The developments of in vitro fertilization-embryo transfer technology have witnessed significant advances since the first in vitro fertilization (IVF) baby was born in the 1970s. Although there are continuous improvements in the technology of embryo culture in vitro, there is still a lack of objective and accurate methods to judge the embryo quality as an important factor, thereby affecting the outcome of clinical pregnancy.[1,2]
In 2011, an expert meeting from Alpha and ESHRE resulted in a consensus paper on morphological criteria for embryo assessment. However, there is no general agreement on the quality classification of day 4 (D4) embryos in the frozen embryo transfer (FET) cycles. Because embryos were usually frozen on the third day, we routinely cultured embryos overnight for transfer on the fourth day during FET cycles. It was a great challenge to select the most viable and potential embryos to transfer for D4 embryos in reducing the number of transplanted embryos while improving the ability of embryo implantation. The objective of this study is to present a novel and practical grading system for D4 embryos which could be applicable in clinical practice conceived via IVF and intracytoplasmic sperm injection (ICSI) for predicting the implantation potential of embryos in FET cycles.
2 Material and methods
This study is a single-center retrospective cohort analysis, which includes the medical records of patients with infertility taking IVF–ICSI cycles and subsequent FET from January 2015 to October 2019. The inclusion criteria for patients were as follows: female age < 40 years old, first controlled ovarian stimulation cycle in lifetime. The ethics research committee at the first hospital of Lanzhou University approved the conduct of this study.
2.1 Study group
This study included patients who received embryo transfer on D4 of FET cycles. The day 3 embryos were thawed and cultured overnight. Then, 1 or 2 embryos were transferred selectively. Thereafter, we assessed whether all the embryos were implanted successfully or not. We excluded the cycles with partial implantation.
In total, 2255 patients with 3448 FET cycles were recruited for the final analysis. Table 1 shows baseline characteristics. There were 1256 cycles of single embryo transfer and 2192 cycles received 2 embryos transfer on D4. According to embryos compaction and different blastomere number ratio (BNR, number of embryo blastomere on day 4/ number of embryo blastomere on day 3, D4/D3), we calculated the implantation rates of different graded D4 embryos.
2.2 Ovarian stimulation
Patients underwent controlled ovarian stimulation in long or short protocols with GnRH agonists (Decapeptyl, Ferring, Germany) and recombinant follicle-stimulating hormone (FSH) (Puregon, Organon, the Netherlands). FSH dosage was adjusted according to the ovarian response, which was evaluated by the assessment of ultrasound and estrogen and progestational hormone levels. Oocyte maturation was induced with 5000 to 10,000 units of human chorionic gonadotropin ( Pregnyl, Saint-Prex, Switzerland). Follicular aspiration guided by ultrasound was retrieved 36 hours after the induction of ovulation.
2.3 Embryo culture
The choice of fertilization method depended on the infertility of substantial male factor. Fertilization status was evaluated at day 1 (18 hours after insemination) for the appearance of 2 polar bodies and 2 pronucleus. Embryos were then cultured with Vitrolife G-I plus Cleavage Medium (Vitrolife, Goteborg, Sweden) in a CO2 incubator (37°C, 6% CO2, and 5% O2). Embryonic development was assessed on day 3 graded with the Gardner method. All embryos were cultured to the D3 stage and vitrified by using the VitriFreeze Media Kit (Kitazato, Japan) and after thawed with VitriThaw Media Kit (Kitazato, Japan). All samples were stored in liquid nitrogen.
2.4 D4 embryo grading
We established a new grading system modified from the 2011 ESHRE Istanbul Consensus for D4 embryos in FET cycles. Usually, the compaction phenomenon took place during the fourth day of human embryo development. Embryos with complete compaction will be rated as grade A. Embryos with partial compaction will be classified as grade B. In our grading system, we divided embryos without compaction into grades C, D, and E according to the different BNRs (D4/D3). Embryos with BNR ≥1.5 were defined as grade C, embryos with BNR ≥1.2, <1.5 were considered as grade D, and while those with BNR ≥1.0, <1.2 were placed in grade E (Table 2). Two embryologists with at least 4 years working experience perform embryo grading at the same time.
2.5 Frozen cycle endometrial preparation
2.5.1 Artificial hormone replacement
The cycle started on day 3, in which the patients were orally administered with 2 to 6 mg of estradiol (Estrace; Allergan Pharmaceuticals) for endometrial preparation. An ultrasound endometrial assessment was conducted to assess the lining for its readiness for the ET procedure. The thickness of the endometrium was evaluated weekly until it was ≥7 mm thick. Based on the proposed day of thawing and transfer of the embryo, the progesterone suppository (200 mg, 3 times daily, tid) was vaginally administered to the women to start luteal support. The embryos were thawed on day 3 of progesterone and transferred overnight culture.
2.5.2 Natural cycles
After spontaneous menstruation, endometrial thickness and the follicular development of patients were monitored regularly by the serial ultrasound procedures. The levels of progesterone and luteinizing hormone (LH) were observed until the LH peak appeared corresponding to a day before oocyte ovulation (LH level exceeded 180% of baseline). On the second day, progesterone suppositories (200 mg tid) started to show their effects. The embryos were thawed on day 3 of progesterone administration and transferred after overnight culture.
2.5.3 Embryo transfer and confirmation of implantation
Based on the patient's age and anamneses, 1 or 2 embryos were thawed and cultured overnight for transplantation during the natural cycle or hormone replacement FET cycle. All ET procedures were performed under ultrasonic guidance by using specified standardized techniques. Clinical pregnancy was confirmed by ultrasound visualization of the gestational sac and the detection of fetal heartbeat under ultrasound at 7 weeks of pregnancy. The total number of fetal heart activity was equal to the total number of transplanted embryos, which was considered complete implantation. Monozygotic twins are excluded.
2.6 Statistical analysis
We performed statistical analysis by using the IBM Statistical Package for the Social Sciences (IBM SPSS v.22; IBM Corporation). Continuous variables were expressed as mean values with standard deviation and analyzed by Student t test. Categorical variables were compared by using a χ2 test. The area under the ROC curve (AUC) was used to test the predictive ability of the receiver operating characteristic (ROC) on the outcome of embryo implantation. A P value of less than .05 was considered statistically significant for this study.
3.1 Demographic data and cycle characteristics
Our study included 2255 controlled ovarian hyperstimulation cycles (1353 cycles in IVF group and 902 cycles in ICSI group). The baseline characteristics and demographics, including female age, anti-müllerian hormone level, E2 and P4 on trigger day, number of metaphase II oocytes retrieved, between the IVF and ICSI groups did not had a statistically significant difference (Table 1).
3.2 Implantation rates of the proposed model
In total, 5640 transferred embryos were retrospectively annotated into 5 grades (A–E) in FET cycles as described in Table 2. Table 3 shows the implantation rates of different embryo grades (embryo implantation rates of IVF and ICSI), whereas Table 4 shows the implantation rates of embryos according to age of female. Among IVF, ICSI, nonelderly and elderly groups, transferred embryos exhibited a similar declining trend in the implantation rates from the top grade to the lowest grade E (P < .01 for all groups). After converting the grades (from E to A) to numeric ranking (from 1 to 5), the ROC analysis showed that the proposed model predicted implantation outcomes in a degree in the IVF group (AUC = 0.65; 95% CI, 0.63–0.67; P < .01), ICSI group (AUC = 0.63; 95% CI, 0.60–0.65; P < .01), nonelderly group (AUC = 0.65; 95% CI, 0.63–0.67; P < .01), elderly group (AUC = 0.64; 95% CI, 0.61–0.67; P < .01), or a combination of all embryos (AUC = 0.65; 95% CI, 0.63–0.66; P < .01).
The primary objective of IVF is to select the best quality embryos for transfer to achieve a healthy single pregnancy. As increasingly global consensus has been achieved to avoid multiple pregnancies after IVF/ICSI–ET treatment, selective singleton ET, supported by improved embryo selection, has been promoted to maintain pregnancy rates while reducing the risk of multiple pregnancies.[5–7]
Li et al reported a consistent success rate between morula embryo transfer on D4 and blastocyst transfer on day 5 in fresh IVF/ET cycles. Additionally, early blastulation was a useful predictor in selecting the best embryo to achieve a higher pregnancy rate in fresh eSET cycles. Here, we proposed a simple embryonic morphological grading system that was developed on a dataset of 5640 embryos transferred on D4 in FET cycles. The fate of each embryo after transplantation (implanted and nonimplanted) was clearly known. The resulting grading system was based on total or partial compaction (grades 1 or 2, respectively) and BNR (D4/D3, grade 3, 4, or 5), modified from the 2011 ESHRE Istanbul Consensus for D4 embryos. Our grading system was feasible and operable in selecting the best quality of embryos on D4, which were classified from grade 1 to 5. The embryologist needed few minutes to observe embryos cultured on D4 in routine duties. The grading system provided by us can be easily applied to daily practice. However, using implantation as endpoint for the new day 4 embryo grading system was an inherent limitation of the present study. Another limitation of the study was that body mass index (BMI) data were not collected. BMI is inversely related to embryo quality and IVF outcome.[10,11] Furthermore, the interobserver and intraobserver variability in grading the embryos in the new day 4 embryo grading system were not determined.
There were several retrospective cohort studies at different reproductive medical centers conducted on patients who underwent in vitro fertilization with preimplantation genetic screening. Minasi et al showed that there appears to be a relationship between the polyploidy and blastocyst morphologies. Excellent- and fair-quality embryos had higher euploidy rates and a higher chance to result in an ongoing pregnancy. As described in the previous literature, embryo contraction, a physiological feature during blastulation, was conditioned by the ploidy status of the embryo. Additionally, the presence of contractions may compromise the implantation rates. Taken altogether, maternal age and some morphological parameters (expansion, time of blastocyst formation, and trophectoderm grade) were associated with euploidy, whereas other parameters, such as embryo sex and biopsy day, were not related to aneuploidy. There was a positive correlation between the speed of D4 embryo development and the time of blastocyst formation. Compaction embryos of D4 (grade 1) may have higher chromosome aneuploidy than other groups.
As compared with cleavage/blastocyst embryos transfer, there were several advantages of morula embryo transfer on D4. D4 embryos transplantation can effectively eliminate the phenomenon of embryos arrest in the cleavage stage.[16,17] It was well known that human embryos entered the uterine cavity on the fourth day after fertilization. To some extent, D4 embryos transfer was more natural than the cleavage embryos transfer of D3. In vitro culture time of the embryos transplanted on D4 was reduced as compared with that of the blastocysts. This reduction decreased the sensitivity of the disruption of epigenetic regulation mechanism.[18,19] Other research works have demonstrated that morula embryo transfer and blastocyst transfer had shown compatible pregnancy rates.
This study excluded the cycles with partial implantation. The embryo implantation results of our included study were clear and provided the personalized information for each embryo. Our data showed that transferred embryos exhibited a similar declining trend in implantation rates from the top grade to the lowest grade E. The implantation rates of grade B and E embryos in the IVF group were significantly higher than that in the ICSI group, thereby suggesting that ICSI fertilization may have a certain impact on embryo development potential. The implantation rates of grade A and grade B embryos in the nonelderly group were 56.15% and 44.39%, respectively. Thus, selective D4 single embryo transfer may be attempted in the nonelderly group in the future during the FET cycles.
In conclusion, our new grading system is a useful tool to assist in embryo selection via embryo morphological changes, and D4 embryo transfer provides a simple and applicable method for daily routine. However, the transferability of this model between laboratories with independent patient populations and large-sized prospective randomized controlled studies are still required to be studied in future research works.
The authors thank the clinical doctors, nursing team, and fellow embryologists at reproductive medicine center of the first Hospital of Lanzhou University for their ongoing support and technical assistance. And the author would like to thank Caixia Ma and Lei Xiao for English language editing.
The authors thank Di Cao at the First Clinical Medical College of Lanzhou University for revising the manuscript and performing the analysis with some constructive discussions.
Hongxing Li and Yuanxue Jing contributed to the conception of the study; Xiaojuan Xu and Hongxing Li contributed significantly to analysis and manuscript preparation; Hongxing Li and Lin Liu performed the data analyses and wrote the manuscript; Di Cao and Yiqing Wang helped perform the analysis with constructive discussions.
. Nasiri N, Eftekhari-Yazdi P. An overview of the available methods for morphological scoring of pre-implantation
embryos in in vitro fertilization. Cell J 2015;16:392–405.
. Fukui Y, Hirota Y, Matsuo M, et al. Uterine receptivity, embryo
attachment, and embryo
invasion: multistep processes in embryo implantation
. Reprod Med Biol 2019;18:234–40.
. Sanchez T, Seidler EA, Gardner DK, et al. Will noninvasive methods surpass invasive for assessing gametes and embryos? Fertil Steril 2017;108:730–7.
. Alpha Scientists in Reproductive Medicine, ESHRE, Special Interest Group of Embryology. The Istanbul consensus workshop on embryo
assessment: proceedings of an expert meeting. Hum Reprod 2011;26:1270–83.
. Tiitinen A. Single embryo
transfer: why and how to identify the embryo
with the best developmental potential. Best Pract Res Clin Endocrinol Metab 2019;33:77–88.
. Feng G, Zhang B, Zhou H, et al. Comparable clinical outcomes and live births after single vitrified-warmed and fresh blastocyst transfer. Reprod Biomed Online 2012;25:466–73.
. Munné S, Kaplan B, Frattarelli JL, et al. Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo
transfer in good-prognosis patients: a multicenter randomized clinical trial. Fertil Steril 2019;112:1071–9.
. Li RS, Hwu YM, Lee RK, et al. Day 4 good morula embryo
transfer provided compatible live birth rate with day 5 blastocyst embryo
in fresh IVF/ET cycles. Taiwan J Obstet Gynecol 2018;57:52–7.
. Hsieh CE, Lee RK, Sun FJ, et al. Early blastulation (EB) of day 4 embryo
is predictive of outcomes in single embryo
transfer (SET) cycles. Taiwan J Obstet Gynecol 2018;57:705–8.
. Carrell DT, Jones KP, Peterson CM, et al. Body mass index is inversely related to intrafollicular HCG concentrations, embryo
quality and IVF outcome. Reprod Biomed Online 2001;3:109–11.
. Garalejic E, Arsic B, Radakovic J, et al. A preliminary evaluation of influence of body mass index on in vitro fertilization outcome in non-obese endometriosis patients. BMC Womens Health 2017;17:112–20.
. Richardson A, Brearley S, Ahitan S, et al. A clinically useful simplified blastocyst grading system
. Reprod Biomed Online 2015;31:523–30.
. Minasi MG, Colasante A, Riccio T, et al. Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study. Hum Reprod 2016;31:2245–54.
. Barash OO, Ivani KA, Willman SP, et al. Association between growth dynamics, morphological parameters, the chromosomal status of the blastocysts, and clinical outcomes in IVF PGS cycles with single embryo
transfer. J Assist Reprod Genet 2017;34:1007–16.
. Viñals Gonzalez X, Odia R, Cawood S, et al. Contraction behaviour reduces embryo
competence in high-quality euploid blastocysts. J Assist Reprod Genet 2018;35:1509–17.
. Lee SH, Lee HS, Lim CK, et al. Comparison of the clinical outcomes of day 4 and 5 embryo
transfer cycles. Clin Exp Reprod Med 2013;40:122–5.
. Murphy BD. Under arrest: the embryo
in diapause. Dev Cell 2020;52:139–40.
. Kohda T. Effects of embryonic manipulation and epigenetics. J Hum Genet 2013;58:416–20.
. Chronopoulou E, Harper JC. IVF culture media: past, present and future. Hum Reprod Update 2015;21:39–55.
. Kang SM, Lee SW, Jeong HJ, et al. Clinical outcomes of elective single morula embryo
transfer versus elective single blastocyst embryo
transfer in IVF-ET. J Assist Reprod Genet 2012;29:423–8.