Introduction Pregnancy is a unique and complex physiological process that involves many events, including successful implantation, decidualization, placental formation, and parturition. The human placenta is a critical organ that grows in the uterus and controls fetal growth and viability, consequently controlling maternal health. Invasion of extravillous trophoblasts into the uterine decidua and myometrium occurs within the first 20 weeks of gestation.[ 1 ] Extravillous trophoblasts migrate and invade the uterine wall, resulting in remodeling of the maternal vasculature. Abnormal trophoblast function leads to serious pathological conditions, such as spontaneous miscarriage , preeclampsia, and intrauterine growth restriction.[ 2 ]
Acetylsalicylic acid, commonly knowns as aspirin , is a drug that is extensively used for various conditions; it possesses the ability to reduce fever and vascular hyperplasia, has anti-inflammatory activity, and can prevent platelet aggregation.[ 3 ] Low-dose aspirin is often considered useful to treat pregnancy-related complications, such as pregnancy complicated by antiphospholipid syndrome (APS), preeclampsia, and unexplained recurrent spontaneous miscarriage .[ 3-7 ] However, a large observational study of the use of aspirin in recurrent miscarriage showed that it did not improve pregnancy outcomes.[ 8,9 ]
During pregnancy, placental syncytiotrophoblasts and corpus luteum synthesize various hormones, including estrogen, progesterone , and human chorionic gonadotropin, all of which play important roles in maintaining normal pregnancy. Endocrine factors of progesterone deficiency may be one of the important causes of recurrent spontaneous miscarriage . Emerging evidence indicates that progesterone supplementation in early pregnancy can reduce the incidence of recurrent miscarriages.[ 10,11 ]
Despite these findings, there is no direct evidence for the possible effects of aspirin and progesterone on trophoblasts function. This study aimed to explore the effect of low-dose aspirin and progesterone on the proliferation , invasion , and apoptotic properties of trophoblasts in vitro. Furthermore, we sought to provide potential theoretical and experimental bases for the prevention of adverse pregnancy outcomes using low-dose of aspirin and progesterone .
Methods Cell culture and treatment The HTR-8/SVneo trophoblast cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (HyClone, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin solution (HyClone, USA). Cell culture was performed at 37°C in an atmosphere containing 5% CO2 .
The HTR-8/SVneo cells were cultured in a 6-well plate (5 × 105 cells/well) and treated with low-dose aspirin (0, 10−5 , 10−4 , 10−3 mol/L; MedChem Express) and progesterone (P4; 0, 10−8 , 10−7 , 10−6 mol/L; MedChem Express), respectively, and the control group was treated with 0.1% DMSO. HTR-8/SVneo cells in the aspirin -treated, progesterone -treated, or control groups were cultured in DMEM/F-12 containing 10% FBS and incubated in a humidified incubator with 5% CO2 at 37°C for 24 h.
Cell proliferation function test To test the cell proliferation ability, we used a cell counting kit-8 (CCK-8) (YEASEN, Beijing, China) according to the manufacturer's instructions. HTR-8/SVneo cells were digested and seeded onto 96-well plates at a density of 1 × 104 cells per well, with each well containing 100 μL medium. The cells were then treated with different concentrations of aspirin and progesterone . After 24 h, the cells were treated with 10 μL/well CCK-8 solution. The cells were then cultured for 3 h at 37 °C before measuring the absorbance at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
Matrigel migration assay Matrigel (BD Biosciences, USA) was diluted at a ratio of 1:8, and 35 μL was added to the Transwell upper chamber (8 μm, Corning, USA). The Transwell chambers were placed in a 24-well plate and incubated overnight at 4°C. Briefly, 200 μL (HTR-8/SVneo, 1 × 105 cells/well) DMEM/F-12 suspension with or without 10% FBS was added to the upper chamber, and 600 μL DMEM/F-12 containing 10% FBS was added to the lower chamber. According to different experimental requirements, cells pretreated with aspirin , progesterone , or vehicle were added in the upper chamber. The cells were cultured for 48 h at 37°C in a 5% CO2 incubator. The 24-well plate was removed, and the upper chamber medium and nonpenetrating cells were gently wiped off with a cotton swab, washed three times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and stained with crystal violet for 20 min. Thereafter, random photographs were acquired under an inverted microscope (×200), and 5 visual fields were counted in each chamber. The number of invaded cells was counted using Image J software (National Institute of Mental Health, USA).
Reverse transcription polymerase chain reaction Total RNA in HTR-8/SVneo cells was extracted (Trizol, TaKaRa, Japan) and reverse transcribed using PrimeScript™ RT Master Mix (RR036A, TaKaRa). Reverse transcription polymerase chain reaction (RT-PCR) was performed using the TB Green™ Premix Ex Taq™ II (RR820A, TaKaRa). Briefly, PT-PCR was conducted in a 10μL reaction system comprising 0.8 μL primers, 5 μL TB GreenPremix Ex TaqII (Tli RNaseH Plus) (2×), 0.2 μL ROX Reference Dye II (50×), 3 μL distilled water, and 1 μL complementary DNA sample (<100 ng). These measurements were performed in triplicate using an ABI Prism 7000 Sequence Detector (Applied Biosystems, Carlsbad, CA, USA). The primers (Shenggong Corp., Shanghai, China) are listed in Supplementary Table 1 .
Supplementary Table 1: Primer sequences of marker of proliferation Ki-67, matrix metalloproteinases 2, matrix metalloproteinase 9, ACTB and progesterone receptor
Hoechst staining HTR-8/SVneo cells were cultured in a 24-well plate (1 × 105 /well) and exposed to aspirin and progesterone for 24 h. Thereafter, the culture medium was removed, and the cells were washed with PBS. The cells were stained with a Hoechst Staining Kit (Solarbio Science and Technology Co., Ltd, Beijing, China) and observed under an Olympus BX51 fluorescence microscope (Tokyo, Japan), and the images were captured with a high-resolution DP70 Olympus digital camera (×200). The percentage of apoptotic cells was determined by observing nuclear morphology using ImageJ software. At least 1,000 cells were counted in each group, and the counter was blinded to the identities of the samples to avoid experimental bias.
Reactive oxygen species detectionReactive oxygen species (ROS) were detected in different groups (control, hydrogen peroxide (H2 O2 ), aspirin + H2 O2 , progesterone + H2 O2 , and aspirin + progesterone + H2 O2 ) using a 2,7-dichlorofluorescin diacetate (DCFH-DA) assay (Beyotime Institute of Biotechnology, Haimen, China). After 6 h of treatment with 250 μmol/L H2 O2 , the cells were treated with low-dose aspirin (10−4 mol/L) and progesterone (10−7 mol/L) for 24 h, followed by seeding onto the wells of 6-well plates. DCFH-DA (10 μmol/L) was added to each well. After incubation for 20 min at 37°C, the cells were rinsed with PBS and analyzed by flow cytometry. ROS levels were analyzed using FlowJo software (Treestar,USA), and the results were calculated relative to the control group.
Statistical analysis The results were representative of multiple experiments and were presented as the mean ± standard error of the mean (standard error of mean). The variables were analyzed by a Student's t -test between two groups or by one-way analysis of variance using Tukey's post hoc -test in multiple groups (STATA, version 15, StataCorp, USA). Differences were considered statistically significant at P < 0.05.
Results Aspirin and progesterone stimulate the proliferation of HTR-8/SVneo cellsTo explore whether aspirin and progesterone affect trophoblast proliferation , the proliferative capacity of HTR-8/SVneo cells was detected by CCK-8 assay after treatment with different concentrations of aspirin and progesterone for 24 h. Low-dose aspirin was used at a concentration equivalent to that detected in the serum of women receiving low-dose aspirin (75-150 mg/day; 10−5 -10−3 mol/L).[ 12 ] As shown in Figure 1a , the results showed that aspirin could promote trophoblast proliferation at a concentration of 10−4 mol/L, but not at 10−3 or 10−5 mol/L. The concentrations of progesterone that enhanced the proliferative ability of trophoblasts were 10−7 and 10−6 mol/L, and there was no significant difference between these two concentrations [Figure 1b ]. Next, we explored whether aspirin combined with progesterone was more effective in promoting the trophoblast proliferation . The results showed that low-dose aspirin (10−4 mol/L) combined with progesterone (10−7 mol/L) could also promote the proliferation of HTR-8/SVneo cells. However, low-dose aspirin and progesterone did not have a synergistic, as there was no significant difference between the combined treatment group and the groups treated with aspirin or progesterone alone [Figure 1c ].
Figure 1::
Aspirin and progesterone promote the proliferation of HTR-8/SVneo cells. (a-c) Effects of aspirin and/or progesterone on the proliferation of HTR-8/SVneo cells according to a CCK-8 assay (n = 6). (d-f) The effect of aspirin and/or progesterone on the expression of proliferation -associated gene MKI67 in HTR-8/SVneo cells was analyzed by RT-PCR (n = 6). The data are expressed as the mean ± SEM. *P < 0.05, †P < 0.01, ‡P < 0.001 compared with the control group; NS: No significance; P4: Progesterone ; SEM: Standard error of mean; RT-PCR: Reverse transcription polymerase chain reaction.
To verify the above results, we detected the expression of the proliferation -related gene marker of proliferation Ki-67 (MKI67 ) by RT-PCR. As expected, low-dose aspirin , progesterone , or a combination of aspirin and progesterone could upregulate MKI67 expression, but there was no significant difference in efficiency among the three groups [Figure 1d-1f ].
In summary, both low-dose aspirin and progesterone promoted the proliferation of trophoblasts in vitro , but combined aspirin and progesterone treatment did not appear to have a better effect. In subsequent experiments, we treated HTR-8/SVneo cells with aspirin (10−4 mol/L) and progesterone (10−7 mol/L) and measured their effects on other functions of trophoblasts .
Aspirin and progesterone enhance the invasiveness of HTR-8/SVneo cellsInvasion of extravillous trophoblasts into the endometrial stroma and inner third of the myometrium is critical for the development of definitive maternal-fetal circulation and for successful pregnancy in humans.[ 13,14 ] Therefore, we next explored the effects of low-dose aspirin and progesterone on trophoblast invasiveness through a Matrigel invasion assay. As shown in Figure 2a and 2b , low-dose aspirin and progesterone were able to promote the invasion of trophoblasts in the presence of FBS in the upper Transwell chambers, and there was no statistical difference between the groups using aspirin or progesterone alone and the combined treatment group. Furthermore, we detected the expression of matrix metalloproteinase 2 (MMP2 ) and matrix metalloproteinase 9 (MMP9 ) genes by RT-PCR, which play a critical role in regulating trophoblast invasion . The results showed that both aspirin and progesterone upregulated MMP2 and MMP9 expression [Figure 2c and 2d ]. Notably, low-dose aspirin combined with progesterone can also facilitate trophoblast invasion , but there was no statistically significant difference among the groups using the above two drugs alone or in combination [Figure 2a-2d ].
Figure 2::
Aspirin and progesterone promote the invasion of HTR-8/SVneo cells. (a and b) The invasion of HTR-8/SVneo cells in the control group (n = 6), aspirin group (n = 6), progesterone group (n = 6), and aspirin plus progesterone (n = 6) group was detected by Transwell assay in the presence of FBS in the upper Transwell chamber. (c and d) The effects of aspirin (n = 6), progesterone (n = 6), and aspirin plus progesterone (n = 6) on MMP2 and MMP9 expression in HTR-8/SVneo cells were analyzed by RT-PCR. Ctrl: control. The data are expressed as the mean ± SEM. *P < 0.05, †P < 0.01, ‡P < 0.001 compared with the control group; NS: No significance; SEM: Standard error of mean; RT-PCR: Reverse transcription polymerase chain reaction; FBS: Fetal bovine serum.
Aspirin and progesterone cooperatively inhibit the apoptosis of HTR-8/SVneo cellsPrevious studies have confirmed that excessive trophoblast apoptosis can be observed in the placentas of patients with pregnancy complications.[ 15,16 ] Therefore, using Hoechst staining as an index, we next explored the changes of apoptosis of trophoblasts after treatment with aspirin and progesterone . As shown, HTR-8/SVneo cells apoptosis decreased after treatment with low-dose aspirin and/or progesterone . Interestingly, low-dose aspirin combined with progesterone inhibited trophoblast apoptosis more significantly than either of the two compounds alone [Figure 3a and 3b ]. Therefore, we sought to determine which potential molecular mechanisms could lead to the observed results.
Figure 3::
Aspirin and progesterone cooperatively inhibit the apoptosis of HTR-8/SVneo cells. (a and b) The apoptosis of HTR-8/SVneo cells in the control group (n = 6), aspirin group (n = 6), progesterone group (n = 6), and aspirin plus progesterone group (n = 6) was detected via Hoechst staining. The apoptotic cell is indicated by the red triangle. The data are expressed as the mean ± SEM. *P < 0.001 compared with the control group; †P < 0.001 represented the comparison of the two groups at the end of the line; NS: No significance; SEM: Standard error of mean.
Aspirin and progesterone cooperatively upregulatePGR expression and reduce oxidative stress-mediated reactive oxygen species production and apoptosis in HTR-8/SVneo cells A previous study has shown that progesterone can induce progesterone receptorPGR ) expression.[ 17 ] Furthermore, progesterone has been shown to regulate apoptosis in cancer cells by modulating ROS levels.[ 18 ] Here, our data suggested that coapplication of low-dose aspirin and progesterone has better efficacy in inhibiting trophoblasts apoptosis compared with using aspirin or progesterone alone. Therefore, we speculated whether low-dose aspirin and progesterone could increase PGR expression and inhibit trophoblast apoptosis . Low-dose aspirin , progesterone , and the dual application of the above two drugs upregulated PGR expression in HTR-8/SVneo cells. Notably, the PGR expression was significantly higher in the low-dose aspirin and progesterone dual application groups than the other groups [Figure 4a ].
Figure 4::
Aspirin and progesterone may cooperatively upregulate PGR expression and inhibit oxidative stress-mediated apoptosis of HTR-8/SVneo cells. (a) The expression of PGR in the control group (n = 6), aspirin group (n = 6), progesterone group (n = 6), and aspirin plus progesterone group (n = 6) was detected by RT-PCR. (b) ROS levels in HTR-8/SVneo cells were detected by a 2,7-dichlorofluorescin diacetate assay. (c and d) Quantitation of the HTR-8/SVneo cells apoptosis based on Hoechst staining of nuclear morphology (n = 6). The apoptotic cell is indicated by the red triangle. The data are expressed as the mean ± SEM. *P < 0.05, †P < 0.01, ‡P < 0.001 compared with the control group; §P < 0.05, ||P < 0.01, ¶P < 0.001 represented the comparison of the two groups at the end of the line. NS: No significance; PGR : Progesterone receptor2 O2 : Hydrogen peroxide; ROS: Reactive oxygen.
Next, we established an oxidative injury model of HTR-8/SVneo cells induced by H2 O2 . Intracellular ROS levels were measured using the oxygen-sensitive fluorescent dye DCTH-DA. The accumulation of ROS in HTR8/SVneo cells was higher in the H2 O2 -treated group than in the control group [Figure 4b ]. However, a significant decrease in ROS formation in HTR8/SVneo cells was detected in the aspirin plus H2 O2 and progesterone plus H2 O2 group compared with the H2 O2 group. More notably, the low-dose aspirin and progesterone dual application group restricted the level of intracellular ROS in trophoblasts better than either compound alone [Figure 4b ].
Finally, we determined whether combined aspirin and progesterone treatment exerted protective effects against H2 O2 -mediated trophoblast apoptosis . Hoechst staining was used to detect the apoptotic status of cells in each treatment group. The apoptosis rate of trophoblasts in the H2 O2 group was higher than that in the control group [Figure 4c and 4d ]. After treatment with low-dose aspirin and/or progesterone , trophoblast apoptosis was significantly decreased compared with the H2 O2 group, especially in the aspirin and progesterone dual application group [Figure 4c and 4d ]. These results indicate that low-dose aspirin plus progesterone inhibits H2 O2 -induced apoptosis in trophoblasts .
Discussion Recurrent miscarriage , also known as spontaneous pregnancy loss, refers to the experience of at least two or three consecutive fetal deaths or spontaneous miscarriages before the 24th gestational week. While recurrent miscarriage has complex causes, including chromosomal abnormalities, anatomical defects, acquired infection, and endocrine disease, its cause remains unexplained in almost half of the patients.[ 19 ]
Aspirin , also known as acetylsalicylic acid, inhibits cyclooxygenase (COX), the first synthetase involved in the synthesis of prostaglandins, by irreversibly acetylating and inactivating the COX group of fatty acids. This inactivation inhibits the synthesis of PGI2 and TXA2.[ 20,21 ] Therefore, aspirin is widely used in clinical practice to inhibit platelet aggregation and prevent thrombosis. Clinically, low-dose aspirin is frequently used to prevent miscarriage . A previous study suggested that triple therapy with aspirin , prednisone, and multivitamins could improve pregnancy outcomes in unexplained recurrent spontaneous miscarriage .[ 22 ] However, a meta-analysis of randomized controlled trials of low-dose aspirin for treating APS-associated miscarriage and a large observational study that investigated its use in recurrent miscarriage have shown no improvement in pregnancy outcomes.[ 8,9 ] Here, we found that low-dose aspirin promoted the proliferation of cultured trophoblasts and inhibited trophoblast apoptosis in vitro . In addition, we found that low-dose aspirin , in the presence of FBS in the upper migration chambers, can also promote the invasion of trophoblasts . Moreover, the above results are consistence with the results of Matrigel migration experiments without adding FBS in the upper Transwell chambers (data not shown). These data suggest that aspirin and/or progesterone rather than FBS are responsible for cell invasiveness of trophoblasts in vitro culture system. In some malignancies, recent research has shown that aspirin regulates cell invasion and reduces the risk of cancer cell metastasis. Moreover, some mechanisms of cell invasion involve MMP2, MMP9, COX2, extracellular signal-regulated kinase, phosphatase, and Akt signaling.[ 23-25 ] We observed that MMP2 and MMP9 expression was increased after treatment with low-dose aspirin , but the specific regulatory mechanism involved needs further study in the future.
During normal pregnancy, the corpus luteum secretes progesterone to establish pregnancy, and the fetoplacental complex, including the endometrium, produces most of the progesterone needed to maintain pregnancy. If the human corpus luteum was removed before reaching 7 weeks of pregnancy, miscarriage would occur; thus, supplement with exogenous progesterone maintains pregnancy.[ 26 ] Progesterone is therefore one of the most important hormones to maintain early pregnancy. Clinically, progesterone is recommended to prevent spontaneous miscarriage . Boza et al . conducted a prospective study to evaluate the effect of progestogen on the pregnancy outcome of threatened miscarriage .[ 27 ] Unexpectedly, although progestogen supplementation led to the increased levels of serum progesterone , this finding did not translate to its beneficial effect on pregnancy outcomes in cases of threatened miscarriage .[ 27 ] Our study observed that trophoblast proliferation and invasion were significantly improved and trophoblast apoptosis was inhibited after treatment with progesterone . Interestingly, the dual application of low-dose aspirin and progesterone showed a more powerful inhibitory effect on trophoblast apoptosis , but there was no significant difference in trophoblast proliferation and invasion compared with the groups treated with aspirin or progesterone alone.
Oxidative stress refers to a pathological state in which the production rate of ROS is greater than the clearance rate. This process results in cell and tissue damage due to an imbalance of the intracellular oxidation and antioxidation systems. Oxidative damage mainly includes lipid peroxidation of biofilms, damage to intracellular proteins, and enzyme-denatured DNA, which ultimately leads to cell death, apoptosis , and disease.[ 28 ] During normal pregnancy, the systems of oxidation and antioxidation maintain a relative balance at the maternal-fetal interface. As placental ischemia and hypoxia occur, trophoblasts produce large amounts of ROS, which exceed the compensation ability of the antioxidation system mentioned above, and cause placental dysfunction.[ 29 ]
Oxidative stress plays an important role in the pathogenesis of pregnancy-associated diseases, such as miscarriage and preeclampsia.[ 30 ] H2 O2 , a key factor in the cellular oxidative stress cascade, has also been reported as an important component of oxidative ischemia/reperfusion stress in the placenta.[ 31 ] Aspirin is a nonsteroidal anti-inflammatory drug that effectively inhibits oxidative stress. Progesterone induction has also been reported to inhibit the relative activity of mitochondria and reduce ROS production.[ 32 ] Our research indicates that low-dose aspirin and progesterone can decrease oxidative stress-induced apoptosis in trophoblasts in vitro , although the mechanism by which this occurs remains to be further clarified.
Many of the functions of aspirin have been associated with the inhibition of ROS levels, and current studies have shown that aspirin can inhibit ROS levels in cells. Moreover, it is widely accepted that aspirin can inhibit COX expression. A previous study reported that aspirin inhibited ROS generation by inhibiting COX-1 and COX-2 expression, especially that of COX-2.[ 33-35 ] A similar effect was observed in trophoblasts , and aspirin also regulates COX expression in trophoblasts .[ 36 ] He et al . reported that storkhead-box protein 1 (STOX1) augmented trophoblast apoptosis by upregulating the expression of COX2, while aspirin restricted the expression of STOX1 and reversed the apoptosis of STOX1-induced trophoblasts .[ 37 ] It is not clear whether progesterone can decrease ROS accumulation by inhibiting COX expression, but progesterone can regulate COX expression and activity in other cells.[ 38 ] Interestingly, COX-2 expression has been shown to be decreased by the action of ROS scavengers.[ 39 ] While more data are clearly necessary to support our hypothesis, we speculate that low-dose aspirin and progesterone , similar to other ROS scavengers, may inhibit the production or accumulation of ROS by regulating the activity of COX in trophoblasts .
Notably, our results showed that low-dose aspirin combined with progesterone had a stronger inhibitory effect on trophoblast apoptosis than either two drugs alone. Previous studies suggest that progesterone can induce PGR expression, which plays a critical role in regulating apoptosis in other cells through the modulation of ROS.[ 17,18,32 ] Our result also showed that progesterone could promote PGR expression in trophoblasts in a concentration dependent manner (data not shown). Therefore, we propose that low-dose aspirin and progesterone may cooperatively inhibit oxidative stress-mediated cell apoptosis of HTR-8/SVneo cells in a PGR -dependent or -independent manner. Further analysis showed that the PGR expression was upregulated in trophoblasts after treatment with low-dose aspirin and/or progesterone , especially in the dual application group. H2 O2 treatment resulted in abundant ROS accumulation in trophoblasts , and low-dose aspirin plus progesterone clearly decreased the levels of ROS and oxidative stress induced by H2 O2 . Therefore, it can be speculated that low-dose aspirin combined with progesterone inhibits the apoptosis of trophoblasts more effectively than either aspirin and progesterone alone. Furthermore, this process may be dependent on the PGR -mediated suppression of ROS production, and more studies are needed to confirm this hypothesis.
In conclusion, as shown in Figure 5 , we propose that low-dose aspirin and progesterone promote the proliferation and invasion of trophoblasts and cooperatively reduce the apoptosis of trophoblasts in vitro . Interestingly, the effect of low-dose aspirin and progesterone dual therapy in inhibiting trophoblast apoptosis was more significant than either compound alone. The specific mechanisms of low-dose aspirin and progesterone in modulating trophoblast function remain unclear. These results suggest that aspirin and progesterone can effectively improve the adverse outcomes associated with pathological pregnancy; therefore, more clinical studies are needed in the future to elucidate their effects and specific mechanisms.
Figure 5::
Protective effect of aspirin and progesterone on trophoblasts . Low-dose aspirin and progesterone promote the proliferation and invasion of trophoblasts and cooperatively inhibit their apoptosis . Oxidative damage could lead to cell apoptosis , and H2 O2 , a key factor in the cellular oxidative stress cascade, and is also reportedly an important component of oxidative stress in the placenta. Excessive ROS accumulation induces trophoblast apoptosis under states of oxidative stress. Low-dose aspirin and progesterone dual therapy may inhibit H2 O2 -induced apoptosis in trophoblasts by upregulating PGR expression. The dotted line indicates that the specific mechanisms are not yet understood. ROS: Reactive oxygen species ; PGR : Progesterone receptor2 O2 : Hydrogen peroxide.
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
Financial support and sponsorship This study was supported by the National Natural Science Foundation of China (No. 31970798, 31671200, 82072872), the Innovation-oriented Science and Technology Grant from NPFPC Key Laboratory of Reproduction Regulation (CX2017-2), the Program for Zhouxue of Fudan University (JIF157602), and the Support Project for Original Personalized Research of Fudan University.
Conflicts of interest There are no conflicts of interest.
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