepts such as: reproductive autonomy, professional autonomy, human rights, children’s rights and best interests, human dignity and instrumentalisation are reflected or validated in governance and supranational norms. Furthermore, new governance frameworks must take account of the different regulatory actors and agencies at play (the state, professional bodies, other intermediaries) and this raises questions of regulatory locus. For example, as international legal structures are so disparate, there is a pressing need to map the governance roles that have been adopted by trans-national professional bodies (e.g. the European Society of Embryology & Human Reproduction [ESHRE]).
Conclusion: The ongoing work discussed in this paper will lay the groundwork for future international policy engagement that will enable dynamic discussions of new regulatory approaches, with a particular emphasis on the growing use of ARTs in LMIC.
OR14: Discussions from ethical or human right aspect may confer women’s hypertension before pregnancy
Shinichiro Koga, Section for Disorders in Endocrine & Kidney Systems, Department of Medicine, Tokyo Metropolitan Police Hospital, Tokyo, Japan; Juri Ogishima; Naoko Nakazawa
Background: Limited evidence could be applied to the management for hypertension before pregnancy.
Methods: This was with descriptive approach. CASE RECORD A 43-year-old null GPAC female was brought to general medicine department with chief complaint of dry cough for two months and general fatigue for one week. Her blood pressure 248/150 mmHg, and the diagnosis of chronic hypertensive heart disease and chronic kidney disease were made with elevated cardiothoracic ratio, presence of pleural fluid, left ventricular ejection fraction ~40%, Cr 5.57 mg/dL (eGFR 7.2 mL/min/1.73 m2), and spot urine Pro/Cr ratio 2.94 g/gCr. Her past 25-year-medical history included IgA nephropathy, histochemically diagnosed on 29 years old. Her deteriorating hypertension control had affected kidney function, because angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) was truncated due to desired pregnancy from 34 years old.
Results: (1) ACEI and ARB need to be withdrawn if a hypertensive women wishes to become pregnant, because ACEI and ARB are teratogenic, with increased risk of congenital abnormalities. (2) Methyldopa, and labetalol, a combined alpha- and beta- blockers, are considered the first choice for medication. (3) There is increasing debate regarding discouraging the use of diuretics. (4) Recent guidelines also encourage woman with chronic hypertension to keep their dietary sodium intake low, either by reducing or substituting sodium salt before pregnancy.
Conclusion: No discussion has had so far on aspect from ethical or human right in limited evidence for this unshed-lighted disorder, women’s hypertension before pregnancy.
Funding: No funding to disclose.
 N Engl J Med 2006;354:2443–51.
 Mol Cell Biol 1997;176:61–71.
 J Hypertens 2013;31:1281–357.
 Cochrane Db Syst Rev 2017:CD04022.
Psychology and Counseling
Friday, 12 April 2019
OR15: Affordability of assisted reproductive treatment (ART): a mixed method comparative study between the UK and Nigeria
Ada Achinanya, University of Sheffield; Robert Akparibo; Dilly Anumba
Background: Most interventions geared at increasing chances of conception through assisted reproductive treatment (ART) have failed to consider the financial implications of the treatment. This study aims to assess the prevalence of catastrophic ART expenditure and the cost-burden of the treatment to the households.
Methods: A mixed methods study utilising qualitative and quantitative designs. A total of 116 (UK=64, Nigeria=52) infertile women took part in the quantitative cross-sectional survey, while a subsection of 32 (UK=15, Nigeria=17) from the 116 participants took part in the qualitative study. Questionnaires were used to capture information on socio-economic status, monthly household non-food expenditure, subjective financial well-being (SFW) and out of pocket costs for the ART cycle. Payments for ART as a percentage of annual non-food expenditure was calculated to estimate catastrophic ART expenditure, and factors associated with SFW were identified. Follow-up interviews were conducted using semi-structured open-ended questions. Descriptive analysis looked at the proportion of households experiencing catastrophic ART expenditure, while interviews were analysed thematically.
Results: Descriptive results showed that most Nigerian households incurred catastrophic expenditures, defined as an OPP of >40%; whereas, this did not occur among the UK cohort. The Nigerian households used various coping strategies which include depleting savings, borrowing money or taking contributions. Among the UK women, household income was predictive of SFW, with higher incomes associated with increased financial well-being. However, this was not observed among the Nigerian cohort. The interview findings supported the descriptive results. Both UK and Nigerian women expressed similar concerns about the financial stress of funding the treatment. A key explanatory factor in both was that the short-term financial constraints affected their quality of life negatively.
Conclusion: Most Nigerian households experienced catastrophic ART expenditures, whereas UK households did not. Not all couples who are unable to afford ART in low-middle income countries, forfeit it. ART counselling should include financial psychoanalysis.
OR16: Adaption vs. ART for non biological child - Indian scenario
Anuj Sharma, Prem Test Tube Baby Centre; Aarti Sharma; Saarthak Bakshi
Background: I wonder why anyone will spend money and time to have non biological child through ART, is it a social concern or a matter of self esteem. Adaption is never considered as an option. Feeling of being the Mother makes female life complete, but sometime we get the cases who want to get non biological child through surrogacy, atrocious i will say. Why not people ready to adapt an orphan which will give a child to them and parent to the needed child, so its like win win situation for both. So we decided to analyse why our society is not in favour of adaption.
Methods: We prepared a questionnaire for the couples seeking ART services for a non biological child weather it is due to multifactorial infertility or due to exhaustion of couples after multiple failed ivf cycles. We have also included couples who says it doesn’t matter whose gamete you use, we just want to have the best chance from our cycle, though after counselling they understood the things.
We have analysed 36 such couples in last one year.
Results: Infertility is considered as a curse not disease, couple want a baby so that family, friend and relative stop cursing them, so they are only concern is to get pregnant. Multiple failed ivf attempts is a big mental, physical and financial trauma and some ART centres also guide them to go for Embryo Donation for cheaper solution and better results.
Adaption policies are not very friendly, waiting time is too long and baby they get might have disabilities. Couple also have a hitch that a grown up child if they get might not get adapted to their rituals and culture.
Conclusion: Education of society about infertility and adaption, counselling should be done not only for couples but also for family and close friends. Policies of adaption should be made simpler. If we can shift couples looking for non biological child via ART to adaption then it will save a huge amount of money loss and also physical and mental stress these couple go through. It will also help in improving the quality of life of many orphans and a little measure to control our population.
OR17: Assessment of stress and anxiety in infertile women commencing assisted reproductive treatment (ART): a comparison of UK and Nigerian women
Ada Achinanya, University of Sheffield; Robert Akparibo; Dilly Anumba
Background: Globally, infertile women experience increased rates of psychological distress all over the world. Certain socio-demographic factors may aggravate this distress, and this might differ between a high income and low-middle income country. This study assessed the prevalence of stress and anxiety and its associated socio-demographic differences in UK and Nigerian women presenting for assisted reproductive treatment (ART).
Methods: A cross-sectional survey involving 116 infertile women (UK=64, Nigeria=52) presenting for In Vitro-fertilisation (IVF) and Intra Cytoplasmic Sperm Injection (ICSI) in public fertility clinics in the UK and Nigeria was conducted. The prevalence of stress was measured using the Perceived stress scale (PSS-10), while anxiety was measured using the Beck Anxiety Inventory (BAI-21). Socio-demographic and fertility data were collected using a structured questionnaire. Associations between stress, anxiety and socio-demographic variables were explored using Pearson’s correlation, independent t-test and regression analyses.
Results: Nigerian women were more likely to be stressed (M=19.7 vs. 16.7, P<0.01) compared to UK women. In both UK and Nigerian women, there was a comparable frequency of negative results for anxiety and its intensity. Among the UK women, higher stress levels occurred most frequently among cohabiting women (P<0.05), diagnosed female infertility (P<0.01), and women with a history of treatment failure (P=0.05). Whereas in the Nigerian women, longer duration of infertility and IVF-recommended procedures were associated with the severity of stress and anxiety symptoms (P<0.05).
Conclusion: Nigerian infertile women were more stressed than UK women. Infertile women are more vulnerable to anxiety, irrespective of the location. There is a need to include psychological screening in the routine care of infertile women especially in developing countries.
Poster Session 2
Male and Female Infertility
Friday, 12 April 2019
PS139: Novel inactivating mutations in the FSH receptor cause premature ovarian insufficiency with resistant ovary syndrome
Wen-Bin He, Institute of Reproductive and Stem Cell Engineering, Central South University; Juan Du; Wen Li; Guang-Xiu Lu; Ge Lin; Fei Gong; Yue-Qiu Tan
Background: Premature ovarian insufficiency (POI) is defined as the depletion or loss of normal ovarian function in women less than 40 years of age, and is an important cause of female infertility. The object of this study is to identify the genetic aetiology of three resistant ovary syndrome (ROS) pedigrees from thirteen Chinese Han families with non-syndromic POI.
Methods: A set of 13 unrelated families with POI were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya, including three pedigrees with ROS. The proband in each family was subjected to whole-exome sequencing (WES). Bioinformatic analysis was employed to predict the pathogenicity of the identified variants, and subsequently cyclic adenosine monophosphate (cAMP) assay was performed for the functional characterization of the FSHR mutations.
Results: Four novel mutations, two homozygous mutations (c.419delA, c.1510C>T), and a compound heterozygous mutation (c.44G>A and deletion of exons 1 and 2) of FSHR were identified in the three non-syndromic POI-with-ROS families. A donor splice-site mutation in STAG3 (c.1573+5G>A) and a missense mutation in DMC1 (c.106G>A) were identified in two of the thirteen families, one in each family. Bioinformatic analysis predicted that the three novel point mutations in FSHR are deleterious and associated with POI in the three families, which was confirmed by in vitro functional analysis in which FSH-induced cAMP production was abolished for all receptors.
Conclusion: The three novel point mutations in FSHR were all functional inactivating mutations, and were the genetic aetiology of the three non-syndromic POI-with-ROS families. We report the first FSHR frameshift mutation, and the first missense mutation in the signal peptide-encoding region of FSHR to be associated with POI. Women affected by ROS should consider undergoing mutation screening for FSHR.
Funding: This work was supported by grants from the national natural science fund of China (No.81471432 and 81771645) and Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-201802).
PS140: DMC1 mutation causes human non-obstructive azoospermia and premature ovarian insufficiency
Wen-Bin He, Institute of Reproductive and Stem Cell Engineering, Central South University; Chao-Feng Tu; Juan Du; Wen Li; Chang-Gao Zhong; Guang-Xiu Lu; Ge Lin; Yue-Qiu Tan
Background: The genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown. The object of this study is to identify the genetic cause of NOA and POI in two affected members from a consanguineous Chinese family.
Methods: We performed whole-exome sequencing of DNA from both affected patients. The identified candidate causative gene was further verified by Sanger sequencing for pedigree analysis in this family. In silico analysis was performed to functionally characterize the mutation, and histological analysis was performed using the biopsied testicle sample from the male patient with NOA.
Results: We identified a novel homozygous missense mutation (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1, which co-segregated with NOA and POI phenotypes in this family. The identified missense mutation resulted in the substitution of a conserved aspartic residue with asparaginate in the modified H3TH motif of DMC1. This substitution results in protein misfolding. Histological analysis demonstrated a lack of spermatozoa in the male patient’s seminiferous tubules. Immunohistochemistry using a testis biopsy sample from the male patient showed that spermatogenesis was blocked at the zygotene stage during meiotic prophase I.
Conclusion: To the best of our knowledge, this is the first report identifying DMC1 as the causative gene for human NOA and POI. Furthermore, our pedigree analysis shows an autosomal recessive mode of inheritance for NOA and POI caused by DMC1 in this family.
Funding: This study was supported by grants from the National Natural Science Foundation of China (81771645 and 81471432 to Y.T.), and Graduate Research and Innovation Projects ofCentral South University (Grant 2017zzts071).
PS141: Growth hormone promotes in vitro maturation of human oocytes
Yue Li, Center for Reproductive Medicine, Shandong University, Jinan, China; Shigang Zhao; Hui Liu
Background: Increasing the success rate of in vitro maturation(IVM) for human oocytes has a major significance. Related studies showed that growth hormone (GH) added into IVM medium could promote maturation of oocytes in non-human beings. However, few studies on systematic IVM for human oocytes with GH have been reported.
Methods: Human germinal vesicle (GV) oocytes were collected for IVM cultured with different concentrations of GH to determine the optimal GH concentration. Metaphase II (MII) stage oocytes obtained from IVM were fertilized by intracytoplasmic sperm injection (ICSI). Maturation rate, fertilization rate, and blastocyst rate were assessed after IVM culture with GH. Further more, gene expression profiles were compared in oocytes after IVM with or without GH using single cell transcriptome sequencing.
Results: The optimal concentration of GH for IVM was 200ng/mL, and the maturation rate of 200ng/mL group reached 70% which was significantly higher than 35% of control group (P=0.004). The fertilization rate (76.9% vs. 59.5%) and blastocyst rate (37.5% vs. 24%) were also higher in the GH group, though the differences were of no statistical significance. RNA-Seq and real-time PCR data showed that GH could significantly enhance the expression of genes associated with meiosis process and embryo development, such as CENPJ (centromere protein J, P=0.039), AURKA (aurora kinase A, P=0.007), PDIA6 (protein disulfide isomerase family A member 6, P=0.007), and LINGO2 (leucine rich repeat and Ig domain containing 2, P=0.007).
Conclusion: GH could promote maturation of human oocytes by adding GH, which might work through accelerating the meiosis progress, balancing redox homeostasis of cellular environment, and promoting full development of oocytes.
Funding: This study was supported by the National Key Research and Development Program of China (2017YFC1001500, 2017YFC1001000), the National Natural Science Foundation of China (31871509, 81622021, 31571548, 31601199), China Postdoctoral Science Foundation (2016M600542) and the National Natural Science Foundation of Shandong Province (JQ201816, ZR2016HQ38).
PS142: Differential expression profile of plasma exosomal microRNAs in PCOS women
Xiao Jiang, Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University; Shigang Zhao; Bingqian Zhang; Linlin Cui
Background: Exosomes are small membrane-bound extracellular vesicles (30-100 nm) secreted by most cell types. Exosomal miRNAs have recently been identified as a novel mechanism of intercellular communication, as well as source of diagnostic and therapeutic biomarkers, due to its stability compared with free miRNAs. The current study aimed to detect the different expression profile of plasma exosomal microRNAs (miRNAs) between polycystic ovary syndrome (PCOS) patients and healthy controls, and to reveal their potential roles in PCOS insulin resistance.
Methods: Plasma exosomes were isolated from either PCOS women or normal controls by ExoQuick Precipitation methods. MiRNA sequencing was conducted among 15 PCOS women and 15 control women, and the expression profile of exosomal miRNAs was further determined by real time PCR in a larger sample size of 75 PCOS and 75 control women. Gene ontology (GO) and Kyoto Encyclopedia of Genomes pathway (KEGG) enrichment analyses were performed using http://www.genome.jp/kegg/ and ftp://ftp.ncbi.nih.gov/gene/DATA/gene2go.gz.
Results: The miRNA sequencing result showed that 35 exosomal miRNAs are significantly differentially expressed in plasma between PCOS women and healthy controls (P<0.05), and 18 new miRNA candidates were found that have not been recorded in miRBase library before. A total of 5 miRNAs were validated differentially expressed by qRT-PCR analysis, among which three were down-regulated in PCOS patients compared with controls while another two were up-regulated (P<0.05). GO and KEGG analysis revealed that the predicted target functions of these differentially expressed miRNAs mainly involved in insulin signaling pathway and MAPK pathway, indicating that plasma exosomal miRNAs may promote the occurrence of insulin resistance in PCOS women, via affecting the proliferation and differentiation of insulin target cells.
Conclusion: Plasma exosomal miRNA may contribute to the development of insulin resistance in PCOS patients. Further functional study was needed in the future.
Funding: The research was supported by National Natural Science Foundation of China (81501223).
PS143: Palmitic acid causes insulin resistance in granulosa cells via activation of JNK
Xu Lan, Center for Reproductive Medicine, Shandong University; Wenting Wang; Xinyue Zhang; Hanni Ke; Yingying Qin; Li You; Shidou Zhao
Background: Obesity is a worldwide health problem with rising incidence and results in reproductive difficulties. Elevated saturated free fatty acids (FFAs) in obesity can cause insulin resistance (IR) in peripheral tissues. The high intra-follicular saturated FFAs, especially palmitic acid (PA), may also account for IR in ovarian granulosa cells (GCs).
Methods: Human and mouse GCs were cultured and treated in vitro. The effects of PA on insulin-induced glucose uptake and lactate accumulation were measured. The activation of insulin signaling pathway and c-Jun NH2-terminal Kinase (JNK) pathway after PA treatment were detected by Western blotting.
Results: The glucose uptake in cultured GCs and lactate accumulation in the culture medium were stimulated by insulin, but the effects of insulin were attenuated by PA treatment. Besides, insulin-induced phosphorylation of Akt was reduced by PA in a dose and time-dependent manner. Furthermore, PA increased phosphorylation of JNK and JNK inhibitor rescued the phosphorylation of Akt which was down-regulated by PA.
Conclusion: PA can interfere with insulin-induced glucose metabolism in GCs and may partially account for the obesity-related reproductive disorders.
PS144: One case report of hyperandrogenism
Weina Du, Dalian Maternal and Child Health Care Hospital; Longqi Wu
Background: The androgens in female mainly include dehydroepiandrosterone sulfate (dhea-s), DHEA, androstenedione (AD), testosterone (T) and dihydrotestosterone (DHT). These androgens play an important role in the body of female and are the precursors of estrogen synthesis. They play a key role in puberty and early period of gonadal differentiation as well as growth of female. Also, they are critical for target organs (muscles, bones, etc.). However, excessive androgen in female body can cause menstrual disorder or amenorrhea, hairiness, androgen alopecia, acne, clitoral hypertrophy and other female masculine manifestations, which seriously affects the normal life and fertility for female. This essay will report a case of female hyperandrogenism caused by granulosa cell tumor of ovary.
Methods: Case report.
A Female. 30 years old. With 10 years of dysmenorrhea, 3 years of amenorrhea, and 2 years of body hair increase. With both clinical and biochemical hyperandrogenism performance. The height of the patient was 170cm, the weight was 82kg, and BMI=28.3kg/m2. She was muscular, with low voice. The development of secondary sex characteristic Tanner is B5P5. The clitoris was slightly longer. Ferriman-gallwey hair score: 16.Accessory examination: T 3.67-4.04ng/mL (<0.1-0.75ng/mL), dhea-s 229.6ng/dl (18-391ng/dL), AND >10ng/mL (0.3-3.3ng/mL); Chromosome: 46, XX. Three-dimensional color Doppler ultrasound: The size of the right ovary is about 55*50*47mm, with a few follicles around, and the rest is hypoechoic light mass, which is about 42*43*42mm in size. CDFI shows abundant blood flow signals in the ovary, RI=0.32.
Results: Postoperative diagnosis: right ovarian granulocytoma (adult), hyperandrogenemia, vaginal septum, endometrial polyps. The patient recovered well after surgery. The serum testosterone dropped to 0.204ng/mL on the fourth day after the first operation, and the postoperative clinical manifestations of hyperandrogen were significantly reduced. The follow-up testosterone level was normal. The patient is now in the treatment of ovulation induction in our center.
Conclusion: To sum up, women with hyperandrogenism need to pay attention to distinguish the source of ovarian source. The source of ovary is the most common one, but other sources must be excluded at the same time. Careful physical examination and auxiliary examination can help to identify the results. Chromosome and gene detection should be performed when necessary.
PS145: Novel circular RNA expression in cumulus cells of patients with polycystic ovary syndrome
Zhi Ma, Medical College of Qingdao University; Huishan Zhao; Yan Zhang; Xiaoyan Liu; Hao Cuifang
Background: Circular RNAs (circRNAs) mediate multiple posttranscriptional gene regulation by founctioning most importantly as microRNA (miRNA) sponges. Lots of works have revealed their significance in the field of oncology and neuroscience but rarely concerning about female endocrinological disorder. This study aims to detect the novel expression of circRNAs in cumulus cells (CCs) of PCOS patients and their potential significance in the pathogenesis of PCOS.
Methods: CircRNAs in the CCs from 6 PCOS patients and 6 normal control individuals were collected for microarray analysis, and an independent cohort study including 25 PCOS patients and 25 normal control individuals was conducted to validate the circRNA microarray results with qRT-PCR. Spearman’s rank correlation and reciever operating characteristic (ROC) were performed to delineate the potential correlation of novel circRNAs and patients’ clinical characteristics, and its efficacy for the diagnosis of PCOS. Also, we predicted their potential significance in the competing endogenous RNAs (ceRNAs) network through several bioinformatic tools.
Results: A total of 286 circRNAs (167 upregulated and 119 downregulated) were identified by microarray to be differentially expressed between PCOS and Non-PCOS group. After further qRT-PCR validation, expression levels of hsa_circ_0043533 (P<0.05) and hsa_circ_0043532 (P<0.01) were significantly higher, while hsa_circ_0097636 (P<0.01) was prominently lower in PCOS group. Spearman’s rank correlation indicated that serum testosterone (T) level positively correlated with the expression of hsa_circ_0043533 and hsa_circ_0097636 in PCOS group. The ROC curve analysis found that combination of hsa_circ_0097636 and T resulted in a larger area under the curve (AUC) (0.893) compared with each circRNA alone (0.709, 0.738 and 0.718 for hsa_circ_0043533, hsa_circ_0097636 and hsa_circ_0043532, respectively). Four targeting genes (DGKI, FOXO3, PCYT1B and CCNB1) in the dysregulated KEGG Pathways of PCOS are predicted to be potentially involved in the ceRNAs network.
Conclusion: CircRNAs expression in CCs are significantly different between PCOS and normal control individuals. We validated three of them and predicted their potential significance in the ceRNAs network, which may offer great promises in understanding the pathogenesis and developing effective therapeutic interventions for PCOS patients.
Funding: This work was funded by National Natural Foundation of China (Grant No. 81671416).
PS146: Effects of oral contraceptives pretreatment on IVF outcomes in women with GnRH agonist protocol
Xu Lan, Center for Reproductive Medicine, Shandong University; Daimin Wei; Yingying Qin
Background: Oral contraceptives (OCs) consisting of oestrogen and progestogen are widely used in IVF–ICSI cycle scheduling. It has been reported that OCs pretreatments prior to GnRH antagonist protocol lead to low rates of pregnancy and poor IVF outcomes. However, the effect of OCs on pregnancy outcome with GnRH agonist (GnRH-a) protocol is inconclusive.
Methods: In this retrospective study, a total of 2052 women undergoing first IVF treatment with long protocol and 3557 women with short protocol between 2012 and 2017 at Reproductive Hospital Affiliated to Shandong University were enrolled. According to OCs pretreatment or not, patients were divided into OCs group and control group. And the ovarian response, outcomes of fertilization and embryo culture, and pregnancy outcomes were compared between OCs group and control group within long protocol and short protocol, respectively.
Results: (1) On the day of hCG trigger, OCs pretreatment was associated with thinner endometrium (long protocol: 11.2±1.7mm vs. 11.4±1.8mm; short protocol: 10.5±1.9mm vs. 10.7±2.5mm) and lower LH level (long protocol: 2.2±1.0IU/L vs. 2.5±1.2IU/L; short protocol: 4.9±2.7IU/L vs. 5.4±2.4IU/L) compared with control group. (2) The numbers of oocyte retrieved, cancellation rate and Gn dose were comparable between OCs group and control group within long protocol and short protocol. (3) No significant differences in clinical pregnancy rate, early pregnancy loss were observed between OCs group and control group within long protocol and short protocol. (4) The rates of live birth did not differ significantly between the OCs group and the control group, both in women using long protocol (59.8% vs. 59.1%, P>0.05) and short protocol (44.6% vs. 42.4%, P>0.05).
Conclusion: Pretreatment with OCs did not affect the pregnancy outcome of IVF-ET cycle, both in GnRH-a long protocol and short protocol, except for low LH level and thin endometrium on the day of hCG trigger.
PS147: The correlation between age and sperm DNA integrity, semen parameters and the impact of sperm DNA fragmentation index on outcomes of IVF/ICSI
Xiangli Pang, Reproductive Medicine Center, Renmin Hospital of Wuhan University; Jing Yang
Background: Objective: To explore the correlation between age and sperm DFI, semen parameters and the effect of DFI on the clinical outcomes of IVF/ICSI.
Methods: Study Design: we analyzed 1218 infertile males, the DFI was detected, and semen routine examination was implemented in accordance with the WHO fifth edition standard. The relationship between age and DFI, normal sperm, morphology rate, sperm motility was analyzed. 1388 couples treated by ART were classified as three groups based on the DFI, each group was divided into two subgroups on the basis of means of fertilization, IVF or ICSI. Then analyzed the Fertilization rate, the rate of high-quality embryo, and clinical pregnancy rate.
Results: We found that DFI was positively correlated with age. The forward motility sperm (PR%) decreased significantly with the increase of DFI. when DFI> 30%, the fertilization rate and the rate of high-quality embryo in the IVF subgroup were significantly lower than those in the other two groups. In IVF subgroup, the rate of clinical pregnancy was lower than that in ICSI subgroup.
Conclusion: Sperm DFI and forward motility sperm will be affected by age; when the DFI> 30%, fertilization rate, high-quality embryo rate will be adversely affected in IVF.
Funding: This project was supported by National Natural Science Foundation of China (No. 81401255, No.81771618, No.81571513).
PS148: Clinical evaluation for the decline of serum estradiol in PCOS patients treated with GnRH antagonist protocol during controlled ovarian hyperstimulation
Jie Li, Reproductive Medical Center, Renmin Hospital of Wuhan University, China; Jing YangDan Cheng; Xiangli Pang; Xueyao Li
Background: In the process of controlled ovarian hyperstimulation (COH), serum estradiol (E2) is widely accepted as a vital indicator for the assessment of the follicular growth and development. The application of GnRH antagonist protocol in patients with PCOS contributed to the effective prevention of premature luteinizing hormone (LH) surge and the occurrence of ovarian hyperstimulation syndrome. However, the decline of serum E2 has been detected in many patients during the COH at times 5. In the present study, the coping measures were reported, in which the dosages of Gonadotropin-releasing hormone antagonists (GnRH-ant) and Human menopausal gonadotropin (HMG) were adjusted. It aimed to ameliorate the impact of the declining serum E2 on IVF-ET outcomes among PCOS patients with GnRH antagonist protocol.
Methods: 120 PCOS patients including 58 cases with the decreased level of E2 (study group) and 62 cases with continuously increased of E2 (control group) were studied retrospectively in this research. The study group was also divided into 2 subgroups: the spontaneous decreased E2 group (n=30) and the group with Gn alterations (n=28). The clinical data and outcomes of IVF-ET were analyzed.
Results: The three groups exhibited no significant differences in female age, infertility duration, basal sex hormone levels, the fertilization rate, 2PN cleavage rate, and the rate of high-quality embryo. The dosage of gonadotropin (Gn) in the decreased E2 group with Gn alterations was significantly higher than that in the spontaneous decreased E2 group and control group. Compared with that in Gn alterations group and the control group, the spontaneous group experienced a significant decrease in the clinical pregnancy rate.
Conclusion: Spontaneous decline of serum E2 indicated the undesirable IVF outcomes in the PCOS patients treated with GnRH antagonist protocol during controlled ovarian hyperstimulation.
Funding: This project was supported by National Natural Science Foundation of China (grant number 81401255, 81771618, 81571513). Hubei Province Technology Innovation Special Project (No. 2017ACA101), Hubei Province Natural Science Project (2016CKB703) National Key Research and Development Program of China (No. 2016YFC1000600 2018YFC1002804).
PS149: The association between TSH level and pregnancy outcomes in euthyroid women undergoing IVF/ICSI: a retrospective study and meta-analysis
Bo Zhang, Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology; Meng Wang; Lei Jin
Background: Thyroid gland is very important for female fertility. Thyroid dysfunction may cause ovulatory disorders, menstrual irregularities, infertility and even increased pregnancy morbidity. The change of TSH level have been indicated to result in sub-optimal environment for the implantation and develop of embryo. Therefore, the level of TSH is very important for the women, especially the euthyroid women undergoing ART. Though, women undergoing with thyroid disorders must receive therapy before COH, which have been confirmed all over the world. However, there is controversy regarding the upper limit of normal TSH in human reproduction societies. Some study suggest a TSH level of 2.5 mIU/L as the upper limit of normal, however, this suggestion is not fully accepted. Hence, the cut-off of TSH concentrations remains controversy. Unfortunately, though some study have revealed the effect of TSH for euthyroid women undergoing ART, there is no data about Chinese women undergoing ART, which have largest population in the world. In addition, few meta-analysis have performed to confirm the effect further.
Methods: A total of 1185 women were included in the retrospective study and twelve studies with a total of 6624 women were included in the meta-analysis (including our data). According to the level of serum TSH, the women were divided into two sub-groups, the TSH≤2.5mIU/L group and the TSH>2.5mIU/L group. All the couples were follow up to determine clinical pregnancy and miscarriage.
Results: In total, 1185 women with euthyroid undergoing IVF/ICSI were recruited in our study. In the serum TSH≤2.5mIU/L group, 441 (53.1%) had got clinical pregnancy, 48 (5.8%) had early pregnancy loss and 12 (1.4%) had ectopic pregnancy. In the serum TSH>2.5mIU/L group, 175 (49.3%) had got clinical pregnancy, 21 (5.9%) had early pregnancy loss and 3 (0.8%) had ectopic pregnancy. No significant difference were revealed between these two group (P=0.126, P=0.512, P=0.297). These findings were consistent with the meta-analysis results.
Conclusion: Our findings suggested the normal high level of TSH concentrations (TSH level>2.5mIU/L) did not decrease clinical pregnancy rate or improved miscarriage rate in euthyroid women undergoing IVF/ICSI. These findings reinforce through our meta-analysis.
PS150: Elevated incidence of monozygotic twinning is associated with extended embryo culture, but not with zona pellucida manipulation or freeze-thaw procedure
Hanyan Liu, Third Affiliated Hospital of Guangzhou Medical University; Jian Qiao; Shiping Chen; Xiangjing Kang; Hongzi Du; Li Lei
Background: To identify the incidence and risk factors associated with IVF-conceived monozygotic twinning (MZT).
Methods: All nondonor, fresh and frozen IVF/ICSI cycles from January 2014 to February 2015 at a single reproductive center were retrospectively analyzed. The measures were the incidence of MZT based on the number of embryos that were replaced, type of insemination method (conventional IVF or intracytoplasmic sperm injection [ICSI]), with or without the use of assisted hatching (AH), and day of embryo transferred in fresh and frozen cycles.
Results: Ninety-three women (2.69%) with MZT were observed. No statistically significant differences were observed in the cycle parameters of fresh or frozen cycles between MZT and other non-MZT pregnancies. Specific IVF procedures or techniques, such as the number of embryo replaced, zona pellucida manipulation (ICSI and AH), and freeze-thaw procedure, did not significantly increase the rate of MZT, except for the day of embryo transferred. Compared with day 3 transferred, day 4 and 5/6 transferred showed an increased probability of MZT (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.16–6.42 for day 4 transferred and OR, 3.68; 95% CI, 2.29–5.93 for day 5/6 transferred).
Conclusion: Extended culture (advanced embryo stage) in fresh and frozen cycles appeared to be associated with increased rates of MZT. The effect of the number of embryos transferred, ICSI and AH, and freeze-thaw procedures on the risk for MZT was not demonstrated.
Funding: Supported by the National Nature Science Foundation of China, grant no. 81502507; and the Scientific and Technological Program of Guangzhou, grant no. 201607010200.
Citation: The article was published in fertility and sterility in June 2018.
PS151: Effect of early cumulus cells removal and early rescue ICSI on pregnancy outcomes in high-risk patients of fertilization failure (Gynecological endocrinology 34(8): 689-693)
Yuxia He, Third Affiliated Hospital of Guangzhou Medical University; Haiying Liu; Haiyan Zheng; Xin Fu; Jianqiao Liu
Background: The objective of this retrospective study was to evaluate clinical outcomes of early cumulus cells removal and early rescue intracytoplasmic sperm injection (ICSI) in high-risk patients of fertilization failure during human in vitro fertilization (IVF).
Methods: A total of 5,518 patients were enrolled between January 2014 to December 2016. Of which 505 couples performed short insemination with >30% fertilization rate were included in short-term IVF group, 102 patients detected total fertilization failure (TFF) were treated with early rescue ICSI (R-ICSI group), and 4911 couples underwent conventional IVF with overnight co-incubation of gametes (traditional IVF group).
The clinical outcomes were analyzed among the three groups.
Results: The embryo implantation rates (40.34%, 39.78% and 42.42% for traditional, short-term IVF and R-ICSI groups, respectively) were comparable in the three groups. The clinical pregnancy rates among traditional IVF group, short-term IVF group and R-ICSI group were 57.95%, 57.03% and 60.78%, respectively, and the difference among three groups didn’t reach significance.
Conclusion: The present study indicated that short insemination had no detrimental effects on clinical outcomes in human IVF and could prevent the occurrence of TFF combined with early rescue ICSI for high-risk patients of fertilization failure, which attained acceptable pregnancy outcomes.
Funding: This study was supported by the National Natural Science Foundation of China (NSFC, 81501327) and Scientific Research Program of The Third Affiliated Hospital of Guangzhou Medical University (2017B04).
PS153: Clinical outcomes of different quantity and quality of frozen blastocyst transfer in patients requiring whole embryo freezing stratified by age
Yuxia He, Third Affiliated Hospital of Guangzhou Medical University; Haiyan Zheng; Haiying Liu; Li Li; Jianqiao Liu
Background: To evaluate the pregnancy outcomes of different quantity and quality of blastocysts stratified by age, using a cutoff of 35 years, in patients requiring whole embryo freezing and underwent a following frozen-thawed transfer cycle.
Methods: A total of 1,477 patients were divided into five groups: group A (n=416), single good-quality blastocyst; group B (n=728), two good-quality blastocysts; group C (n=190), one good and one average-quality blastocysts; group D (n=89), two average-quality blastocysts; group E (n=54), one average-quality blastocyst.
Results: For women less than 35 years of age, the clinical pregnancy rate (PR) was slightly, but significant lower in group A compared to group B (70.26% vs. 77.20%), and no statistical difference between group A and group C or D. However, the multiple pregnancy rate (MPR) was significant lower in group A compared to the groups B-D. In women 35 years of age and over, there was no significant difference in the clinical PR among groups A-D (P=0.087), and the MPR was significant lower in group A compared with group B or C. Moreover, the PR was comparable between group D and E when people in the same age category, and no multiple pregnancy happened in Group E.
Conclusion: When good-quality blastocysts are available, single blastocyst transfer (SBT) is a preferable option for patients irrespective of age, because it significantly reduces the risk of multiple pregnancy without compromising PR. For patients who only have average quality blastocyst, no advantage was found in women undergoing double blastocyst transfer (DBT) compared with SBT.
Funding: This study was supported by the Science and Technology Planning Project of Guangdong Province (2014A020213017) and Scientific Research Program of The Third Affiliated Hospital of Guangzhou Medical University (2017B04).
PS154: Lymphoma is the most malignant common blood tumor in the world and its incidence is on the rise. This paper reports a case of successful pregnancy in 30-year-old women with amenorrhea for seven months after chemotherapy for lymphoma
Sushi Jiang, Department of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital; Xiaozhu Zhong; Yang Ye; Min Tan; Weicheng Yao; Xiaomiao Zhao
Background: This paper mainly reports a case of successful pregnancy in 30-year-old women with amenorrhea for seven months after chemotherapy for diffuse large B-cell lymphoma.
Methods: After chemotherapy, this woman with amenorrhea, thin internal membrane, but there are dominant follicles, so treated with the artificial cycle of internal membrane and related adjuvant treatment.
Results: This woman had natural pregnancy and childbirth.
Conclusion: The case of the diagnosis and treatment of the patients with colleagues to share, in order to strengthen the attention and management of the fertility of this type of patient.
PS155: Effect of surgery and conservative treatment on IVF-ET pregnancy outcome in patients with caesarean scar diverticula and endometrial cavity fluid
Weifen Deng, Reprodutive Center, Dongguan Guangji Hospital, Guangdong, China; Feng Wang; Huidong Long
Background: Patients with caesarean scar diverticula often complicated with endometrial cavity fluid, which would reduce the IVF-ET (in vitro fertilization-embryo transfer) pregnancy rate and increase the miscarriage rate. This study aimed to evaluate a better treatment for IVF-ET patients with caesarean scar diverticula and endometrial cavity fluid.
Methods: 152 patients with caesarean scar diverticula complicated with endometrial cavity fluid underwent IVF-ET in the reproductive center of Shenzhen Armed Police Hospital from January 2015 to December 2017 were included. 62 cases underwent transvaginal repair of caesarean scar diverticula with 88 frozen embryo transfer cycles as group A, the patients in group A were divided into two subgroups according to existence of uterine cavity effusion after surgery. 90 cases treated conservatively with 178 frozen embryo transfer cycles as group B, conservative treatment including traditional Chinese medicine, acupuncture, intrauterine perfusion of ozone and physical therapy. The general condition and clinical outcome of the two groups were compared.
Results: There was no significant difference between the two groups in general condition, number of embryos transferred, good quality embryo rate and thickness of endometrium on the day of transfer (P>0.05). The average age of the two groups was (34.74+3.91) vs. (35.26+4.59) years, of no statistically difference. The clinical pregnancy rates of group A and group B were 28.4% and 48.9% respectively, the difference was statistically significant (P<0.05). The miscarriage rate was 29.9% in group A, while 12% in group B, there was no significant difference between the two groups (P>0.05). 56 cases in subgroup A1 had intrauterine effusion after surgery and 19 cases in subgroup A2 normal. The clinical pregnancy rate and miscarriage rate of the two subgroups were 17.85% vs. 73.68% and 40% vs. 0%, respectively, there was significant difference between the two subgroups (P<0.05).
Conclusion: Caesarean scar diverticula causes endometrial cavity fluid, which would decrease pregnancy rate and increase miscarriage rate in IVF patients. Compared with transvaginal repair, conservative treatment such as traditional Chinese medicine, acupuncture and other drugs can better clear uterine effusion, lighten the burden of patients, and improve the clinical outcome in IVF-ET, which is worthy of clinical application.
PS156: Nomogram to predict pregnancy rate after laparoscopic surgery in women with minimal to mild endometriosis
Bin Luo, West China Second University Hospital; Guojun Zhou; Wei Huang
Background: Present study had assessed the predictive valueof endometriosis fertility index (EFI) for either in vitro fertilization and embryo transfer (IVF-ET) or spontaneous pregnancy rates (PRs) after surgery. Prediction models provide information that can aid evidence-based management of unexplained subfertile couples. But no study had developed a model and nomogram to predict the spontaneous pregnancy rate in infertility women with minimal to mild endometriosis.
Methods: The retrospective study including 270 infertility women who were postoperative diagnosed of stage 1 and 2 endometriosis between January 2016, and July 2016. A multivariate logistic regression analysis of selected factors and a nomogram to predict the subsequent pregnancy rate was constructed.
Results: A nomogram built according to the woman’s age, AMH level, history of infertility, history of pregnancy, r-AFS scores, intraoperative findings and the use of Gn-RHa after surgery can accurately predict the probability of natural pragnancy in infertility women with minimal to mild endometriosis. 156 women had spontaneous pregnancy (53.4%) after laparoscopic surgery. Age, AMH, history of infertility, history of pregnancy, R-AFS-Score, endometrial polyps, ovarian reduction, and Gn-RHa use after surgery were included in the nomogram. The sensitivity of our model had reached 72.4%.
Conclusion: According to the development of prediction model, our results suggest that our nomogram predicting the pregnancy rate after laparoscopic surgery for women with endometriosis could be a useful tool in helping clinicians and patients decide on a treatment option.
PS157: Effects of preventing premature luteinizing hormone surges and clinical outcomes between cetrorelix and ganirelix in gonadotropin-releasing hormone antagonist cycles
Jun Zhang, Center for Reproductive Medical Center, Dept. Ob/Gyn, Nanfang Hospital, Southern University; Shiling Chen
Background: Gonadotropin-releasing hormone antagonists(GnRH-ant) are currently used to suppress endogenous LH secretion during ovarian stimulation. Our intention was to investigate the effect of cetrorelix and ganirelix in preventing premature luteinizing hormone (LH) surges and clinical outcomes in GnRH-ant cycles.
Methods: We retrospectively analyzed 2566 GnRH-ant cycles of in vitro fertilization and embryo transfer from January, 2013 through December, 2016, including 1365 cycles in the cetrorelix group and 1201 cycles in the ganirelix group. And the baseline characteristics of the patients and clinical outcomes of the two groups were compared.
Results: The age (33.08 vs. 33.88 years, P<0.001) and body mass index (BMI) (21.59 vs. 21.83kg/m2, P=0.049) of the patients were slightly lower in the cetrorelix group than in the ganirelix group. After adjusting the age of the patients, there were no significant differences in BMI (21.59 vs. 21.83kg/m2, P=0.270), follicle-stimulating hormone (19.97 vs. 19.80mIU/mL, P=0.431), LH (3.37 vs. 3.48mIU/mL, P=0.932), estradiol (2489.09 vs. 2395.14pg/mL, P=0.858), progesterone (1.00 vs. 0.98ng/mL, P=0.551) levels on the day of hCG triggering and the number of oocytes retrieved (11.04 vs. 10.63, P=0.879) between the two groups. Also, clinical pregnancy rate (48.1% vs. 45.9%, P=0.821) of fresh embryo transfer between the two groups was comparable.
Conclusion: Cetrorelix and ganirelix can achieve similar effects of preventing premature LH surges and similar clinical outcomes in GnRH-ant cycles.
PS158 – (Chinese language abstract – see Chinese supplement)
Wang Wei, Center for Reproductive Medicine, The Second Clinical Medical College, Yangtze University, Jingzhou, China; Hong Liu; Yuhua Shi
PS159 – (Chinese language abstract - see Chinese supplement)
Wang Wei, Center for Reproductive Medicine, The Second Clinical Medical College, Yangtze University, Jingzhou, China; Yuhua Shi; Yao Chen
PS160: Development and validation of a prediction model for predicting cumulative live birth rate after IVF-ET cycle
Meng Zhang, Xinjiang Medical University Affiliated First Hospital; Haiqing Tian; Xiaolin La
Background: The aim of this study was to develop a nomogram based on an association of patients’ characteristics to predict the cumulative live birth rate in Xinjiang area, with intention to provide more information for doctors to comprehensively evaluate the condition of patients.
Methods: A retrospective study was conducted on a cohort of patients between January 2014 to December 2014, 1141 women who had firstly underwent IVF treatment at the First Affiliated Hospital of Xinjiang Medical University. Logistic regression analysis was used for univariate and multivariate analyses. Nomogram was formulated based on the results of multivariate analysis by applying the package of rms in R version 3.3.1. The concordance index (C index) was used to evaluate the performance of the nomogram.
Results: The model was built from a retrospective cohort of 1159 patients (1141 first IVF-ET cycles) from January 2014 to December 2014, a minority (18 patients) still had cryopreserved embryos and no live birth at the end of the trial. There were 556 women had live birth after their first cycle, the cumulative live birth rate was 48.7%.
The female age, male age, antral follicle count (AFC), basal FSH, basal T and tubal factor had statistical significance between the live birth group and the non-live birth group. The Gn dose, E2 level on HCG day, number of 2PN and retrieved oocytes were statistically significant in these two groups, and the live birth group had a higher number of 2PN and retrieved oocytes.
A final model selection was performed by a stepwise selection process, clinically confirmed factors were using enter selection process in final model, female age (OR=1.087, 95% CI: 1.054 to 1.121), AFC (OR=0.902, 95% CI: 0.856 to 0.949) and tubal factor (OR=1.395, 95% CI: 1.087 to 1.806) were statistically significant associated with live birth. The final model was then organized as a nomogram designed to calculate patient-specific probabilities of cumulative live birth, the C-index (0.6584, 95% CI: 0.6564 to 0.6604) indicates that the nomogram had good performance.
Conclusion: It is possible to predict the individual probability of cumulative live birth before underwent IVF-ET treatment using a nomogram. Applying this nomogram in the clinic may be a step forward in optimizing the decision-making process in the treatment of infertility. Our results suggest that our nomogram could be a useful tool in helping clinicians and infertile women to decide a treatment option.
Funding: Funding ID:2018d01C205.
PS161: Influence of different ovulation induction protocols on AID pregnancy outcome of ovulatory women
Tang Xianan, Tongji Hospital; Yue Jing
Background: AID refers to an assisted reproductive process that donor’s sperms are well placed into female’s reproductive duct artificially for successful pregnancy, and mainly benefits males diagnosed azoospermia or genetic diseases. It can also be a good option for severe blood-type-incompatible couples failure to repeated treatment. It’s generally accepted that ovulation induction plus AID should be displayed in dis-ovulatory women. But for women with normal ovulation, whether AID should be operated plus ovulation stimulation or not remains an open question.
Methods: Clinical information of partients with normal ovulation visiting our centre for AID treatment were analysed retrospectively from 2013 January to 2016 December. According to the protocols, participates were divided into natural cycle (NC) group and controlled ovulation stimulation (COS) group, the latter were further divided into LE group and LE+HMG group according to the medicine. Basic information, cycle information and pregnancy related outcome were compared among these three groups.
Results: Women in NC group possessed more advanced age than COS group (28.34±4.21 vs.27.41±4.04, P<0.05). After a ratio of I:2 matching based on elementary and cycle information, COS group displayed more dominant follicles (1.09±0.29 vs. 1.47±0.66, P<0.001), clinical pregnancy rate (22.55% vs. 26.47%) and live birth rate (15.69% vs. 21.57%) of both groups were shown no significant difference, decreased multiple pregnancy rate (4.35% vs. 9.26%), ectopic pregnancy rate (0 vs. 3.7%) and increased miscarriage rate (30.43% vs. 14.81%) were observed in NC group, though with no statistical significance. We further discussed effects of distinct COS protocols, elevated BMI were discovered in patients of LE+HMG group. After matching, LE group performed uprising clinical pregnancy rate (34.31% vs. 18.63%, P<0.05) and live birth rate (27.45% vs. 15.69%,P<0.05), but no significant difference of multiple pregnancy rate (11.43% vs. 5.26%), ectopic pregnancy rate (5.71% vs. 0) and miscarriage rate (14.29% vs. 15.79%) were indicated between both groups.
Conclusion: NC protocol is recommended for ovulatory women seeking for AID treatment due to its comparative clinical pregnancy rate and live birth rate as well as less occurrence of multiple pregnancy and ectopic pregnancy to some extent. However, it might be difficult for us to draw a conclusion which COS protocol is better due to our small-size study.
PS162: Qualitative study of psychological experience of women who receive AIH
Hu Qinbo, No.1 Hospital; Xu Jingjing
Background: The aim was to provide a better holistic care for the women who received artificial insemination by husband (AIH) by understanding their real psychological feelings.
Methods: Purposive sampling method was used, and in-depth interviews were carried out for 16 cases of women who received AIH. The obtained data was analyzed and combined with social reality to extract the theme.
Results: Four main themes were obtained: the intense stress caused by the treatment of AIH; the desire to a successful AIH; the reduction in social support and self-efficacy; the disharmony of their marriages and their sexual life.
Conclusion: The psychological needs for women who received AIH were more, so they should be provided with more emotional and medical support, to improve the quality of life. Also propaganda and popularization of reproductive knowledge should be paid highly attention to, and social support should be appealed.
PS163: Putrescine improves oocyte quality of aging mice and its mechanism
Mengting Hu, Nanjing Medical University; Lingjun Li; Lingbo Cai; Jiayin Liu; Yugui Cui
Background: The oxidative stress in aging oocyte is significantly increased, while the developmental potential of embryo derived from aging oocyte is decreased. Putrescine improves the cell viability by down-regulating the level of intracellular reactive oxygen species (ROS). This study was designed to explore the positive effects of putrescine on oocyte quality by the anti-oxidation mechanism in aging oocyte.
Methods: The GV oocytes obtained from 9-month-old C56/B6J9 female mice were in vitro cultured in the IVM medium (as control group). The GV oocytes were cultured in the IVM medium with 0.5 mmol/L putrescine as the putrescine group, while treated with 0.5 mmol/α-difluoromethylornithine (DFMO), an inhibitor of putrescine production, as the DFMO group. After cultured for 16-18 hours, MII oocytes were collected. The maturity rate was calculated, and the levels of mitochondrial DNA (mtDNA) copy numbers was detected by RT-PCR. The mitochondrial membrane potential was tested by JC-1 fluorescence staining.
Results: The maturity rate of oocytes in the putrescine group was significantly higher than that in the control group (22%, P<0.001). The mtDNA copy numbers of putrescine group was lower than control group (31.1%, P<0.05). Compared with the control group, the putrescine group had the decreased ROS (37%, P<0.01), and the DFMO group had the increased ROS (179%, P<0.01). The mitochondrial membrane potential in the putrescine group was significantly increased by 253%, and that in the DFMO group decreased by 46.7% when compared with the control group (both P<0.01).
Conclusion: In the in vitro cultured aging oocytes, putrescine improves the oocyte quality and developmental potential of embryo derived from aging oocytes by increasing antioxidation effect, decreasing oxidative stress and promoting mitochondrial function.
PS164: Excess androgen inhibiting lactate production in granulosa cells
Qing Zhu, Lianyungang Maternal and Child Health Hospital; Jia yin Liu; Yugui Cui; Jiayin Liu
Background: Excess androgen can cause hyperinsulinemia and insulin resistance in many tissues, which results in the disorder of energy metabolism. There are the co-existence of hyperandrogenism and insulin resistance, and retardant follicle development.
Methods: The KGN cell, a granulosa cell line derived from human ovarian cancer, was cultured in vitro and treated with different concentration of testosterone (10-10-10-6 mol/L) for 12 to 48 h. The lactate level in the culture supernatant was measured by ELISA. The levels of LDHA and LDHB mRNAs were quantified by real-time PCR. The protein expression of LDHB was detected by Western blotting.
Results: The lactate level was significant decreased by 10-6,10-7, 10-8, 10-9 mol/L testosterone treatment for 24 h, and decreased by 10-8, 10-9mol/L testosterone treatment for 36 h and 48 h (P<0.05). The expression level of LDHB mRNA was decreased by 10-7 mol/L testosterone treatment for 24 h (P<0.05), while the LDHA was decreased by 10-9 mol/L testosterone treatment for 24 h (P<0.05). The protein expression of LDHB was significantly inhibited by 10-7 and 10-9mol/L testosterone treatment for 24 h (P<0.05).
Conclusion: Excess androgen can suppress the lactate production in human granulosa cell by inhibiting LDHB, suggested a disorder of energy metabolism induced by excess androgen in PCOS ovary.
PS165: SET protein inhibited the acetylation of histone H3 and H4 in spermatid
Bei Zhang, The Tenth People’s Hospital of Shanghai; Qian Zhu; Weiwei Ma; Jiayin Liu; Yugui Cui
Background: Spermiogenesis refers to the haploidic round spermatids undergo dramatic morphological changes, including cytoplasmic dropping and the form of compact nuclear chromatin, and transform into spermatozoa. During which, hyperacetylated nuclear histones are replaced by more basic transition proteins, and ultimately protamines to facilitate denser packaging of the paternal genome into the sperm head. SET protein is called the inhibitor of acetyltransferase (INHAT) and is the component of the INHAT complex, which can inhibit histone acetylation. Our previous study showed that SET protein is mainly expressed in spermatogonia and spermatocytes, with low expression in spermatid. This study was designed to explore the effect of SET protein in regulating histone acetylation during spermiogenesis.
Methods: The GC-2 spd cell was cultured in vitro, and transfected with AdH1-siRNA/SET to knockdown the expression of SET protein. Western blot and Real time RT-PCR was used to detect the expression levels of SET protein, Ac-histone, histone acetyltransferases and histone deacetylases, HAT Activity Assay Kit and HDAC Activity Assay Kit to assess the HAT and HDAC activity. The combination of SET and H4 or SET and H3 were detected by an immunoprecipitation kit from the protein extracted from in vitro GC-2 spd cells.
Results: In GC-2 spd treated with AdH1-siRNA/SET, knockdown of SET protein significantly decreased the cell number, inhibited the cell proliferation (P <0.05). The acetylation level of H3 and H4 was significantly increased (P<0.05) by 2~3 fold. However, there were no significantly differences in the mRNA and protein expressions of HATs and HDACs, the same with HAT activity/HDAC activity. SET protein combined with H3 and H4 in the protein extracted from in vitro GC-2 spd cells.
Conclusion: SET protein regulats spermatogenesis by promoting spermatocytes proliferation. SET protein inhibited the acetylation of histone H3 and H4 by combining and masking the lysine acetylation sites in spermatid, thus regulating the spermiogenesis.
PS166: Promotion of SET protein on spermatogonia proliferation
Qian Zhu, The Affiliated Jiangning Hospital of Nanjing Medical University; Bei Zhang; Weiwei Ma; Siliang Xu; Jiayin Liu; Yugui Cui
Background: SET protein was previously found in the spermatogonia of the seminiferous epithelium. We hypothesized that SET protein expressed in the seminiferous epithelium could improve the cell viability and proliferation of spermatogonia. This study was designed to explore the regulatory effects of SET protein on the cell viability and proliferation of spermatogonia.
Methods: The in vitro cultured GC-1 spg cell, a spermatogonial cell line, was transfected with AdH1-siRNA/SET adenoviruse to knock down the expression of SET protein. The GC-1 spg cell was verified the positive expression of OCT4 and VASA by immunofluorescence.
Results: SET protein expression was located in both the cytoplasm and nucleus of GC-1 spg cell. The cell viability and proliferation were inhibited significantly when SET protein was knocked down. The percentage of cells in G2/M phase also increased significantly. The level of phosphated serine/threonine protein kinase (p-AKT) (Ser473) decreased. The apoptosis index was significantly increased, while the expression of cleaved caspase 3, cleaved caspase 9 and cleaved caspase 8 increased and the Bcl2/Bax ratio decreased.
Conclusion: SET protein promotes the cell proliferation of spermatogonia by the AKT-related pathway in an in vitro cell model, suggesting that in the seminiferous epithelium SET protein could be a potential regulator of male fertility.
PS167: Putrescine delays postovulatory aging of mouse oocytes by upregulating PDK4 expression and improving mitochondrial activity
Wendan Xu, Nanjing Medical University; Lingjun Li; Jingwen Sun; Songyue Zhu; Zhengjie Yan; Li Gao; Chao Gao; Yugui Cui; Caiping Mao
Background: Putrescine, one of the abundant biogenic amines, improves the oocyte quality of aged mice during IVM, suggesting that those aging postovulatory oocytes could be reactivated by some small molecules. The mitochondrial pyruvate dehydrogenase kinase-4 (PDK4) is one of key factors in the regulation of energy homeostasis and is increased during oocyte meiotic maturation. The study was designed to explore the effect of putrescine on postovulatory aging and the potential mechanism associated with PDK4.
Methods: The denuded MII oocytes were layed aside for 0 and 24 h postovulation in the medium-M2 with or without 0.5 mM putrescine. The morphological changes were explored by the optical microscope. The expressions of Sirt1, FOXO3a, SOD2, LC3 I/II, p-Mtor, p-AKT, P-ERK1/2, Bcl2, and PDK4 were analyzed by Western blot (WB). SiRNA-PDK4 was microinjected into oocytes in the culture medium with or without putrescine. Reactive oxygen species (ROS) production was evaluated by the carboxy-H2DCF diacetate labelling. Mitochondrial density was detected by 200 nM MitoTracker Red. The distribution of spindle and chromosome was analyzed by immunocytochemistry and DNA fluorescent labelling, respectively. The apoptosis were measured by TUNEL, and autophagy by immunocytochemistry. The mitochondrial membrane potential (MMP) was observed using JC-1 staining.
Results: The rate of oocyte morphological defects was significantly decreased in the putrescine treatment group (P<0.05), while level of ROS accumulation also decreasd (P<0.05). Analysis of MII oocytes after 24 h of aging showed that the proportion of abnormal mitochondrial distribution were significantly reduced in the putrescine treatment group (P<0.05). The percentages of TUNEL-positive oocyes and autophagy were significantly lowered (P<0.05 ). The increased MMP in the putrescine treatment group showed the mitochondrial activation. Meanwhile, down-regulation of SOD2, Sirt1, and PDK4 in the aged oocytes was prevented by putrescine. After PDK4 was downregulated by SiRNA-PDK4, ROS production and the apoptosis rate were significantly increased while the mitochondrial function was decreased in the postovulatory aged oocytes with the putrescine treatment.
Conclusion: Putrescine, as a potential additive reagent of IVM medium, can prevent the deterioration of the postovulatory oocytes following prolonged culture by regulating PDK4 expression and mitocondrial activity.
PS168: The influence of growth hormone supplementation in patients with thin endometrium undergoing frozen-thawed embryo transfer
Junyi Yang, Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University; Lu Li; Junyi Yang; He Li; Nan Lu; Xiaoxi Sun
Background: Patients with thin endometrium receive various adjuvant therapies in order to maximize its growth and guarantee efficient contraception, but the true benefit is actively debated. Growth hormone (GH) is a peptide hormone secreted by the anterior pituitary gland. Apart from promoting the metabolism of our body, it also regulates the reproductive process. However, the review of published data does not identify its effect on endometrial receptivity. This study applied GH to the endometrial preparation of frozen-thawed embryo transfer (FET) in thin endometrial patients to investigate its influence on endometrial receptivity.
Methods: A retrospective research was conducted among 225 patients who underwent artificial cycle FET in Shanghai, China, between January 2016 and November 2017. Data was analyzed from 245 FET cycles, where 184 cycles received GH (GH group) and 61 did not (control group).
Results: 119 of 184 GH cycles achieved clinical pregnancy (64.7%), which was quite higher than that in control group (30 of 61, 49.2%, P=0.032). In addition, the implantation rate was significantly greater in GH group than in control group (44.8% vs. 32.8%, P=0.019). After logistic regression analysis, we found the administration of GH was the only significant variable that influenced clinical pregnancy rate, increasing by 1.89-fold (95%CI 1.05 to 3.40, P<0.05). On the other hand, the presence of GH did not seem to affect the early pregnancy loss (14.3% vs. 10.0%, P=0.23>0.05).
Conclusion: These data provided substantial evidence to indicate that simultaneous addition of GH could improve clinical outcomes after FET, particularly in those patients older than 30 but younger than 35 years. Since we exclude the impact of embryo quality and maternal age, it appears that the biological mechanism may be related to endometrial receptivity, which means GH might improve the endometrial receptivity through blood flow or molecular level without increasing its thickness.
PS169: A randomized triple blind controlled trial comparing the live birth rate of IVF following brief incubation versus standard incubation of gametes
Zhiqin Chen; Yu Wang; Ernest Ng; Mei Zhao; Jia Ping Pan; Haixia Wu; Xiaoming Teng
Background: Some small randomized studies showed that brief incubation was associated with a significantly higher ongoing pregnancy rate than standard incubation.
Methods: This is a randomized triple blind study of 320 infertile women for their first or repeated cycles undergoing IVF between September 2015 and October 2016. Women were randomized into the brief incubation group (n=160) or the standard incubation group (n=160) according to a computer-generated randomization list. Oocytes were incubated with spermatozoa (0.3–1.2 million motile sperm/mL) for 3–4 h in the brief incubation group while oocytes were incubated with spermatozoa at similar concentration for 20h in the standard incubation group. The primary outcome was the LBR (a baby born alive after 22 weeks gestation) in the fresh cycle.
Results: There was no significant difference in the LBR between the brief and standard incubation groups based on both intention-to-treat [33.0% (53/160) versus 36.8% (59/160), relative risk (RR) 0.898 (95% CI=0.666–1.212), P=0.482] and per protocol [41.4%(53/128) versus 41.0% (59/144), RR1.011 (95% CI=0.760–1.343), P=0.942] analyses. Clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy and implantation rates were comparable for the two groups. Similar results were found with subgroup analysis of advanced maternal age, abnormal semen analysis and repeated IVF cycles. No differences were observed in cumulative LBR between two groups.
Conclusion: The present study demonstrated that a brief incubation of gametes had no significant beneficial effect on the LBR when compared with the standard incubation. The practice of brief incubation of gametes is not necessary and this can save the already tight manpower in many laboratories.
Funding: The study was supported by the Merck-Serono China Research Fund for Fertility Experts (2015), which was not involved in study design, execution, data analysis and manuscript preparation. There are no conflicts of interest for all authors.
PS170: Mitochondrial dysfunction and endoplasmic reticulum stress involved in oocyte aging: an analysis using single-cell RNA-sequencing of mouse oocytes
Tao Zhang, Reproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology; Lixia Zhu; Lei Jin
Background: To explore ovarian aging is still not well understood, we performed overall analysis on the age-related alterations of gene expression profiles in mouse Germinal vesicle (GV) stage oocytes by means of single-cell RNA-sequencing method (scRNA-seq).
Methods: Two age groups 5-week-old and 32-week-old female KM mice were used as young and old models. Subsequently, GV oocytes were collected for scRNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between GV oocytes of young and old mice.
Results: The analysis of scRNA-seq data showed that there were 624 differentially expressed genes (DEGs) between two age groups of mouse GV stage oocytes. 449 DEGs were up-regulated while 175 DEGs were down-regulated in the GV oocytes of the old group. KEGG pathway analysis revealed that the genes involved in mitochondrial function including oxidative phosphorylation and ATP production pathway were significantly down-regulated in GV oocytes of 32-week-old mice, especially the mitochondrial encoded NADH dehydrogenase (mt-Nd), including mt-Nd2, mt-Nd3, mt-Nd4, mt-Nd4L and mt-Nd5. Analysis of DEGs revealed that endoplasmic reticulum stress-related genes including AdipoR2, IRAK-1, RCAN1 and MsrB1 were significantly down-regulated in GV oocytes of 32-week-old mice. Also, analysis of DEGs demonstrated that antioxidation-related genes including Erbb3, Rcan1, Gsto2 and Msrb1 were also significantly down-regulated in GV oocytes of 32-week-old mice.
Conclusion: The disorder of mitochondrial function and endoplasmic reticulum stress might be involved in the progression of oocyte aging. Especially, the down regulation of mitochondrial encoded subunits of respiratory chain complexes may play critical roles in the relevant mechanisms.
Funding: This work was supported by the research grants from the Chinese Medical Association (16020520668) and the Natural Science Foundation of Hubei Province (2017CFB752).
PS171: BMI might be a risk factor for low prognosis in patients with PCOS undergoing ART
Yanxiu Guo, Reproductive Medical Center, Peking University People’s Hospital, Department of Obstetrics and Gynecology; Yuan Fan; Li Tian
Background: Polycystic ovary syndrome (PCOS) affects 5–10% of women in reproductive age and is characterized by chronic anovulation and hyperandrogenism. IVF-ET is the main treatment for ovulation induction therapy failure of PCOS patient, but there is still a few part of patients present as a poor ovarian response (POR), which always leads to a “low prognosis”. What is the risk factor of these patients for the POR remains unclear. This retrospective study is going to find the possible risk factors for POR of women with PCOS.
Methods: 334 patients with PCOS who underwent assisted reproductive technology (ART) in Peking University People’s Hospital Reproductive Center from Jan 1st, 2016 to Dec 31st, 2017 were enrolled into this study. The number of oocytes were calculated, and the pregnancy rate and live birth rate related to the oocytes number were also estimated. Patients with POR were grouped according to the POSEIDON stratification. Multivariate logistic regression analysis were used to value the risk factors for the POR of patients with PCOS.
Results: The mean number of oocytes in the enrolled 334 patients was 16.28. Forty-two patients were diagnose as POR with the mean oocytes number of 2.8. The pregnancy rate in patients with POR was similar with other patients (56.5% vs. 41.3%, P=0.194), but the live birth rate was significantly decreased (13.0% vs. 21.3%, P=0.047). The duration of infertility (P=0.022), BMI (P=0.010) and duration of GnRH use (P=0.027) was significantly higher in patients with POR than that in other patients. After the multivariate logistic regression analysis, BMI remained to be the risk factor for the POR of patients with PCOS (RR=1.101, 95%CI: 1.002-1.209, P=0.045).
Conclusion: PCOS patients with POR had a poor live birth rate. BMI might be the possible risk factor for POR in patients with PCOS.
PS172: Live birth rate (LBR), ongoing pregnancy rate (OPR) and ovarian hyperstimulation syndrome (OHSS) risk with originator versus biosimilar recombinant follitropin alfa: a pooled analysis of clinical trial data
Ruediger Papsch, Merck KGaA, Darmstadt, Germany; Claudia Roeder; Thomas D’Hooghe; Salvatore Longobardi
Background: To investigate whether LBR, OPR and OHSS, and the benefit-risk balance of these outcomes, differ between originator and biosimilar recombinant human follicle-stimulating hormone preparations.
Methods: Pooled analysis of Phase III clinical trial data for Ovaleap® and Bemfola® versus GONAL-f®. The primary endpoint was LBR per patient. Other endpoints were OPR per patient, and OHSS incidence (any grade). The null hypothesis was no difference between originator follitropin alfa and biosimilars. Pooled data were analysed, and biosimilars were also evaluated separately to confirm trends. Endpoints were evaluated over one cycle. LBR per patient, OPR per patient and OHSS incidence were compared between originator and the biosimilars (pooled) using a logistic regression model, with treatment (two categories; biosimilar or originator) and study (Ovaleap or Bemfola) included as categorical covariates.
Results: Data were included for 269 patients who received originator and 402 patients who received biosimilars (Ovaleap, 153; Bemfola, 249). The maximum likelihood estimate (standard error) and P-value were: 0.3105 (0.1699), P=0.0338 for LBR per patient; 0.3019 (0.1686), P=0.0367 for OPR per patient; and –0.9916 (0.4323), P=0.0109 for OHSS incidence. Differences between originator and biosimilars were independent of the study (Ovaleap or Bemfola). These results show that different outcomes were observed with originator compared with biosimilars and suggest that there may be improved outcomes (greater likelihood of LBR and OPR) with less risk of OHSS if originator is used instead of a biosimilar preparation. Evaluation of individual studies confirmed this finding.
Conclusion: This pooled analysis highlights that biosimilar preparations are not identical to the originator preparation, and suggests that the benefit-risk balance, based on LBR, OPR and OHSS incidence, is better with the originator preparation. Prospective clinical trials and/or analysis of real-world data powered to compare the LBR, OPR and OHSS risk with all the available products are required to support these findings.
Funding: Research was funded in full by Merck KGaA, Darmstadt, Germany. This abstract was presented at the ISPOR Europe 2018 conference and the 12th Joint Conference of the UK Fertility Societies, 2019.
PS173: Chronic unpredictable mild stress down-regulates nerve growth factor leading to premature ovarian failure in rats
Qun Zheng, Jinhua People’s Hospital, Jinhua, Zhejiang Province, P.R. China; Xiaoyan Fu; Libo Zou; Mingxing Ding; Haohao Chen
Background: Premature ovarian failure (POF) is caused by multiple factors, the pathogenesis is unclear. The aim of the study was to investigate whether chronic unpredictable mild stress (CUMS) is mediated by the nerve growth factor (NGF)/high affinity nerve growth factor receptor (TrkA) pathway and af-fects follicular development in the ovary leading to the onset of POF.
Methods: Rats were divided into control and CUMS group. Rats in CUMS group were exposed to a set of chronic unpredictable mild stressors. Then the serum levels of gonadotropin releasing hormone (GnRH), follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) were detected. The expression levels of NGF, TrkA, p75 and FSHR in ovarian tissue were de-tected, immunofluorescence was used to detect the above proteins, and numbers of small follicles were counted. Ovarian tissues of CUMS rats were cultured in vitro, β-NGF and FSH were added. Then the expression levels of NGF, TrkA, p75, FSHR, E2 and AMH were detected.
Results: In CUMS group, the serum levels of E2, AMH and GnRH, and the number of small follicles de-creased significantly. Expression of NGF, TrkA, p75 and FSHR decreased in the ovarian tissues. Exogenous NGF up-regulates the expression of TrkA, p75 and FSHR of CUMS ovarian tissue in vitro, and increased E2 expression of tissue culture supernatant when combined with FSH.
Conclusion: CUMS down-regulates NGF and TrkA, which leads to premature ovarian failure in rats. Exoge-nous NGF and FSH could up-regulate the expression of their receptors, E2 and AMH in vitro.
Funding: The present study was supported by grants from Experimental Animal Science and Technology Project of Zhejiang Province (grant no. 2015C37104), and Science and Technology Project of Jinhua City in P.R. China (grant no. 2017-4-069).
PS174 – (Chinese language abstract – see Chinese supplement)
Chen Xiao, JinHua people’s Hospital; Ouyang Luan
PS175: Study on the improvement of IVF outcome in insulin-resistant PCOS patients by acupoint embedding based on PI3K/Akt signaling pathway
Xiang Shan, Shandong University of Traditional Chinese Medicine; Lian Fang
Background: To observe the effect of acupoint embedding method on the regulation of the key effector molecule Akt of PI3K/Akt signaling pathway and the level of GLUT4 associated with pathway on the quality of IVF egg cells in patients with insulin resistance PCOS.
Methods: Sixty patients with insulin resistance PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) were randomly divided into acupoint embedding treatment group (acupoint embedding combined with GnRH-a+Daying-35 double descending treatment). The basic treatment group (GnRH-a+Daying-35 double-dose treatment), 30 cases in each group, the acupoint catgutization treatment group (GnRH-a) decreased the regulation of the 10th day of the previous cycle. On the day of the down regulation, the COH cycle was treated by the first day of the net. The changes of insulin resistance index HOMA-IR before and after treatment in the acupoint embedding treatment group were observed. The fertilization rate, the rate of excellent embryos, the clinical pregnancy rate, the time parameters, and the p-Akt and GLUT4 protein levels in the PI3K/Akt signal transduction pathway.
Results: (1) The HOMA-IR of the acupoint embedding treatment group was significantly lower than that before treatment. (2) Compared with the basic treatment group, the 2PN fertilization rate and clinical pregnancy rate of the acupoint embedding treatment group were not statistically significant (P>0.05), but there is an increasing trend, the rate of excellent embryos is significantly increased P <0.05); t2, t3, t5, time period comparison, the difference between the two groups is not statistically significant (P>0.05), in the t4, cc2 time period comparison. The development time of the acupoint embedding treatment group was significantly faster than that of the basic treatment group (P<0.05). (3) The expression of p-Akt and GLUT4 protein was up-regulated in the acupoint embedding group compared with the basic treatment group (P<0.05).
Conclusion: Acupoint embedding may be through PI3K/Akt signal transduction pathway, up-regulate GLUT4 protein expression, improve insulin resistance, thereby improving IVF egg cell quality in PCOS patients, and ultimately improving the outcome of pregnancy.
PS176: Influence of mouse defective zona pellucida in folliculogenesis on apoptosis of foliicles and developmental competence of oocytes
Yan Wang, Institute of Reproductive & Stem Cell Engineering, School of Basic Medical Science, Central South University; Hong-Mei Xiao
Background: First of all, we must declare that this abstract was accepted for presentation as a poster at at the 12th annual meeting of the Chinese Society of Reproductive Medicine (CSRM).
Complex bilateral signaling between the oocyte and surrounding granulosa cells is important for the development of both cells, especially for the oocyte’s fully developmental competence, i.e., the ability to be fertilized and generate an individual. Filamentary Transzonal projections (TZPs) mediate the bilateral signaling and are maintained by the Zona Pellucida (ZP), an “egg-shell” of the oocyte, composed of zona pellucida proteins (ZPs). In 2014, we reported failure of IVF of ZP free eggs caused by mutation of human ZP1. Then using CRISPR/Cas9 system, we obtained a mouse strain carrying the resembling mutation. Eggs of mutant mice showed two types: ZP thinned and ZP free, originating from follicles with thinned ZP and cracked ZP respectively. Here, we examined the morphology of TZPs and apoptosis of follicles, as well as the developmental potential of eggs in order to explore the effects of different degrees of ZP defects in oogenesis on the quality of eggs.
Methods: (1) Phalloidin staining and TUNEL assay were performed to reveal the morphology of TZPs and apoptosis of follicles respectively. (2) Transfer of blastocysts after in vivo fertilization and in vitro culture was performed to check the developmental potential of eggs. To eliminate the favorable impacts of ZP itself on embryogenesis, IVF-ET of wild type eggs with artificially removed ZP was performed. These eggs were set as the control of mutant ZP free eggs.
Results: (1) In the ZP-cracked rather than the ZP-thinned growing follicles, TZPs became sparse and the apoptosis of granulosa cells instead of oocytes increased significantly. (2) ZP-thinned (loosened but intact) eggs had normal developmental competence, while ZP-free eggs had impaired competence characterized by reduced live birth rate.
Conclusion: Zona pellucida integrity rather than thickness or porosity in folliculogenesis is important for maintaining transzonal projections (TZPs), and ensuring the survival of granulosa cells and the establishment of the fully developmental competence of oocytes.
Funding: This study was supported by National Key R&D Program of China (2016YFC1201805 and 2017YFC1001100), National Natural Science Foundation of China (81471453 and 81501248), and Natural Science Foundation of Hunan Province of China (2015JJ2166 and 2017JJ3425).
PS177: Influences of different treatment methods and degree of disease control satisfaction on IVF/ICSI outcomes in infertile patients with adenomyosis (adenomyoma)
Yuan Zhang, The First Affiliated Hospital with Nanjing Medical University; Yundong Mao; Yuan Zhang; Yundong Mao; Chunxiang Wu; Jie Huang; Li Shu; Zhen Hou; Wei Ding; Jinyong Liu; Xiang Ma; Jiayin Liu
Background: To investigate whether adenomyosis (adenomyoma) affects the outcome of IVF-ET/ICSI, and to explore different treatment methods for different types of adenomyosis (adenomyosis), with emphasis on the influences of degree of disease control satisfaction (DDCS) on IVF/ICSI outcomes in infertile patients with adenomyosis (adenomyoma).
Methods: 571 cycles/226 patients with adenomyosis (adenomyosis) were included to compare the outcome of IVF/ICSI for these patients among different groups. According to different treatment methods before IVF/ICSI and different standard of DDCS in this study, patients were divided into three groups: treatment-satisfaction group (Group A, 208 cycles), treatment-unsatisfaction group (Group B, 219 cycles), no treatment group (Group C, 144 cycles). Patients with denomyoma (338 cycles) and diffuse adenomyosis (233 cycles) were distinguished according to the diagnosis of vaginal ultrasonography.
Results: (1) There were significant differences in the number of retrieved oocytes, implantation rate, clinical pregnancy rate, miscarriage rate, cycle cancellation rate and delivery rate among groups A, B and C (P<0.05), with no significant difference in the 2PN rate, high quality embryo rate and pre-term birth rate (P>0.05). (2) The IVF/ICSI outcomes of adenomyoma were better than those of diffuse adenomyosis, but there was no significant difference (P>0.05). (3) For patients of adenomyoma, surgical excision of uterine adenomyoma combined with GnRHa treatment seems have better IVF/ICSI outcomes than those received GnRHa treatment alone, however the difference was not significant (P>0.05).
Conclusion: Adenomyosis (adenomyosis) affects the outcome of IVF/ICSI and needs to be treated before IVF/ICSI, and DDCS is closely related to the outcome of IVF/ICSI. IVF/ICSI outcomes could be obviously improved when adenomyosis (adenomyosis) were under satisfactory disease control. The best standard for the treatment of adenomyosis before IVF/ICSI is: uterus shrinkage, Ca125<20IU/mL, VAS score of gynecological tenderness<3-4. Adenomyosis mainly affects embryo implantation, closely related to increased abortion rate and decreasesd delivery rate, but might have little effect on fertilization and embryo quality. Adenomyoma may have a slightly better IVF/ICSI outcome than diffuse adenomyosis, and surgical excision together with medication treatment may be more conducive to get pregnant.
PS178: Clinical outcomes analysis of minimal-stimulation used clomiphene in dysfunction ovary patients
Chunyan Jiang, State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital; Yan Gao; Jiayin Liu; Yi Qian
Background: Ovarian dysfunction are significant cause of infertility. In vitro fertilization (IVF) in natural cycles or with minimal stimulation has gained worldwide attention and interest in infertile patients. Here, we investigated the clinical parameter of the ovarian dysfunction patients who received minimal stimulation protocol with clomiphene in IVF-ET/ICSI.
Methods: The clinical date of a total 971 IVF cycles used clomiphene minimal stimulation in dysfunction ovary patients undergone from Jan 2015 to Dec 2016 in Center for Clinical Reproductive Medicine, the First A ffiliated Hospital of Nanjing Medical University. The patients whose antral follicle diameter of day 3 less than 8mm, FSH less than 16IU/L and estradial less than 280 pmol/L took clomiphene citrate and/or HMG stimulation protocol. According to the different dosage of clomiphene, the patients were divided in to 2 groups: Group A with 533 cycles of persistent using clomiphene, while group B with 438 cycles of early 3 days using clomiphene.
Results: The pregnancy rate per cycle was about 30% and comulative pregnancy rate was about 46%. The AFC and pregnancy rate gradually diminished with age, but there is no statistical change in basal FSH level, the oocyte retrieval rate and the fertilization rate. The basic background data, including age, basic FSH and AFC numbers in 2 groups showed no significant difference (P> 0.05). Compared to groups A, Group B patients had thicker endometrium on trigger day, lower embryo frozen rate, higher pregnancy rate in fresh embryo transfer cycle (P<0.05); but risk of preovaluation was higher in Group B (P<0.05).
Conclusion: Minimal stimulation using clomiphene as short as 3 days could increase the rate of pre-ovalution, but decrese the infect of endometrium and get more chance of fresh embryo transfer and clinical pregnancy in fresh cycles.
PS179: Aminopeptidase N expression in the endometrium could affect endometrial receptivity
Lijun Shui, The First Affiliated Hospital of Nanjing Medical University; Yan Meng; Yi Qian; Jiayin Liu
Background: Aminopeptidase N (ANPEP) is a membrane-bound zinc-dependent peptidase. It is widely believed that ANPEP acts as an important angiogenesis regulatory factor; however, there are few studies about its function in the female reproductive system.
Methods: In this study, we observed the expression of ANPEP in the endometrium of mice at different ages (3wk, 8wk, 8mo, 16mo). And further dectected the expression of ANPEP in the endometrium of IVF-ET patients in the proliferative, preimplantation and implantation stages. While the human primary endometrial stromal cells were infected with an adenovirus expression vector containing the ANPEP gene and a green fluorescent protein (GFP) fusion protein, the expression of endometrium receptivity factors were detected.
Results: The results showed the expression of ANPEP was highest in the mature period in mice. Further we found the highest expression level of ANPEP in the implantation group. The mRNA levels of endometrial receptivity factors, such as HOXA-10, LIF, and integrin β3, in endometrial cells improved with the upregulation of ANPEP.
Conclusion: Therefore, we conclude that ANPEP was involved in the regulation of endometrial receptivity.
Funding: The Science Foundation of Jiangsu Province (BK20161067) and the National Science Foundation (81701517).
PS180 - (Chinese language abstract – see Chinese supplement)
Chaoyi Shi, The International Peace Maternity & Child Health Hospital of Shanghai Jiao Tong University School of Medicine, Shanghai, China; Jingjing Xu; Hefeng Huang
PS181: Identification of novel mutation sites of DNAH1 gene in multiple morphological anomalies of the sperm flagella (MMAF) patients
Yan-Wei Sha; Wen-sheng Liu
Background: Abnormality of sperm is the main reason for male infertility that troubled approximately seven percent of man in the world (Krausz & Riera-Escamilla 2018). Severely incompetent of sperm motility in clinic was known as severe asthenospermia, which contributes to male absolute infertility (Bonanno et al 2016). One of the common reasons for severe asthenospermia is multiple morphological abnormalities of the flagella (MMAF), which is characterized by several morphological defects of sperm flagella, including absent, short, bent, coiled, and/or irregular flagella (Ben Khelifa et al 2014).
Methods: Whole genome sequencing and Sanger sequencing, Papanicolaou staining, Transmission Electron Microscopy, Western blot analysis, Immunostaining.
Results: Here, we report several novel mutation sites of DNAH1 gene in three infertility patients with MMAF. Papanicolaou staining and transmission electron microscopy showed several morphology and ultrastructure defects of sperm flagella in patients with MMAF. Using whole genome sequencing and Sanger sequencing, we identified five novel heterozygous mutations of DNAH1 gene: c.5626G>C:p.A1876P and c.11779G>A:p.D3927N in P1 patient, c.7471C>A:p.Q2491K and c.9505C>T:p.R3169C in P2 patient, c.G8151-1C:3p21.1 in P3 patient. These mutations are located in the exons of DNAH1 genome as well as the functional domains of the DNAH1 protein and distincted from previous reports and strongly associated with severe asthenospermia through bioinformatics analysis. Furthermore, DNAH1 protein is nearly undetectable in the sperms from MMAF patients. Fortunately, two of the patients successfully fertilized and raised their own babies by intracytoplasmic sperm injection (ICSI). These discoveries provide more valuable information for clinical diagnosis and efficient therapeutic approach for MMAF.
Conclusion: DNAH1 gene mutations may be the main cause of MMAF.
Funding: The National Natural Science Foundation of China (Grant No. 31171375, No. 81871200); the Science Technology Guidance Project of Fujian Province [Grant No. 2017D018]; the Linqiaozhi Funding Supporting Youth Project of Xiamen Maternity and Child Care Hospital [Grant No. FYLQZ2015004].
PS182: Impact of supraphysiologic estradiol during controlled ovarian hyperstimulation on the perinatal outcomes of frozen embryo transfer
Nan Lu, Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University; Hua Chen; Junyi Yang; Xiaoxi Sun
Background: Numerous studies have shown great impact on health from Assisted Reproductive Technologies (ART) including higher risk of adverse pregnancy and perinatal outcomes. During IVF-ET, Controlled Ovarian Hyperstimulation (COH) plays a crucial role. COH will cause a significant rise of estradiol in patients, creating an adverse environment of ongoing pregnancy. In fresh transfer cycles, supraphysiologic estradiol is an independent predictor of low birth weight, whereas in frozen transfer cycles, estradiol level is similar with natural pregnancy.
Methods: All patients undergoing FET during January 1st, 2014- December 31, 2017 in Shanghai JiAi genetics and IVF institute were assessed for potential inclusion. Baseline characteristics, IVF characteristics for COH and frozen transfer cycles and perinatal outcomes were recorded for all patients. Patients were divided into four groups according to the distribution of E2 level on day of HCG trigger: E2≤25% percentile, 25% percentile<E2≤50% percentile, 50% percentile<E2≤75% percentile, and ≥75% percentile. Statistical analyses and multivariable logistic regression analysis was performed.
Results: A total of 8843 patients, 11823 cycles were included into our study.
With the rise of E2 level on the day of HCG triggering, patients showed a lowering trend of age, basal FSH level and BMI. For patients with higher E2 level, less Gn dosage was used.
As E2 level rises, clinical pregnancy rate and live birth rates first rise then drop. Clinical pregnancy rate reaches 53.99% and live birth rates reaches 48.34% at 50-75% percentile of E2 distribution (4240<E2≤s5903pg/mL) in frozen transfer cycles.
There was no difference in gestational age and the occurrence rate of gestational complications including gestational diabetes mellitus, and postpartum hemorrhage.
A significant rise in LBW incidence was apparent with the elevation of E2 level on day of triggering in frozen transfer cycles, and birth weight was significantly lower as well. When E2 level exceeds 5903pg/mL, LBW is 2.1 times(RR 2.165, 95%CI 1.409 to 3.327) higher than when E2 level is less than 2950pg/mL.
Conclusion: Patients’ age, basal FSH and BMI may affect the level of E2 on day of triggering. Embryos obtained in supraphysiologic estradiol environment may still be affected even transferred in frozen transfer cycles as the prevalence of LBW increase and birthweight diminishes in patients with higher E2 level on day of HCG triggering in COH.
PS183: The difference on the cumulative live-birth rate of fresh and subsequent frozen cycles in poor ovarian responders using mild and controlled ovarian stimulation protocolsï¼ša prospective randomized study
Xiao-ping Liu, Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University; Bo Wang; Ting-ting Li; Xue-fen Xiao; Rui Huang
Background: To compare the “mild” protocol with letrozole, low-dose gonadotropins and a GnRH-antagonist (LE/Gn/GnRH-ant protocol) with the “modified long” protocol with a GnRH-agonist (GnRH-a) discontinuation before administration of gonadotropins and high-dose Gn for the controlled ovarian stimulation (COS) of poor ovarian responders (PORs) undergoing IVF/ICSI.
Methods: A prospective randomized study was performed in 191 patients with PORs undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The patients were randomized into two groups, modified long protocol was applied in COS group (n=94), while letrozole compensated with low dose gonadotropin protocol was applied in mild stimulation group (n=97). The main outcome measure was cumulative live births among the fresh and all vitrified blastocyst transfers combined after the same stimulation cycle.
Results: The “mild” stimulation led to significantly lower consumption of exogenous Gn and lower peak estradiol level than the “modified long” regimen (939.43±264.52 vs. 3117.82±749.56 U, 563.69±465.06 vs. 1195.10±701.45pg/mL, respectively; P<0.001). With the “modified” long protocol, it obtained significantly more oocytes (2.77±1.89 vs. 4.01±2.81; P<.001), more fertilized oocytes (1.74±1.52 vs. 2.53±2.01; P<.001) and more available embryos (1.50±1.41 vs. 1.97±1.61; P=.038). As for the pregnancy outcomes, the two stimulation regimens had similar implantation rate (27.27% vs. 21.70%), ongoing pregnancy rate per embryo transfer (23.21% vs. 22.95%), clinical pregnancy rate per embryo transfer (37.50% vs. 32.79%), and per initiation cycle (21.65% vs. 21.28%), live birth rate (23.21% vs. 22.95%), cumulative pregnancy rate (23.71% vs. 25.53%) and cumulative live birth rate (15.46% vs. 18.09%).
Conclusion: For POR patients, the “modified long” protocol and the mild stimulation protocol yield a similar cumulative live birth rate. Considering the fewer Gn dosage and shorter ovarian stimulation length associated with the mild stimulation, it can be concluded that the mild stimulation protocol is a valid alternative to the COS protocol.
PS184: Participation of Sialidase-1/3 (NEU1/3) in sperm function and its implication in idiopathic male infertility
Ying Feng, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University; Yilun Wu; Lin Wang; Fang Ma
Background: Sialic acids mediate many biological functions, including molecular recognition during development and immune response and fertilization. A sialic acid-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their sialic acids. In the somatic cells, sialidases hydrolyse sialic acid moities from glycoproteins and glycolipids, and these enzymes were traditionally thought to be in the acrosome of the sperm. However, the detailed role of sialidase in the sperm function is yet to be elucidated.
Methods: Sialidase 1 and 3 (NEU1 and 3) on human and mouse sperm in different stages of sperm en route to fertilization were investigated via glycobiological, biochemical and cell biological methods.
Results: Our research found: ① Both human and mouse sperm bears tens of millions of Sia molecules; ② Neu5Ac is transferred to mouse sperm from luminal CMP-Neu5Ac during epididymal transit; ③ Sias shed from capacitated mouse sperm; ④ Neu1and Neu3 were found on both mouse and human sperm with distinct localization patterns, moreover the localization pattern is different from the classic acrosome location; ⑤ Semen samples from infertile male showed abnormal levels of NEU1/3 on sperm surface and in the seminal plasma.
Conclusion: The unique localization of Neu1 and Neu3 in sperm implies these two sialidases may participate the sperm function more than just enzymes in the acrosome. Moreover, they may connect to the idiopathic male infertility according to the distinct protein levels between normal and infertile male.
Funding: This research was supported by the National Natural Science Foundation of China (NSFC) (No. 31470797, No. 31771662 and No. 31800677).
PS185: Effect of vitamin D status on normal fertiziation rate following in vitro fertiliazation
Liu Xuemei, Yantai Yuhuangding Hospital, Affiliated Hospital of Qingdao University; Zhang Wei; Wang Xinrong
Background: Vitamin D plays critical role in the system of female reproduction. It was supposed that vitamin D is associated with clinical pregnancy outcome of ART. The study is aimed to establish whether vitamin D was associated with clinical outcomes of in vitro fertilization (IVF).
Methods: The cross-sectional study was carried out from January 1st 2017 to December 31st 2017. A total of 848 patients who had an indication for IVF were enrolled. The patients were classified by serum 25OHD quartiles (group 1 to group 4). The outcome parameters of IVF were compared in each group, including normal fertilization rate, high quality embryo rate, clinical pregnancy rate, implantation rate and live birth rate.
Results: Serum 25OHD levels in women change with the seasons. We found that serum 25OHD levels were higher in autumn (from August to October) than other seasons, and they were lowest in spring (from February to April). All of the patients’ characteristics were comparable between the four groups, as no significant differences were observed. Normal fertilization rate was significantly difference among four groups. The group of women with the highest serum 25OHD levels had the highest normal fertilization rate. However, the clinical pregnancy rate, implantation rate and live birth rate were not significantly differences among four groups. In addition, we found that serum 25 OHD levels were significantly higher in patients received IVF than patients received R-ICSI.
Conclusion: Vitamin D deficiency may affect the normal fertilization rate after IVF. However, vitamin D was not associated with the clinical pregnancy and live birth rate after IVF.
Funding: This work was supported by National Natural Science Foundation of China (81601276), the special fund for clinical research of the Chinese Medical Association (16020220638).
PS186: Study on the effect of high blood estradiol concentration combined with absorbable adhesion barrier in preventing re-adhesion of patients with intrauterine adhesions after TRCA
Qiuping Li, The Affiliated Wuxi Maternity and Child Health Hospital of Nanjing Medical University; Yun Zhang
Background: Nowadays, the morbidity of intrauterine adhesion after transcervical resection of adhesions(TRCA) is higher and higher, especially medium and severe types, which leads to infertility of childbearing aged women, and ii’s a tough task for gynecologist. This research is designed To observe the effect of high blood concentration estradiol combined with anti-adhesion barrier in preventing re-adhesion of patients with intrauterine adhesions after transcervical resection of adhesions.
Methods: A total of 60 patients hospitalized in our hospital during 2015.4-2018.4 diagnosed as IUA were enrolled in this study. They were randomly divided into 2 groups. Intrauterine device (IUD) encapsulated by absorbable adhesion barrier (polylactic acid membrane) and vaginal 17-beta estradiol 2mg (Group A), IUD encapsulated by absorbable adhesion barrier and oral estradiol valerate (E2V) 3mg (Group B). Side effects, blood concentration of estradiol and herapeutic effects were observed
Results: There was no obvious side effects in both groups. The blood concentration of estradiol in Group A is significantly higher than that in Group B (P<0.05). In aspect of menstrual improvements, postoperative menstrual models were both better than that before operation, the differences were significant (P<0.05). Comparing menstrual improvement rates of two groups, Group A is significantly higher than that in Group B, difference was statistically significant (P<0.05). A comparison of postoperative prognosis score, improve score (preoperative-postoperative) in two groups, the differences were significant (P<0.05). There was differenc in treatment effects of two groups, which was statistically significant (P<0.05). There was no significant difference in total treatment efficacy (P=0.04), but the significant treatment efficacy was different in two groups, which of Group A was greater than that of Group B, the differences was significant (P<0.05).
Conclusion: Vaginal 17-beta estradiol combined with IUD encapsulated by absorbable adhesion barrier which made higher blood estradiol concentration is as fine as IUD encapsulated by absorbable adhesion barrier and oral estradiol valerate in preventing re-adhesion after TRCA, but in the aspect of improving the uterine cavity morphology and menstrual model, the effect of the former is better than the latter.
PS187: Clinical observation on the treatment of LUFS with adding Bushen Huoxue medicine at different stages of follicular development:late follicle development and ovulation
Yuling Li, Nanjing University of Traditional Chinese Medicine; Yong Tan; Qianqian Luo; Jiaqi Shuai; Ling Li
Background: This study was designed for evaluation and comparison of LUF incidence in eligible infertile women adding Bushen Huoxue medicine at two different stages (late follicle and ovulation) in the controlled ovulation stimulation cycles. comparison of LUF incidence in the ovulation period of eligible infertile women were treated by two different methods (Bushen Huoxue method and Huoxue method) as secondary outcomes.
Methods: The study was designed as a prospective randomized controlled trial. For group A, (n=30) Bushen Huoxue medicine was administrated orally in late follicle, group B (n=30) Bushen Huoxue medicine was administrated orally in ovulation, and group C (n=30) Huoxue medicine was administrated orally in ovulation. The optimum size of follicles for the late follicle was considered 14 mm and the ovulation was considered 18 mm. Eligible patients are treated continuously for 3 menstrual cycles of controlled ovulation stimulation (COS: CC/LE+HMG+HCG). The ultrasound examination was performed to observe the evidence of LUF syndrome at the time of 24 hours after HCG.
Results: The differences among the rates of ovulation and recurrence rates of LUF were statistically significant (P<0.05).The rate of follicular development and the Estrogen level of single dominant follicle on the trigger day in group A were significantly higher than the group B (the former P<0.05, the latter P<0.01). There was no significant difference between group B and group C.The clinical pregnancy rates in the three groups were: 33% in group A, 13.3% in group B, 6.7% in group C, the difference wasn’t statistically significant (P>0.05). The scores of TCM symptoms in three groups were lower than before (P<0.05).
Conclusion: Late follicle, with lower incidences of LUF, is more effective than ovulation period administrated orally Bushen Huoxue medicine in the COS cycles. Adding Bushen Huoxue medicine during late follicle can significantly improve the speed and quality of follicle development and alleviate the clinical symptoms of patients. During ovulation period adding Bushen Huouxue medicine is more effective than simple Huouxue medicine.
Funding: Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
PS188: Infertility treatment of Y123F female mice by assisted reproductive technology and gene expression changes of Y123F mouse oocytes
Xiaoyu Tu, Women’s Hospital, School of Medicine, Zhejiang University; Zhaogui Sun; Runju Zhang
Background: Obesity and related reproductive endocrine diseases, such as PCOS, result in the increased incidence of infertility. Therefore, it is of great clinical significance to study the molecular mechanisms of impaired fertility in association with obesity and to investigate rational treatment. Leptin controls energy balance and body weight as well as a permissive signal to regulate female reproduction. Y123F mice are transgenic mice that are expressed mutant leptin receptors with phenylalanine (F) substitution for all three tyrosine (Y) residues. They are infertile, but their symptoms of obesity, hyperphagia, hyperinsulinemia and impairment in glucose tolerance are less severe than db/db mice and they have a longer life span.
Methods: 12-week-old Y123F mice and WT mice were superovulated, then eggs were collected from the fallopian tube and co-cultured with wild-type mouse sperms. The number of oocytes, the rates of fertilization and blastocyst formation were evaluated. Fertilized eggs of both genotype mice were mixed and transplanted into the recipient mouse fallopian tubes to find out whether F1 mice could be obtained. Then heterozygous F1 mice were isolated by genotyping and crossbred with normal fertility heterozygous Y123F mice to explore the availability of F2 mice. Oocyte gene expression changes of two genotype mice were analyzed by transcriptome sequencing. The expression of related genes was verified by RT-qPCR.
Results: Y123F mice showed a significant decrease in the number of oocytes and the rates of fertilization and blastocyst formation compared to WT group. Heterozygous F1 mice were obtained through ART, and then F1 mice could breed F2 mice. Transcriptome sequencing results showed that 78 genes were up regulated and 48 genes were down regulated in Y123F mice. GO analysis showed that ATP biosynthesis GO belonged to the enrichment of the smallest category of highly enriched GO. The oxidative phosphorylation pathway was highly enriched. RT-qPCR validation showed that ATP synthesis and oxidative phosphorylation related genes expression were decreased significantly.
Conclusion: ART treat the infertility of homozygous female Y123F mice effectively. OB-R tyrosine site mutations impaired mitochondrial oxidative phosphorylation process and ATP production capacity in Y123F mouse oocytes, which did not affect the generation of the offspring, but affected the number of oocytes and the rates of fertilization and blastocyst formation.
PS189: Effect of oocyte activation with calcium ionophore on embryo development and pregnancy outcome
Yinyang Bai, Wuxi Maternity and Child Health Hospital Affiliated to Nanjing Medical University; Fang Xiong; Jie Chen; Liyi Cai
Background: In the assisted reproductive technology, there are still some problems such as fertilization failure and retardation of embryo development even after intracytoplasmic sperm injection (ICSI). This study is to investigate the effect of calcium ionophore (A23187) on embryo development and outcome in patients with a history of developmental problems.
Methods: Sixty patients with embryo developmental delay, arrest or low blastocyst formation in a previous cycle were recruited. The total of sixty immediately preceding cycles in the same patients constituted the control group. All metaphase II (MII) oocytes were exposed to A23187 for 15 min immediately after ICSI. After a regular washing procedure, in vitro culture was performed as in the control cycles. Embryo development and blastocyst formation, as well as clinical pregnancy were observed.
Results: Although fertilization rate did not differ (78.5% vs. 76.2%), the cleavage rate of embryo on day 3 was significantly increased (P<0.05) in the ionophore group (95.3%) compared to that in the control cycle (88.1%). In addition, much more quality embryos (31.8% vs. 19.0%, P<0.05) and blastocysts on day 5 and 6 (30.7% vs. 5.7%, P<0.01) were formed in the treatment cycles than that in the controls. This was associated with a significant increase in biochemical pregnancy rate (65.5% vs. 23.1%, P<0.01) and clinical pregnancy rate (48.3% vs. 10.7%, P<0.01).
Conclusion: Application of A23187 leads to increased rates of embryo cleavage and blastocyst formation as well as clinical pregnancy. It brings certain practicability and effectiveness to patients with poor embryo development and pregnancy outcome.
Funding: No funding.
PS190: Retrospective analysis of the prevalence and drug resistance of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis infections in women with tubal pregnancy compared with early pregnancy
Yang Liu, Fudan University Zhongshan Hospital; Yushi Meng
Background: Chlamydia trachomatis (CT) infection is an important factor for tubal pregnancy. However, it is not clear if Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) infections are also involved. This study detected CT, UU, and MH in the cervical secretions of patients with tubal pregnancy and early pregnant women, to explore their prevalence and drug susceptibilities.
Methods: The study was made of data from patients with tubal pregnancy and patients requiring termination of early pregnancy at <12 weeks from July 2013 to March 2014. Cervical secretions were tested with a UU/MH isolation and culture kit and for CT antigen by immunochromatographic assay. Mycoplasma samples were tested for resistance to 12 antibiotics.
Results: There were no cases of CT infection detected. Mycoplasma infection rates were similar in the tubal pregnancy and control groups, but the total rate of infection was higher for tubal pregnancy. All MH samples were sensitive to tetracyclines and to josamycin and azithromycin. Josamycin and clarithromycin were effective against all UU cultures. Over 50% of all samples tested were resistant to ciprofloxacin.
Conclusion: CT infection was not detected but the total rate of infection with UU and MH was higher in women with tubal pregnancy.
Funding: This work supported by the National Nature Fund (grant number 81860515); Health Science and Technology Plan Project of Yunnan (grant numbers 2014NS092, 2016NS286, 2016NS287 and 2017NS277); Yunnan Health Training Project of High Level Talents: (grant number H-201629); Applied Basic Research Joint Special Fund Project of Yunnan Provincial Science and Technology Department-Kunming Medical University (The mechanism of miR-214/Sema4D in angiogenesis of ovarian carcinoma) 2018FE001 (-055).
PS191: SPEF2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella (MMAF)
Wen-sheng Liu; Yan-Wei Sha
Background: Multiple morphological abnormalities of the sperm flagella (MMAF) is a kind of severe teratozoospermia. Patients with MMAF phenotype are infertile and present aberrant spermatozoa with absent, short, coiled, bent and/or irregular flagella. Mutations in several genes can explain approximately 30% to 50% of MMAF cases and more genetic pathogeny needs to be explored.
Methods: Whole genome sequencing and Sanger sequencing, Papanicolaou staining, Transmission Electron Microscopy, Western blot analysis, Immunostaining.
Results: By screen the gene variants in two patients with MMAF using whole exome sequence, we identified c. 12delC, c. 1745-2A>G, c. 4102G>T and c. 4323dupA mutations in the SPEF2 gene from 2 patients. Both of these mutations are rare and potentially deleterious. TEM analysis showed disrupted axonemal structure with mitochondrial sheath defection in patient’s spermatozoa. No dysfunctional SPEF2 protein was localized on patient’s spermatozoa revealed by WB and IF analysis.
Conclusion: Our experimental findings indicate that loss-of-function mutations in SPEF2 gene can cause MMAF phenotype in human.
Funding: The open project of Key Laboratory of Male Reproduction and Genetics, National Health and Family Planning Commission (Grant No. KF201704); the Medicine and Health Science Technology Development Project of Shandong Province [Grant No. 2016WS0704]; Natural Science Foundation of Shandong Province [Grant No. ZR2017LH012]; the National Natural Science Foundation of China (Grant No. 31171375, No. 81871200); the Science Technology Guidance Project of Fujian Province [Grant No. 2017D018]; the Linqiaozhi Funding Supporting Youth Project of Xiamen Maternity and Child Care Hospital [Grant No. FYLQZ2015004]
PS192: Preliminary investigation of the function of hsa_circ_0049356 in azoospermia patients
Lin Liu, The Reproductive Medicine Special Hospital of the 1st Hospital of Lanzhou University; Xuehong Zhang
Background: Azoospermia, the most severe form of male infertility affecting about 1% of all males and 10% to 15% of all infertile males. Spermatogenesis is a complex and accurate regulated process. Abnormality of genetic and epigenetic factors can lead to abnormal spermatogenesis and eventual male infertility. The aim of this study was to investigate the function of circ_0049356 in azoospermia patients.
Methods: The expression of hsa_circ_0049356 in whole blood and seminal plasma were analyzed by qRT-PCR in healthy volunteers and azoospermia patients, indicating that the expression of hsa_circ_0049356 in the whole blood was higher than seminal plasma of azoospermia patients. Further, the relative expression of circ_0049356 host gene (CARM1) was confirmed by qRT-PCR. After that, bioinformatics analysis was performed to predict the target miRNAs of circ_0049356 and investigate the circ_0049356-miRNA-mRNA interactions. Reviewing the literature, we ultimately identified that hsa_circ_0049356 played a significant role in spermatogenesis.
Results: In this study, we identified the expression of hsa_circ_0049356 in the whole blood and seminal plasma of azoospermia patients by qRT-PCR. Given the pivotal biological roles, the host gene of circ_0049356 was investigated both in the whole blood and seminal plasma of azoospermia patients. After that, bioinformatics analysis was performed to predict miRNAs targeted by hsa_circ_0049356 and investigate the hsa_circ_0049356-miRNA-mRNA interactions. In view of their important biological roles, we investigated and identified circ_0049356 in azoospermia patients to offer new idea for diagnosis and treatment.
Conclusion: Our findings laid the foundation for in-depth mechanistic studies on spermatogenesis, which can strengthen the reliability of the circ_0049356 as diagnostic and therapeutic targets.
Funding: Research was funded by Gansu Province Health Industry Research Plan (GSWSKY-2015-50) and Gansu Province Youth Science and Technology Fund (1606RJYA270).
PS193: Discussion on ovulation-promoting methods for refractory ovulatory disorder in patients with polycystic ovary
Background: To investigate the hormonal changes, the growth and development of follicular and ovulation in PCOS patients with high levels of LH (LH> 10mIU/mL) who underwent the intermittent use of clomiphene (CC).
Methods: 24 patients diagnosed with PCOS-refractory ovulatory disorders in Qilu Hospital of Shandong University were treated with intermittent and repeated use of CC for ovulation induction.Patients were tested basal E2, FSH, LH level and underwent transvaginal B-ultrasound. Patients started CC from the 3-5th day of the menstrual cycle. The daily dose of CC was 50-100mg according to the patient’s BMI. The first time to take CC is 5-7 days. Then CC is stopped and begins to take Estradiolvalerate until the end of the treatment cycle. After taking 2-4 days of Estradiolvalerate, the patient was reexamination. If follicles or hormones were not activated, CC was applied again for 3-5 days. After the stop of CC 2-4 days, the patient was referred again.When there are follicle diameter of 9 mm or serum E2 levels of 100pg/mL, CC is completely discontinued and Gn was added to continue ovulation therapy. The maximum times of each patient to use CC are 3-4 times, Gn is continued. GnRH-a or HCG is selected for ovulation induction when follicle matured, Dydrogesterone was used for luteal support, test of serum HCG after ovulation 14 days. We collected dates of patient’s time and dose of CC, Gn, mature follicles number, the state of ovulation and pregnancy, follicles numbers, follicle diameter, endometrium thickness. The data collected were statistically expressed in the form of mean±standard deviation.
Results: (1) There were three significantly increased serum LH fluctuations, and the highest LH (19.57±8.45mIU/mL) appeared in 9≤d<12mm group. (2) The LH reached the highest value during the whole ovulation induction treatment (20.52±7.8mIU/mL) when the clomiphene phase was stopped. (3) Patients withbasal LH>10mIU/mL: LH has two significantly higher fluctuations in the groups 9≤d<12mm and 16≤d<18mm. The highest LH appeared in the discontinuation of CC.
Conclusion: (1) When E2 reaches ≥100pg/mL or the follicular diameter is ≥9mm, the type of ovulation-promoting drugs should be promptly adjusted. (2) A apparently increasing in LH that occurs before immature follicles does not absolutely predict impending ovulation or follicle luteinization, and can continue ovulation therapy at high LH. (3) Intermittent repetitive CC ovulation stimulation program can effectively induce single follicle development.
PS194: Obesity-associated elevated chemerin/CMKLR1 may be involved in oxidative stress and apoptosis in ovaries and granulosa cells
Junning Yao, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology; Hanwang Zhang
Background: Sub-optimal fecundity of obese female has been attributed to abnormal inflammatory responses, exceed oxidative stress, mitochondrial dysfunction and cell apoptosis in ovary. Chemerin and its receptor CMKLR1 has been reported to be expressed in ovary and associated with follicle function.
Methods: In this study, chemerin and CMKLR1 were evaluated in ovaries and granulosa cells of high-fat diet induced obesity (OB) mice and control group. Oxidative stress and apoptosis related biomarker were also assessed. Additionally, mouse granulosa cells (mGCs) were cultured and treated with different dose of chemerin in order to investigating viability, reactive oxygen species (ROS) and apoptosis, as well as alter of p-AKT, p-AMPKα and NF-κB p-p65.
Results: Compared with control group, chemerin/CMKLR1 axis were elevated in serum, ovaries and granulosa cells of OB mice. Apoptotic ovarian follicles, oxidative stress and apoptosis biomarkers were also increased in ovary of OB mice. In vitro study, chemerin suppressed mGCs viability with or without gonadotrophin, induced ROS accumulation and apoptosis increased in mGCs. Moreover, AMPKα and p65 were activated by chemerin, while AKT was suppressed. The phosphorylation change recovered with CMKLR1 knockdown.
Conclusion: Our present study discovered chemerin contributed to ROS accumulation and apoptotic cell proportion increasing through three possible signaling pathway, speculating that the upregulated chemerin/CMKLR1 was one of the reasons for imbalanced oxidative stress and apoptosis in ovaries of OB mice.
Funding: The present study was supported by grants from the National Natural Science Foundation of china (grant no. 81471459) and the National Natural Science Foundation of china (grant no. 81771582).
PS195: Conditional effects of fertility-related stress on psychological distress among infertile female: the moderating role of family resilience
Xiaofei Kang, Reproductive Hospital Afliated to Shandong University; Mei Fang; Guopeng Li; Zhenhua Jiang
Background: Infertility affects approximately 25% of Chinese couples of reproductive age in which nearly 80% infertile female were accompanied by psychological distress. Studies have demonstrated a direct relationship between fertility-related stress and psychological distress. Family resilience could provide a supportive environment that might have helped individuals experience less stress and more family support. Family resilience might buffer the positive relationship between fertility-related stress and psychological distress.
Methods: A total of 492 infertile female participated in this study, and completed self-reported measures, including the kessler 10 psychological distress scale (K10), the fertility problem inventory (FPI), and the family resilience assessment scale (FRAS). Our data analysis included descriptive statistics, the independent-sample t-test, analysis of variance (ANOVA), Pearson’s bivariate correlations, and hierarchical regression analysis.
Results: Fertility-related stress was positively correlated with the K10 score (r=0.551, P<0.01). The family resilience moderated the relationship between fertility-related stress and psychological distress. The simple slope analysis indicated that individuals with higher levels of family resilience demonstrated a weaker positive relationship between fertility-related stress and psychological distress.
Conclusion: Family resilience could buffer the relationship between fertility-related stress and psychological distress. The findings are valuable for identifying those who are more vulnerable to higher psychological distress and may aid in targeted intervention planning.
PS196 - (Chinese language abstract – see Chinese supplement)
Wang Jing, The Zhongda Affiliated Hosopital with Southeast University
PS197: Down-regulation of Gremlin 1 in the cumulus cells of young women with diminished ovarian reserve and its mechanism
Luna Mao, Women’s Hospital School of Medicine Zhejiang University; Quanmin Kang; Fan Jin
Background: Ovarian reserve is reflecting a woman’s reproductive potential with respect to quantity and quality of residual ovarian follicles and oocytes. There are up to 10% of women in the general population have experienced an accelerated decline of their fertility before the age of 32. However, the mechanism of ovarian reserve declining is remaining unclear. A bidirectional communication between oocytes and Cumulus granulosa cells (GCs) is essential for oocyte and follicular growth and is crucial for fertility. Recent gene expression analyses showed Gremlin 1 (GREM1) in GCs may assoicated with the poor reproductive capacity in young women with diminished ovarian reserve.
Methods: 22 women with diminished ovarian reserve and 22 women with normal ovarian reserve (NOR) were examined. We measured the gene expression of GREM1 and cumulus expansion-related transcripts PTX3, HAS2, PTGS2, and TNFAIP6 in granulosa cells from these 44 infertile women under 38 years old. The doses of gonadotropin used, the number of follicles and eggs, the fertilization rate and the rate of high quality embryos were recorded, and the pregnancy rate was followed up. In vitro, the KGN cells strains (n=3) were treated with follicle-stimulating hormone (FSH) and recombinant gremlin for 0, 4, 6 and 24 h to evaluate the effects of exogenous gremlin on the mRNA expression of PTX3, HAS2, PTGS2 and TNFAIP6. We next used a small molecule inhibitor to explore the involvement of TGF-β signaling pathway in gremlin-mediated cumulus expansion-related transcripts induction. SB431542 is a inhibitor of SMAD2/3 signaling pathway. We pretreated KGN cell strains (n=3) for 1 h with or without SB431542 followed by treatment with FSH and recombinant gremlin for 6h to detect the effect of SB431542 on the expression of PTX3, HAS2, PTGS2 and TNFAIP6.
Results: GREM1, PTX3, HAS2, PTGS2, and TNFAIP6 were down-regulated in women with DOR versus NOR (P<0.01). In vitro, Gremlin significantly (P<0.01) induced PTX3, HAS2, PTGS2 and TNFAIP6 mRNA expression, although the time course of induction varied for each gene. By six hours, gremlin significantly induced all genes. And SB431542 blocked gremlin-mediated HAS2 induction in all KGN cell strains tested (P<0.01).
Conclusion: Decreased GREM1 expression in granulose cell has an underlying defect in cumulus cell expansion, and resulted in diminished ovarian reserve.
PS198: Review: exosomes and diseases of female infertility
Sun Xiaoyan, The Reproductive Medicine Special Hospital of the First Hospital of Lanzhou University; Ma Xiaoling; Yang Xia; Xuehong Zhang
Background: Exosomes are small extracellular vesicles (EVs) (40-100nm) secreted by living cells and mediate the transmission of information between cells. The number of exosomes and their contents are associated with human diseases such as inflammatory diseases, cancer, metabolic diseases and infertility.
Results: This review focuses on the role of exosomes in oocyte development, declined ovarian function, PCOS, uterine diseases, endometrial receptivity and fallopian tube dysfunction in female. Moreover, some studies have reported that exosomes can be as a new and potencial therapeutic approach to treat female infertility.
Conclusion: However, the pathophysiological mechanism of exosomes in female infertility has not been entirely elucidated. Further researches should be conducted to explored the etiology and potential clinical treatment possibilities.
PS199: Comparison of the clinical efficacy between uhpFSH and uhpHMG in advanced age women used antagonist protocol in vitro fertilization-embryo transfer
Zhang Yini, Tongji Hospital; Lai Qiao’Hong; Jin Lei
Background: To explore the clinical efficacy of different gonadotropins in advanced age women patients (≥36y old) in the antagonist protocol of vitro fertilization-embryo transfer (IVF-ET).
Methods: Prospective analysis was performed on the advanced age infertility women patients who received the antagonist protocol treatment of IVF-ET with a total 85 cycles in our center. The patients were divided into high-purified urinary follicle stimulating hormone (uhpFSH) with 44 cycles and high-purified urinary human menopausal gonadotropin (uhpHMG) with 41 cycles. The ages, BMI, basic FSH level, AMH, dosage of Gn, days of Gn, average follicles, average obtained follicles, average embryos, clinical pregnancy rate and early abortion rate were compared in the two groups.
Results: There was no significant difference between the two groups with regard to the ages, BMI, basic FSH level, AMH, average follicles, average embryos, clinical pregnancy rate and early abortion rate (P>0.05). The average obtained follicles in group uFSH were significantly higher than those of the uHMG group (P<0.05). The dosage of Gn and the days of Gn in group uHMG were significantly less than those in the uFSH group (P<0.05).
Conclusion: In advanced age women with infertility used the antagonist protocol of IVF-ET, the dosage of uHMG and the days of uHMG were low and the average obtained follicles were higher in uFSH group than those in uHMG group. There was no significant difference in the clinical pregnancy rate and early abortion rate between the two groups.
PS201: Leydig cell tumor of testis leading to male infertility: a case report
Zhangshun Liu, Department of Reproductive Medicine, Shanghai Jiaotong University Affiliated Sixth People’s Hospital; Jiong Zhang; Changying Xing; Minfang Tao
Background: To report a case of male azoospermia caused by Leydig cell tumor of the testis, and to pay attention to the effect of Leydig cell tumor on male fertility.
Methods: A 30-year-old male patient with normal sexual life came to see a doctor because his wife had never been pregnant for 2 years without contraception. Three semen examinations revealed azoospermia. Semen volume and PH value were normal. Serum sex hormone test showed that FSH<0.3IU/L, LH<0.07IU/L, T 7.41nmol/L, E 2 144.94pmol/L, physical examination revealed a 2.5cm mass in the left testis. Ultrasound showed a solid mass of the left testis. Tumor resection with testicular preservation and ipsilateral testicular biopsy were performed.
Results: The pathological finding of the mass was Leydig cell tumor of the testis. The biopsy pathology showed that there were a few spermatogenic cells in the seminiferous tubules of the testis, but no spermatogenic function. One month after operation, serum sex hormone results showed that FSH 2.65IU/L, LH 2.37IU/L, T 7.41nmol/L, E2 110.1pmol/L. At 2 months after operation, the semen examination revealed the presence of sperm and the sperm concentration was 0.72*106/mL. At 3 months after operation, the sperm concentration was 16.48*106/mL. Six months after operation, the wife became pregnant.
Conclusion: Leydig cell tumors of the testis are rare, which can cause changes in endocrine hormones and inhibit testicular spermatogenesis. They are generally benign. Tumor resection with testicular preservation is feasible and spermatogenesis can be restored after operation.
PS202: Pregnancy and perinatal outcomes after frozen and fresh embryo transfer: a retrospective cohort study
Zhe Li, The Third Affiliated Hospital of Zhengzhou University; Lijun Sun; Lulu Wang
Background: Frozen embryo transfer (FET) has been progressively used during the past few decades and it becomes an essential part of assisted reproductive technology. Extra good quality embryos are preserved in FET for women with good response to ovarian stimulation. However, whether FET can improve pregnancy and perinatal outcomes remains inconclusive. Therefore, our objective was to investigate the impact of frozen and fresh embryo transfers on pregnancy and perinatal outcomes.
Methods: A retrospective cohort study was conducted at the reproductive center of The Third Affiliated Hospital of Zhengzhou University between July 2008 and September 2016, which included 8370 FET cycles and 8573 fresh cycles. Pregnancy and perinatal outcomes were assessed, including the rate of implantation, clinical pregnancy, abortion and the incidence of preterm delivery, live birth, ectopic pregnancy, pregnancy-induced hypertension (PIH), gestational diabetes mellitus (GDM), mean birth weight, low birth weight (LBW), macrosomia and congenital malformations.
Results: Significant differences were not observed in implantation rate, GDM and congenital malformation rate between FET group and fresh group (P>0.05). Clinical pregnancy rate [AOR (95% CI) 1.23 (1.14-1.28), P=0.000] and live birth rate [AOR (95% CI) 1.37 (1.26-1.49), P=0.000] were significantly elevated in the FET group compared to fresh group. Also, FET decreased incidences of ectopic pregnancy [AOR (95% CI) 0.71 (0.57-0.92), P=0.004] and LBW [AOR (95% CI) 0.83 (0.82-0.96), P=0.000]. Neonatal mean birth weight of FET group was higher than that of fresh group (3064.88±11.27, 2967.64±11.05, P=0.000). The incidences of preterm delivery [AOR (95% CI) 1.28 (1.16-1.50), P=0.000], abortion [AOR (95% CI) 1.11(1.02-1.25), P=0.005], PIH [AOR (95% CI) 1.63 (1.29-2.12), P=0.000] and macrosomia [AOR (95% CI) 2.03 (1.64-2.47), P=0.000] after FET were increased.
Conclusion: FET improved the rates of clinical pregnancy and live birth and decreased the risks of ectopic pregnancy and LBW. Meanwhile, FET can increase the incidences of preterm delivery, abortion, macrosomia and PIH. Hence, whether whole embryo freezing can replace fresh embryo transfer requires further study.
PS203: Chronological age-specific reference ranges for low anti-mÃ¼llerian hormone in women undergoing IVF/ICSI cycle
Depeng Zhao, Shenzhen Maternity and Child Healthcare Hospital; Xuemei Li
Background: Not much is known on the chronological age-specific definition of low anti-müllerian hormone (AMH) in women undergoing IVF/ICSI cycle. The purpose of this study was to establish a chronological age-specific reference range for low AMH and to evaluate the clinical outcome following IVF/ICSI treatment in patients with low AMH concentration.
Methods: All patients undergoing IVF/ICSI cycles at the Shenzhen maternity and child healthcare hospital from September 2015 to September 2018 were included in this study. Patients without AMH concentration available were excluded. The AMH concentrations were log-transformed. The multilevel modeling was used to estimate the age-specific reference ranges for AMH. An AMH concentration equal or less than the age-specific 10th centile was defined as low AMH concentration. The outcome following IVF/ICSI cycles in patients with low AMH concentration were further stratified by age younger than 35 years or older.
Results: A total of 934 patients with AMH concentration available were included in the present study. The linear regression model was chosen to estimate the relationship between female age and AMH concentration, yielding an age-specific equation for mean as follows: mean of LnAMH=(−0.085×age)+3.722. Sixty-four patient with low AMH concentration were identified, including 35 younger patients undergoing 71 cycles of oocyte retrieval and 29 older patients with 59 cycles of oocyte retrieval. The mean number of MII oocyte per cycle was significantly higher in the younger group compared to the older group, 1.8±2.2 vs. 1.2±1.7, respectively, P=0.03. The cycles of embryo transfer in younger and older groups were 24 and 9, respectively. The rate of clinical pregnancy tended to be higher in younger patients (42%, 10/24) than older patients (11%, 1/9), P=0.10.
Conclusion: We propose that the age-specific 10th centile be used to define low AMH concentration. The prognosis of Women with low AMH levels undergoing IVF/ICSI cycles is significantly affected by chronological age.
Funding: No funding.
PS204: Rosiglitazone promote endometrial angiogenesis via up-regulation of ANGPTL4 during implantation
Mingyang Li, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Jinwen Hu; Lihua Yao; Minzhi Gao
Background: Insufficient angiogenesis during pre-implantation affects endometrial receptivity (ER) leading to recurrent implantation failure (RIF). Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional secretory protein involved in regulating lipid metabolism and angiogenesis in various tissues. So we analyzed the role of ANGPTL4 in endometrial angiogenesis during implantation and its possible regulation mechanism.
Methods: Vaginal ultrasound monitors endometrial thickness and blood flow to the uterine artery. Peripheral blood and endometrial biopsies were obtained from infertile women on days 6 after ovulation in 18 recurrent implantation failure (RIF) women with high uterine artery blood flow resistance and 18 control women respectively. Different concentrations of rosiglitazone were added to observe the expression changes of ANGPTL4 and upstream peroxisome proliferator-activated receptor γ (PPARγ), which were analyzed by RT-PCR, Western-blot and ELISA. Cell proliferation assays, wound-healing assays and tube formation assay were performed to observe the effects of ANGPTL4 on proliferation, migration and angiogenesis of human endometrial stromal cells (hESC) and human umbilical vein endothelial cells (HUVECs).
Results: ANGPTL4 expression and secretion were decreased in endometrium and serum during peri-implantation period of RIF, compared with control. PPARγ and ANGPTL4 expression were up-regulated upon decidualization of hESC. Rosiglitazone promoted the expression and secretion of ANGPTL4 in hESC and HUVEC via PPARγ. Rosiglitazone can promote proliferation, migration and angiogenesis of hESC and HUVEC via ANGPTL4.
Conclusion: Recurrent implantation failure women with high blood flow resistance in uterine arteries had decreased expression of ANGPTL4 in endometrial tissue and serum. Rosiglitazone promotes angiogenesis by improving the expression of ANGPTL4 via PPARγ, which improves endometrial receptivity thus benefits embryo implantation.
Funding: This study was supported by grants from the National Natural Science Foundation of China (81300546) and was supported in part by grants from the Shanghai Commission of Science and Technology (17DZ2271100).
PS205: Dysregulation of collagen expression in peri-implantation endometrium of women with high ovarian response
Mingyang Li, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Lihua Yao; Meizhen Xin; Minzhi Gao
Background: To analyse the effects of high ovarian response on endometrial collagen synthesis and related gene expressionduring the peri-implantation period in patients undergoing in vitro fertilization-embryo transfer (IVF-ET).
Methods: Peripheral blood and endometrial biopsies were obtained from infertile women on days 6 after oocytes retrieval or ovulation in 16 stimulated cycles (SC) and 16 natural cycles (NC) respectively. Serum estrogen (E2), progesterone (P4), histological staging, endometrial collagen, matrix metalloproteinases (MMP2, 9) and tissue inhibitors of metalloproteinases (TIMP1, 3) were assayed.
Results: Serum levels of both E2 and P4 were significantly higher in the SC group than those in the NC group. All endometrial samples were in the secretory phase. The collagen in the stroma of the SC group was more dense and higher than that in the NC group. MMP2 and MMP9 were detected significantly lower in the SC group than those in the NC group, while TIMP1 and TIMP3 were significantly higher. MMP2, 9 expressions are increased by estrogen and reduced by progesterone in dose-dependent manner through estrogen receptor (ESR) and progesterone receptor (PR). Correspondingly, TIMP1, 3 expressions decreased by estrogen dose-dependently while progesterone played the opposite role.
Conclusion: High levels of P4 could stimulate excessive synthesis of collagen in peri-implantation endometrium of women with high ovarian response, and the mechanisms may be related to the decrease of MMP2, 9 and the increase of TIMP1, 3 through P4 receptor.
Funding: This study was supported by grants from the National Natural Science Foundation of China (81300546) and was supported in part by grants from the Shanghai Commission of Science and Technology (17DZ2271100).
PS206: Peroxiredoxin 4, a new oxidative stress marker in follicular fluid may predict IVF outcomes
Yi Qian, The First Affiliated Hospital of Nanjing Medical University; Yugui Cui; Jiayin Liu; Yan Meng
Background: Studies indicated that oxidative stress status in patients was closely associated with IVF outcomes, while the results are still controversial. Prexiredoxin 4 as one member in Prdx family, can catalyze the reduction of reactive oxygen species. While little data on the relationship of Prdx4 and female reproduction were demonstrated.
Methods: Our study is a prospective clinical study. All participants were recuited in the center of clinical reproductive medicine from September 2016 to December 2017. Infertile women with either tubal factor or male factor (n=256) undergoing controlled ovarian hyperstimulation and IVF were recruited in our study. Follicle fluid samples from patients were collected on the day of oocyte collection and then centrifuged and frozen up for analysis. Prdx4 concentration in FF were measured in each participant. Furthermore, the correlation between Prdx4 level and IVF outcomes, such as clinical pregnancy rate and oocyte quality was analyzed.
Results: Pregnant women had higher levels of Prdx4 in FF than non-pregnant women. Prdx4 levels were positively correlated with oocyte fertilization rates (r=0.326; P=0.013) and good quality embryo rates (r=0.334; P=0.011).
Conclusion: Our results provide evidence that the upregulated expression of antioxidants in IVF patients follicular fluid, such as Prdx4, tend to increase the potential pregnancy via oocyte quality mechanism.
Funding: The present work was supported by grants from the Science Foundation of Jiangsu Province (BK20161067) and the National Science Foundation (81701517).
PS207: The analysis on the pregnancy outcome of patients with poor ovarian response who received natural cycle/minimal stimulation
Yan Gao, The First Affiliated Hospital with Nanjing Medical University; Fei yang Diao; Yundong Mao; Jiayin Liu
Background: In China, the trend to delay childbirth increased for the past few years, and the age of mothers increased (especially the second-child-mothers), which is a challenging work for doctors of reproductive medicine. To investigate the clinical parameter of the poor-responder patients who received natural cycle/minimal stimulation protocol in IVF-ET/ICSI.
Methods: The patients whose antral follicle diameter of day 3 is less than 8mm, FSH is less than 16IU/L and estradial is less than 280 pmol/L take clomiphene citrate and/or HMG stimulation protocol, the others take natural cycle. When the follicular diameter is more than 15mm and estradial is about 1100 pmol/L, the patients receive 0.1mg GnRH-a injection at 20:30. We do the ovum pick-up after 34-36 hours and embryo transfer when the endometrial thickness reach 8-14mm otherwise the embryo is vitrificated.
Results: The pregnancy rate per cycle was about 30% and cumulative pregnancy rate was about 46%. The AFC and pregnancy rate gradually diminished with age, but there is no statistical change in basal FSH level, the oocyte retrieval rate and the fertilization rate.
Conclusion: The natural cycle/minimal stimulation protocol is suggest for poor-responder patients. Age is crucial for the successful of IVF/ICSI-ET treatment. Donated oocytes is the better choice for infertile women age older than 45 years.
PS208: HDAC2 mediated H3K27Ac is essential for gene expression turnover during LH surge
Jiamin Jin, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University; Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province; Yerong Ma; Weijie Yang; Yinli Zhang; Songying Zhang
Background: During the estrous cycle, the ovarian granulosa cells have a gene expression transition in response to the change from follicle stimulating hormone (FSH) to luteinizing hormone (LH). However, the specific mechanism is still elusive. Our study aimed to confirm that H3K27Ac is involved in LH-mediated gene expression changes and explore its mechanism of action.
Methods: The different protein expression levels of H3K27Ac were detected in ovaries of three weeks old C57BL/6 female mice through WB and IF, which were injected hCG 1-8h after PMSG 48h. In order to find out potential manipulators of H3K27Ac, the histone deacetylase HDAC1/2 inhibitor Romidepsin was injected into mice before hCG injection. Ovaries were collected for further detection of H3k27Ac and LH target gene expression through IF, WB and qPCR. At the same time, the expression level of H3K27Ac was detected in mouse granulosa cells cultured in vitro added with forskolin 0.5h-4h. Furthermore, Romidepsin’s effects on H3K27Ac expression and gene expression related with forskolin were also analyzed by WB and qPCR.
Results: We found that H3K27Ac protein expression showed a significant decrease at hCG 0h and 1h in mouse ovarian tissue and then gradually increased after hCG injection. IF of mouse ovarian tissue also confirmed that H3K27Ac expression was at lower places at PMSG 48h and 1h of hCG compared with PMSG 24h and hCG 4h. Futhermore, after injection of HDAC1/2 inhibitor Romidepsin before hCG injection, LH-related genes, such as AREG, EREG, btc, and PTX3 were significantly decreased compared with controls. Moreover, in the primary culture experiment of mouse ovarian granulosa cells, the expression of H3K27Ac decreased after 0.5 hours of forskolin addition, and then gradually increased. Romidepsin also disrupts original fluctuation of H3K27Ac and inhibits the enhancement expression of LH-related genes after forskolin was applied.
Conclusion: In summary, our experiments confirmed that H3K27Ac plays an important role in LH-mediated gene expression switching and H3K27Ac could be regulated by HDAC2.
PS209: Clinical observation on the addition of kidney-tonifying and yang-strengthening traditional Chinese Medicine herbs during follicular phase in the treatment of ovulatory-disordered infertility
Qianqian Luo, Nanjing University of Traditional Chinese Medicine; Yong Tan
Background: Observe the clinical efficacy and mechanism of supplementing kidney-tonifying and yang-strengthening TCM herbs in the middle and late follicular phase of ovulatory-disordered patients.
Methods: A total of 51 cases of infertility patients due to ovulatory disorder in the Reproductive Medicine Department of Jiangsu Province Hospital of TCM were selected from March 2017 to March 2018 and randomly divided into two groups. Both groups were treated by the combined treatments of traditional Chinese and Western medicine including CC and Gn and Ziyin Recipe. The distinction is the addition of kidney-tonifying and yang-strengthening TCM herbs in the middle and late of follicular phase in the experimental group. The differences in ovulation rate, LUFS rate, dominant follicle quality, follicle development rate, Gn dosage, follicle development duration, clinical pregnancy rate, early abortion rate were observed.
Results: The dosage of Gn and the time taken for follicular development were significantly lower in the experimental group (P<0.01). The rate of follicular development and E2 level of single dominant follicle on the trigger day were significantly higher than the control group (P<0.05). The rate of follicular development increased in both groups after treatment (P<0.01). In the experiment group, the ovulation rate was 79.4%, the LUFS rate was 14.7%, and the uninitiated cycle rate was 5.9%; while in the control group, the numbers were 59.3%, 30.5%, and 10.2% accordingly. The ovulation rate and LUFS rate were significant different between the two groups (P<0.05) while the uninitiated cycle rate was not (P>0.05). In the experimental group, clinical pregnancy rate was 46.2% and the early abortion rate was 8.3%. In the control group, clinical pregnancy rate was 26.1% and the early abortion rate was 33.3%. They all have no significant difference (>0.05).
Conclusion: Supplementing kidney-tonifying and yang-strengthening TCM herbs in the middle and late follicular phase for ovulatory-disordered patients can improve the rate of follicular development, shorten follicular phase, reduce the dosage of Gn, improve follicular quality, increase ovulation rate and also reduce LUFS rate. In conclusion, tonifying Yang and nourishing Yin in follicular phase have impacts on regulating the balance of Yin and Yang in body, and improving the overall outcome of follicular development.
PS210: The clinic effect of spermatozoa cryopreservation in assisted reproduction
Gao Zhi, Reproductive Medicine Research Center, the Sixth Affliated Hospital, Sun Yat-sen University; Guihua Liu; Yang Xing; Wei Na
Background: Spermatozoa cryopreservation is used to prevent cancellation of oocyte pick-up when sperm cannot be obtained and to prevent repeated testicular biopsies. However, there isn’t an consistency that whether testicular spermatozoa cryopreservation compromises clinical pregnancy outcomes in assisted reproduction. When frozen-thawed or fresh testicular sperm are both available, it is of great importance to determine which is preferable for intracytoplasmic sperm injection (ICSI). The aims of our study is to compare the clinical pregnancy outcomes of fresh or frozen-thawed testicular sperm collected by testicular sperm aspiration (TESA) in ICSI.
Methods: A retrospective study was conducted to compare the outcomes of couples who underwent the first ICSI cycle with fresh or frozen spermatozoa at our center between January 1, 2014, and December 31, 2017. To decrease the potential difference between different ovarian stimulation protocols, we included 347 couples who accepted gonadotropin releasing hormone (GnRH) agonist long protocol. The basic information, embryonic development and clinical outcomes were compared between the two groups.
Results: 232 cycles with the use of fresh testicular sperm, 115 cycles with frozen testicular sperm were included. The rates of fertilization significantly decrease with using frozen-thawed testicular sperm compared with the fresh group (76.2% vs. 69.7%).Furthermore, fresh testicular sperm were found to have better rates of good-quality embryos per oocyte than frozen testicular sperm (49.3% vs. 42.7%, respectively).
However, there were no significant differences in the rates of good-quality embryos (66.7% vs. 63.6%). And, no significant differences between the fresh testicular sperm group (n=114) and the frozen testicular sperm (n=52) who accepted fresh embryo transfer after oocyte extraction in the rates of clinical pregnancy (64.9% vs. 51.9%), and live delivery (54.4% vs. 46.2) (P>0.05 for all comparisons).
Conclusion: In case of GnRH agonist long protocol, no significant differences were found in clinical pregnancy outcomes between the frozen testicular sperm group and the fresh testicular sperm. Spermatozoa cryopreservation is a recommended method in assisted reproduction to avoid repeated testicular biopsies.
PS211: Reversible m6A RNA methylation: A new realm of post-transcriptional gene regulation in eukaryotes
Jianqi Lu; Xuehong Zhang
Background: N6-methyladenosine (m6A) is a modified base which has been known to be present in non-coding RNAs, ribosomal RNA, and at least one mammalian mRNA. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. However, our understanding of the prevalence m6A is present in a large subset of the transcriptome in specific regions of mRNA. This suggests that mRNA may undergo post-transcriptional methylation to regulate its fate and function. The identification and functional characterization of proteins that specifically recognize m6A unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. m6A and other mRNA modifications, including N1-methyladenosine(m1A) and pseudouridine form the epitranscriptome and collectively code a new pathway of information that controls protein synthesis.
N6-methyladenosine (m6A) is a modified base which has been known to be present in non-coding RNAs, ribosomal RNA, and at least one mammalian mRNA.
PS212: The role of GPER and GLUT1 in oocyte aging
Yi Zhou, Qingdao Women and Children’s Hospital; Lili Zang; Chunshuang Xu; Zhou Jiang; Zhen Peng; Lie Wang; Shuhua Zou
Background: Oocyte aging is the main cause of fertility difficulties in elderly women, but the mechanism of oocyte aging has not been fully elucidated. It has been reported that abnormal energy metabolism and low ATP (adenosine triphosphate) content can cause low mitochondrial enzyme activity and antioxidant stress in oocyte, leading to oocyte aging and loss of normal reproductive capacity. Studies suggest that estrogen can inhibit oocyte aging through membrane receptor GPER. Therefore, it is far-reaching significance to study the effects of estrogen and energy metabolism on oo