Melatonin regulates autophagy in granulosa cells from patients with premature ovarian insufficiency via activating Foxo3a.

Premature ovarian insufficiency (POI) is a diverse form of female infertility characterized by a decline in ovarian function before the age of 40. Melatonin (MT) is a potential clinical treatment for restoring or safeguarding ovarian function in POI. However, the specific therapeutic mechanism underlying this effect remains unclear. To address this, we conducted experiments using human granulosa cells (GCs) from both POI and normal patients. We examined the expression levels of autophagy-related genes and proteins in GCs through qRT-PCR and western blot analysis. Autophagy flux was monitored in GCs infected with GFP-LC3-adenovirus, and the regulatory function of MT in autophagy was investigated. Additionally, we employed pharmacological intervention of autophagy using 3-Methyladenine (3-MA) and RNA interference of Forkhead box O-3A (FOXO3A) to elucidate the mechanism of MT in the autophagy process. Compared to GCs from normal patients, GCs from POI patients exhibited irregular morphology, decreased proliferation, increased apoptosis, and elevated ROS levels. The expression of autophagy-related genes was downregulated in POI GCs, resulting in reduced autophagic activity. Furthermore, MT levels were decreased in POI GCs, but exogenous MT effectively activated autophagy. Mechanistically, melatonin treatment downregulated FOXO3A expression and induced phosphorylation in POI GCs. Importantly, silencing FOXO3A abolished the protective effect of melatonin on GCs. These findings indicate that autophagy is downregulated in POI GCs, accompanied by a deficiency in MT. Moreover, we demonstrated that supplementing MT can rescue autophagy levels and enhance GC viability through the activation of FOXO3A signaling. Thus, MT-FOXO3A may serve as a potential therapeutic target for POI treatment.

The influence of polycystic ovary syndrome on abortion rate after in vitro fertilization/intracytoplasmic sperm injection fresh cycle pregnancy.

There are many reports on clinical pregnancy outcomes in polycystic ovary syndrome (PCOS) patients receiving vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), but little research about abortion has been done and there is a debate on whether the abortion risk increases in PCOS patients receiving IVF/ICSI. Therefore, the aim of this study was to investigated the abortion in PCOS patients. Clinical data of 12055 IVF/ICSI fresh cycles performed in our hospital from January 2015 to December 2020 were collected. Based on the Rotterdam diagnostic criteria of PCOS and after propensity score matching (PSM) for baseline data of clinical pregnancy cycles, matched 599 PCOS (PCOS group) and Non-PCOS (non-PCOS group) cycles were obtained. Abortion and abortion-related outcomes were compared between the two groups. Risk factors for late abortion in twins were analyzed using binary Logistics regression. Post-PSM data showed that the late abortion rate was significantly higher in the PCOS group than in the non-PCOS group only in twin pregnancy (9.50% vs. 3.96%, OR: 2.55, 95%CI 1.10-5.89). There were no statistical differences in other pregnancy outcomes. The etiological distribution for late abortion were not statistically different between the two groups in both singletons and twins. Logistics regression indicated that PCOS and obesity [pregnancy-assisted body mass index (BMI) ≥ 28] were risk factors for late abortion in twin pregnancy. In twin pregnancy, PCOS and obese patients are more likely to have late abortion. In twin pregnancy, the late abortion risk significantly increased in the PCOS patients as compared with non-PCOS patients (OR: 2.59, 95%CI 1.11-6.03, P < 0.05), as well as in the patients with obesity (BMI ≥ 28) as compared with the patients with normal BMI (OR: 4.17, 95%CI 1.59-10.90, P < 0.05). PCOS does not significantly affect early and overall late abortion rates after IVF/ICSI fresh cycle pregnancy.

The Upregulation of HAS2-AS1 Relates to the Granulosa Cell Dysfunction by Repressing TGF-ß Signaling and Upregulating HAS2.

HAS2 antisense RNA 1 (HAS2-AS1) is a long noncoding RNA that has increased expression in mature granulosa cells (GCs) and contributes to cumulus expansion by regulating HAS2 expression. However, the roles of HAS2-AS1 during the pathological process of polycystic ovary syndrome (PCOS) are still unclear. This study investigated the roles of HAS2-AS1 in patients with PCOS. Here, a significant upregulation of HAS2-AS1 was found in the primary GCs from patients with PCOS, which was positively correlated with the level of the protein HAS2. The knockdown of HAS2 restored the upregulation of HAS2-AS1 in promoting migration but could not restore the effects of HAS2-AS1 overexpression in promoting proliferation and repressing apoptosis. Transforming growth factor ß (TGF-ß) upregulated HAS2-AS1 levels, while HAS2-AS1 functioned as a feedback inhibition factor repressing TGF-ß signaling by inhibiting TGF-ß receptor type 2 (TGFBR2) expression. HAS2-AS1 bonded with EZH2 and guided the polycomb complex 2 to the TGFBR2 promoter region. HAS2-AS1 overexpression induced H3K27 hypermethylation in the TGFBR2 promoter region and then repressed TGFBR2 transcription in KGN cells and primary GCs. In conclusion, we identified for the first time that HAS2-AS1 is upregulated in patients with PCOS and represses TGF-ß signaling via inducing TGFBR2 promoter region hypermethylation, which allowed us to explore the pathological processes of PCOS.

[Influencing Factors of Pregnancy Outcome of in vitro Fertilization-Embryo Transfer].

OBJECTIVE: To analyze the effect of factors relevant to blastocyst transfer on the pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ET). METHODS: The clinical data of 790 pregnant women who underwent IVF-ET in our hospital from July 2015 to July 2020 were retrospectively analyzed. The pregnancy outcome of blastocysts transferred on day 5 (D5, n=705) and those transferred on day 6 (D6, n=85) were compared. According to the pregnancy outcome, the cases were divided into a live birth group ( n=322) and a non-live birth group ( n=468), and multivariate logistic regression was conducted to study the effect of factors relevant to blastocyst transfer on the live birth outcome of IVF-ET. RESULTS: In the D5 group, the biochemical pregnancy rate, clinical pregnancy rate and live birth rate of blastocyst transfer were 69.93%, 64.96%, and 41.84%, respectively, which were significantly higher than those of the D6 group at 50.59%, 45.88%, and 30.59%, respectively. The difference was statistically significant ( P<0.05). There was no statistically significant difference in the miscarriage rate between the D5 group and the D6 group ( P>0.05). Multivariate logistic analysis revealed that age>35 years, years of infertility>5 years, endometrium thickness<9 mm on the day of blastocyst transfer, trophoblast cell rating of C, blastocyst transfer performed on D6, and multiparity were all risk factors for non-live birth outcome of IVF-ET ( P<0.05). CONCLUSION: The adverse pregnancy outcomes of IVF-ET were found to be associated with age, duration of infertility, endometrial thickness on the day of to blastocyst transfer, trophoblast cell rating, and blastocyst transfer performed after how many days of embryo development, and multiparity, which should be closely monitored, and effective measures should be adopted accordingly to prevent adverse outcomes of pregnancy.

MicroRNA-664a-3p inhibits the proliferation of ovarian granulosa cells in polycystic ovary syndrome and promotes apoptosis by targeting BCL2A1.

BACKGROUND: To investigate whether micro ribonucleic acid-664a-3p (miR-664a-3p) targeting BCL2A1 affects the proliferation and apoptosis of ovarian granulosa cells. METHODS: Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-664a-3p in granulosa cells, granular tumor cell lines (KGN), and normal ovarian epithelial cell lines (IOSE80) in the polycystic ovary syndrome (PCOS) group and the control group. After overexpressing miR-664a-3p or inhibiting its expression in KGN cells, qRT-PCR and Western blotting were used to detect the messenger RNA (mRNA) and protein levels of related genes. At the same time, a cell counting kit-8 (CCK-8) and flow cytometer were used to detect cell proliferation and apoptosis. The TargetScan website was used to predict the potential binding sites of miR-664a-3p and B-cell lymphoma 2-related protein A1 (BCL2A1), which was further verified by qRT-PCR, Western blotting, and the luciferase reporter gene method. RESULTS: The expression of miR-664a-3p was significantly decreased in both PCOS tissues and KGN cells (both P<0.05), and the overexpression of miR-664a-3p inhibited the proliferation of KGN cells and induced their apoptosis. Moreover, our results confirmed that miR-664a-3p directly targets BCL2A1 (P<0.05), and the inhibitory effect of miR-664a-3p on KGN cells was reversed by BCL2A1 overexpression (both P<0.05). The up-regulation of BCL2A1 promotes cell proliferation and reduces cell apoptosis by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (both P<0.05). CONCLUSIONS: The up-regulation of miR-664a-3p inhibits the proliferation of KGN cells and increases apoptosis by down-regulating the expression of BCL2A1 and blocking the MAPK/ERK signaling pathway.

Downregulation of DNA ligases in trophoblasts contributes to recurrent pregnancy loss through inducing DNA damages.

INTRODUCTION: As key components of DNA repair pathways, DNA ligases catalyze the formation of phosphodiester bonds between DNA single strands, which function as a "glue" to seal the DNA breaks. DNA ligases play important roles in almost all the normal physiological processes for maintaining the stability of genomic DNA, but their functions in recurrent pregnancy loss (RPL) are still unclear. METHODS: Immunoblotting was used to determine protein level. DNA damages were examined by comet assay and cell viability was quantified by MTT assay. The cell apoptosis and cell cycle were examined by flow cytometry. The LIG4 mRNA degradation was quantified by qRT-PCR after actinomycin D treatment. The interactions between miRNAs and LIG4 were predicted by TargetScan and confirmed by dual luciferase assay. RESULTS: LIG1 and LIG4 were downregulated in RPL patients, while γH2AX level was upregulated. Knockdown LIG1 and LIG4 increased DNA damages in trophoblasts, which further induced apoptosis and cell cycle arrest. Serine/arginine-rich splicing factor 1(SRSF1) was reduced in RPL patients and positively correlated with LIG1. Knockdown SRSF1 increased the degradation of LIG1 mRNA which further repressed LIG1 expression. MiR-383 was upregulated in RPL patients and repressed LIG4 expression through interacting with 3'UTR of LIG4 mRNA. The level of miR-383 was found negatively correlated with LIG4 protein level in trophoblasts from RPL patients. DISCUSSION: LIG1 and LIG4 are downregulated in patients with RPL owing to abnormal RNA degradation and dysregulated miRNA expression. LIG1 and LIG4 downregulation might contribute to the pathophysiological processes of RPL by increasing DNA damages.

Metformin and pioglitazone combination therapy ameliorate polycystic ovary syndrome through AMPK/PI3K/JNK pathway.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder, which results in health problems such as menstrual disorders, hyperandrogenism and persistent anovulation. Hyperandrogenism and insulin resistance are the basic characteristics of PCOS. To investigate the combined effect of metformin and pioglitazone on POCS and the potential mechanisms, a rat model of PCOS was established by intramuscular injection of estradiol valerate (EV). The effect of metformin and pioglitazone monotherapy or combination therapy in control rats and PCOS rats was evaluated, involving the testosterone level, follicular development and insulin resistance. The potential mechanism for the therapeutic effect of metformin and pioglitazone on POCS was explored through using three inhibitors of the 5'adenosine monophosphate-activated protein kinase (AMPK)/phosphoinositide-3 kinase (PI3K)/c-Jun N-terminal kinase (JNK) pathway (Compound C, Wortmannin and SP600125). The results showed that EV-induced PCOS rats demonstrated hyperandrogenemia, hyperinsulinemia and follicular dysplasia. Metformin or pioglitazone monotherapy significantly suppressed the high level of testosterone, reduced the raised percentage of cystic follicles and primary follicles, promoted the number of early antral follicles, and markedly decreased the high concentration of fasting insulin and homeostatic model assessment for insulin resistance index in PCOS rats. In addition, metformin and pioglitazone combination therapy demonstrated greater efficacy than its individual components. Furthermore, individual or joint treatment with metformin and pioglitazone affected the phosphorylation level of JNK in PCOS rats. Compound C and Wortmannin eliminated the effect of metformin and pioglitazone combination therapy on improving the follicular growth in PCOS rats, whereas SP600125 treatment enhanced this combination therapy effect. These data suggested that metformin and pioglitazone combination therapy demonstrated great efficacy in ameliorating PCOS through regulating the AMPK/PI3K/JNK pathway.

Effects of high progesterone on outcomes of in vitro fertilization-embryo transfer in patients with different ovarian responses.

The data of 3,841 cycles undergoing in vitro fertilization-embryo transfer (IVF-ET) in our reproductive Center between January 2003 and December 2013 were retrospectively analyzed. According to the number of oocytes retrieved, this study was divided into the high ovarian response group (oocyte retrieval≥20, 842 cycles), the moderate ovarian response group (50.05). The increased level of P on the day of hCG may affect the treatment outcomes of IVF-ET. The cut-off values of serum P seem to be associated with ovarian response. Increased ovarian response causes the cut-off values of serum P to rise.

Analysis of blastocyst culture of discarded embryos and its significance for establishing human embryonic stem cell lines.

In recent years, applications of stem cells have already involved in all domains of life science and biomedicine. People try to establish human embryonic stem cell lines (hESCs) in order to carry out hESC-related studies. In this study, we explored what embryos are conducive to the establishment of hESCs. The discarded embryos from in vitro fertilization-embryo transfer (IVF-ET) cycles were sequentially incubated into blastocysts, and then the inner cell mass (ICM) was isolated and incubated in the mixed feeder layer. The cell lines which underwent serial passage were identified. After a total of 1,725 discarded embryos from 754 patients were incubated, 448 blastocysts were formed with 123 high-quality blastocysts. The blastulation rate was significantly higher in the discarded embryos with non-pronucleus (0PN) or 1PN than in the discarded embryos with 2PN or ≥3PN. The blastulation rate of the D3 embryos with 7-9 blastomeres was higher. Among the originally incubated 389 ICMs, 22 hESCs with normal karyotype were established, and identified to be ESCs. Therefore, in establishing hESCs with discarded embryos, D(3) 0PN or 1PN embryos with 7-9 blastomeres should be first selected, because they can improve high-quality blastulation rate which can increase the efficiency of hESC establishment.

Establishment of polycystic ovary syndrome-derived human embryonic stem cell lines.

BACKGROUND: Recently, human embryonic stem cells (hESCs) of some genetic diseases have been established, but little research has been done on polycystic ovary syndrome (PCOS)-derived hESCs. The establishment of PCOS-derived hESCs provides a biological basis for exploring the pathogenesis, gene mapping and gene therapy of PCOS. METHODS: Discarded fresh embryos were collected and cultured until the blastocyst stage, and then inner cell masses (ICM) were isolated by mechanical methods and incubated in the mixed feeder layer containing human stem cell medium. hESCs were identified whether to maintain normal karyotype and pluripotency by alkaline phosphates (AKP), stage-specific embryonic antigen-4 (SSEA-4), NANOG, SOX2 and TRA-1-60, octamer binding protein 4(OCT-4), and in vivo and in vitro differentiation. RESULTS: Of the 11 passaged ICM, nine showed adherent growths within 48 h with an adherence rate of 81.8% (9/11). Five PCOS-derived hESCs were established and all of them have the characteristics of pluripotent differentiation. One was from 2PN embryo which was retarded in the cleavage stage, one was from 1PN embryo and others were from 0PN embryo. They were named p-hES-1, p-hES-2, p-hES-3, p-hES-4, p-hES-5, respectively. CONCLUSION: We provide biological models for studying the pathogenesiss of PCOS.

[Analysis of 1540 neonates with birth defects related to different assisted reproductive technique].

OBJECTIVE: To investigate the incidence of and clinical factors influencing neonatal birth defects from different assisted reproductive technology. METHODS: Between October 1998 and December 2006, 1271 newborns from mothers treated by in vitro fertilization techniques [including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and thaw embryo transfer (Thaw-ET)] matched with 269 newborns from mothers treated by artificial insemination were enrolled in Reproductive Medicine Center in First Hospital Affiliated to Zhengzhou University. Their medical information was analyzed retrospectively to compared neonatal characteristics, the incidence of birth defect and anomalous organs involved between in vitro fertilization group and artificial insemination group. RESULTS: In group of in vitro fertilization, those newborns with low birth weight from IVF, ICSI and Thaw-ET were 20.0% (134/671), 22.4% (92/410), 18.9% (36/190) respectively, which were more than 11.5% (31/269) cases in group of artifical semination with statistical significance (P < 0.05). The rates of multiple pregnancy of 23.8% (160/671), 25.4% (104/410), 21.1% (40/190) in subgroup of IVF, ICSI and Thaw-ET were significantly higher than 10.0% (27/269) in group of artifical insemination (P < 0.05). The rate of macrosomia in group of in vitro fertilization was significantly lower than that of artificial insemination group (3.9% vs 8.2%, P < 0.05). However, the incidence of birth defect involved in various organs did not show significant difference between two groups (P > 0.05). CONCLUSIONS: The incidence of multiple pregnancy demonstrated obviously increasing trends born with various In Vitro Fertilization techniques, which pave the way to high risk pregnancy. However, the incidence of newborn birth defect was not increased significantly. Thus, to lower occurrence of multiple pregnancy was the key approach to obtain neonates health.