Deconvolution at the single-cell level reveals ovarian cell-type-specific transcriptomic changes in PCOS.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine disorders in females of childbearing age. Various types of ovarian cells work together to maintain normal reproductive function, whose discordance often takes part in the development and progression of PCOS. Understanding the cellular heterogeneity and compositions of ovarian cells would provide insight into PCOS pathogenesis, but are, however, not well understood. Transcriptomic characterization of cells isolated from PCOS cases have been assessed using bulk RNA-seq but cells isolated contain a mixture of many ovarian cell types. METHODS: Here we utilized the reference scRNA-seq data from human adult ovaries to deconvolute and estimate cell proportions and dysfunction of ovarian cells in PCOS, by integrating various granulosa cells(GCs) transcriptomic data. RESULTS: We successfully defined 22 distinct cell clusters of human ovarian cells. Then after transcriptome integration, we obtained a gene expression matrix with 13,904 genes within 30 samples (15 control vs. 15 PCOS). Subsequent deconvolution analysis revealed decreased proportion of small antral GCs and increased proportion of KRT8high mural GCs, HTRA1high cumulus cells in PCOS, especially increased differentiation from small antral GCs to KRT8high mural GCs. For theca cells, the abundance of internal theca cells (TCs) and external TCs was both increased. Less TCF21high stroma cells (SCs) and more STARhigh SCs were observed. The proportions of NK cells and monocytes were decreased, and T cells occupied more in PCOS and communicated stronger with inTCs and exTCs. In the end, we predicted the candidate drugs which could be used to correct the proportion of ovarian cells in patients with PCOS. CONCLUSIONS: Taken together, this study provides insights into the molecular alterations and cellular compositions in PCOS ovarian tissue. The findings might contribute to our understanding of PCOS pathophysiology and offer resource for PCOS basic research.

Associations between vaginal microbiota and endometrial polypoid lesions in women of reproductive age: a cross-sectional study.

RESEARCH QUESTION: What are the different characteristics of vaginal microbial composition between patients with endometrial polypoid lesions and controls? DESIGN: This cohort study compared the pre-operative microbial compositions of vaginal samples in a cohort of 703 women with endometrial polypoid lesions [293 and 410 women diagnosed and not diagnosed with polyps pathologically (polyps group and not-polyps group, respectively] and 703 women in the control group. Bacterial abundance, diversity, differential taxa and microbial network structure were assessed using 16S rRNA gene sequencing. Predictive algorithms were used to determine the functional pathways of vaginal microbiota within the cohort. RESULTS: The control group exhibited higher relative abundance of Lactobacillus crispatus in comparison with the polypoid lesions group (P = 0.0427). Beta diversity of vaginal microbiota differed significantly between the groups (P < 0.05). Comparing the polyps group with the not-polyps group, Leptotrichia spp. and Cutibacterium spp. were more abundant in the polyps group, and Fannyhessea spp., Acinetobacter spp. and Achromobacter spp. were more abundant in the not-polyps group. The control group exhibited higher abundance of Bifidobacterium spp., Achromobacter spp. and Escherichia/Shigella spp. (false discovery rate < 0.05). Furthermore, the polyps group and not-polyps group displayed more complex co-occurrence networks compared with the control group. CONCLUSIONS: The results of this study provide compelling evidence supporting associations between vaginal microbiota and endometrial polypoid lesions, highlighting the potential relationship between a well-balanced vaginal microbial ecosystem and a healthy intrauterine environment.

Variations in mitochondrial DNA coding and D-loop region are associated with early embryonic development defects in infertile women.

Mitochondrial DNA (mtDNA) plays a critical role in oocyte maturation, fertilization, and early embryonic development. Defects in mtDNA may determine the alteration of the mitochondrial function, affecting cellular oxidative phosphorylation and ATP supply, leading to impaired oocyte maturation, abnormal fertilization, and low embryonic developmental potential, ultimately leading to female infertility. This case-control study was established to investigate the correlation between mtDNA variations and early embryonic development defects. Peripheral blood was collected for next-generation sequencing from women who suffered the repeated failures of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) cycles due to early embryonic development defects as well as in-house healthy controls, and the sequencing results were statistically analyzed for all subjects. This study found that infertile women with early embryonic development defects carried more mtDNA variants, especially in the D-loop region, ATP6 gene, and CYTB gene. By univariate logistic regression analysis, 16 mtDNA variants were associated with an increased risk of early embryonic development defects (OR > 1, p < 0.05). Furthermore, we identified 16 potentially pathogenic mtDNA variants only in infertile cases. The data proved that mtDNA variations were associated with early embryonic development defects in infertile Chinese women.

Novel variants in ACTL7A and PLCZ1 are associated with male infertility and total fertilization failure.

Total fertilization failure (TFF), which refers to fertilization failure in all mature oocytes, accounting for 5%-10% of in vitro fertilization (IVF) cycles and 1%-3% of intracytoplasmic sperm injection (ICSI) cycles in human. In this study, we recruited three unrelated primary infertile men with repeated cycles of TFF and performed whole-exome sequencing to identify the potential pathogenic variants. We identified homozygous or compound-heterozygous variants of paternal-effect genes ACTL7A and PLCZ1 that followed a Mendelian recessive inheritance pattern. Novel homozygous nonsense variant in ACTL7A [c.C146G: p.S49*] was identified in case 1, who came from a consanguineous family. Ultrastructural observation of ACTL7A-mutated spermatozoa by transmission electron microscopy (TEM) indicated that apparent increased thickness of perinuclear matrix and the acrosome was detached from the nuclear envelop. Besides, two novel compound-heterozygous variants in PLCZ1 were identified in case 2 [c.1174+3A>C:p.?; c.A1274G:p.N425S] and case 3 [c.136-1G>C:p.?; c.G1358A:p.G453D]. Mutated spermatozoa from case 2 with reduced expression of PLCZ1 showed apparent acrosome detachment by TEM analysis. And ICSI with assisted oocyte activation (ICSI-AOA) treatment can partly rescue the TFF. Taken together, our findings revealed that novel biallelic variants in the paternal-effect genes ACTL7A and PLCZ1 were associated with human TFF, which expanding the spectrum of genetic causes and facilitating the genetic diagnosis of male infertility with TFF.

Evidence of positive selection of genetic variants associated with PCOS.

STUDY QUESTION: Was polycystic ovary syndrome (PCOS), which impairs fertility and adheres to the evolutionary paradox, subject to evolutionary selection during ancestral times and did rapidly diminish in prevalence? SUMMARY ANSWER: This study strengthened the hypothesis that positive selection of genetic variants occurred and may account for the high prevalence of PCOS observed today. WHAT IS KNOWN ALREADY: PCOS is a complex endocrine disorder characterized by both reproductive and metabolic disturbances. As a heritable disease that impairs fertility, PCOS should diminish rapidly in prevalence; however, it is the most common cause of female subfertility globally. Few scientific genetic studies have attempted to provide evidence for the positive selection of gene variants underlying PCOS. STUDY DESIGN, SIZE, DURATION: We performed an evolutionary analysis of 2,504 individuals from 14 populations of the 1000 Genomes Project. PARTICIPANTS/MATERIALS, SETTING, METHODS: We tested the signature of positive selection for 37 single-nucleotide polymorphisms (SNPs) associated with PCOS in previous genome-wide association studies using six parameters of positive selection. MAIN RESULTS AND THE ROLE OF CHANCE: Analyzing the evolutionary indices together, there was obvious positive selection at the PCOS-related SNPs loci, especially within the original evolution window of humans, demonstrated by significant Tajima's D values. Compared to the genome background, six of the 37 SNPs in or close to five genes (DENN domain-containing protein 1A: DENND1A, chromosome 9 open reading frame 3: AOPEP, aminopeptidase O: THADA, diacylglycerol kinase iota: DGKI, and netrin receptor UNC5C: UNC5C) showed significant evidence of positive selection, among which DENND1A, AOPEP, and THADA represent the set of most established susceptibility genes for PCOS. LIMITATIONS, REASONS FOR CAUTION: First, only well-documented SNPs were selected from well-designed experiments. Second, it is difficult to determine which hypothesis of PCOS evolution is at play. After considering the most significant functions of these genes, we found that they had a wide variety of functions with no obvious association between them. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide additional evidence for the positive evolution of PCOS. Our analyses require confirmation in a larger study with more evolutionary indicators and larger data range. Further research to identify the roles of the DENND1A, AOPEP, THADA, DGKI, and UNC5C genes is also necessary. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2021YFC2700400 and 2021YFC2700701), Basic Science Center Program of NSFC (31988101), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), National Natural Science Foundation of China (82192874, 31871509, and 82071606), Shandong Provincial Key Research and Development Program (2020ZLYS02), Taishan Scholars Program of Shandong Province (ts20190988), and Fundamental Research Funds of Shandong University. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Deciphering the DNA methylome in women with PCOS diagnosed using the new international evidence-based guidelines.

STUDY QUESTION: Is there any methylome alteration in women with PCOS who were diagnosed using the new international evidence-based guidelines? SUMMARY ANSWER: A total of 264 differentially methylated probes (DMPs) and 53 differentially methylated regions (DMRs) were identified in patients with PCOS and healthy controls. WHAT IS KNOWN ALREADY: PCOS is a common endocrine disorder among women of reproductive age and polycystic ovarian morphology (PCOM) is one of the main features of the disease. Owing to the availability of more sensitive ultrasound machines, the traditional diagnosis of PCOM according to the Rotterdam criteria (≥12 antral follicles per ovary) is currently debated as there is a risk of overdiagnosis. The new international evidence-based guidelines set the threshold for PCOM as ≥20 antral follicles per ovary when using endovaginal ultrasound transducers with a frequency bandwidth that includes 8 MHz. However, current DNA methylation studies in PCOS are still based on the Rotterdam criteria. This study aimed to explore aberrant DNA methylation in patients diagnosed with PCOS according to the new evidence-based guidelines. STUDY DESIGN, SIZE, DURATION: This cross-sectional case-control study included 34 PCOS cases diagnosed using new international evidence-based guidelines and 36 controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 70 women, including 34 PCOS cases and 36 controls, were recruited. DNA extracted from whole blood samples of participants were profiled using array technology. Data quality control, preprocessing, annotation, and statistical analyses were performed. Least absolute shrinkage and selection operator (LASSO) regression were used to build a PCOS diagnosis model with DNA methylation sites. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 264 DMPs between PCOS cases and controls, which were mainly located in intergenic regions or gene bodies of the genome, CpG open sea sites, and heterochromatin of functional elements. Pathway enrichment analysis showed that DMPs were significantly enriched in biological processes involved in triglyceride regulation. Three of these DMPs overlapped with the PCOS susceptibility genes thyroid adenoma-associated protein (THADA), aminopeptidase O (AOPEP), and tripartite motif family-like protein 2 (TRIML2). Fifty-three DMRs were identified and their annotated genes were largely enriched in allograft rejection, thyroid hormone production, and peripheral downstream signaling effects. Two DMRs were closely related to the PCOS susceptibility genes, potassium voltage-gated channel subfamily A member 4 (KCNA4) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1). Finally, based on LASSO regression, we built a methylation marker model with high accuracy for PCOS diagnosis (AUC=0.952). LIMITATIONS, REASONS FOR CAUTION: The study cohort was single-center and the sample size was relatively limited. Further analyses with a larger number of participants are required. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to identify DNA methylation alterations in women with PCOS diagnosed using the new international evidence-based guideline, and it provided new molecular insight into the application of the new guidelines. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2021YFC2700400), Basic Science Center Program of NSFC (31988101), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), National Natural Science Foundation of China (32370916, 82071606, 82101707, 82192874, and 31871509), Shandong Provincial Key Research and Development Program (2020ZLYS02), Taishan Scholars Program of Shandong Province (ts20190988), and Fundamental Research Funds of Shandong University. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Comparative analysis of the vaginal microbiome of healthy and polycystic ovary syndrome women: a large cross-sectional study.

RESEARCH QUESTION: What are the different features of the vaginal microbiome (VMB) between patients with polycystic ovary syndrome (PCOS) and healthy women? DESIGN: A cross-sectional study was conducted at a single academic university-affiliated centre. A total of 1446 participants were recruited (PCOS group, n =713, control group, n = 733). Vaginal swabs were analysed using 16S rRNA gene sequencing. The diversity and composition of the microbiome were compared between the PCOS group and the control group. Microbial interaction networks and functional prediction were investigated. RESULTS: The PCOS group had a higher alpha diversity than the control group (Shannon P = 0.03, Simpson P = 0.02), and higher intra-group variability was observed in PCOS group (P < 2.2E-16). At the genus level, the proportion of Lactobacillus decreased (85.1% versus 89.3%, false discovery rate [FDR] = 0.02), whereas the proportion of Gardnerella vaginalis and Ureaplasma increased in the PCOS group (5.1% versus 3.3%, FDR = 0.006; 1.2% versus 0.6%, FDR = 0.002, respectively). Lactobacillus acidophilus, Prevotella buccalis and G. vaginalis were identified as the main differential species. L. acidophilus was positively correlated with serum levels of anti-Müllerian hormone (AMH), and triglyceride (P = 2.01E-05, P = 0.004, respectively). P. buccalis was negatively correlated with serum levels of AMH and testosterone (P = 0.002, P = 0.003, respectively). G. vaginalis was positively correlated with serum levels of AMH, oestradiol and progesterone (P = 0.004, P = 0.005, P = 0.03, respectively). The VMB interaction network indicated that Lactobacillus crispus, Prevotella timonensis, and P. buccalis could be key drivers in the PCOS group. Overall, 55 predicted genes were found to be differentially abundant between PCOS and the control (FDRs < 0.25). CONCLUSIONS: The PCOS group had a higher diversity of vaginal microbiome and showed an enhanced level of heterogeneity. The proportion of Lactobacillus in the PCOS group decreased, whereas the proportions of Gardnerella and Ureaplasma increased. These results warrant further research that can validate the correlation between PCOS and VMB.

The effect of different endometrial preparations on women with polycystic ovary syndrome undergoing initial frozen embryo transfer: A historical cohort analysis.

INTRODUCTION: Frozen embryo transfer is associated with a higher rate of live birth and a lower risk for ovarian hyperstimulation syndrome in women with polycystic ovary syndrome (PCOS) compared with fresh embryo transfer. The aim of this study is to assess the optimal endometrial preparation protocol for women with PCOS undergoing frozen embryo transfer. MATERIAL AND METHODS: We conducted a historical cohort analysis of 1720 women with PCOS who underwent the "freeze-all" strategy between August 2014 and August 2017 because of their high risk for ovarian hyperstimulation syndrome. Three endometrial preparation protocols were used: natural cycle (NC; n = 191), which relies on the dominant follicle to secrete estrogen that then promotes endometrial growth; ovarian stimulation (OS; n = 96), which induces follicle growth using low doses of human menopausal gonadotropin; and hormone replacement (HRT; n = 1433), which uses exogenous estradiol to promote endometrial growth. The primary outcome was live birth. RESULTS: For women who received a single embryo transfer, the live birth rates for the NC, OS, and HRT groups were 62.4%, 65.0%, and 52.2%, respectively. The live birth rate in the HRT group was significantly lower than that seen in the OS and NC groups (P = .009). The clinical pregnancy rates of the three groups were 72.3%, 73.8%, and 64.9%, respectively; this difference did not reach statistical significance (P = .071). CONCLUSIONS: The rate of live birth with the NC and OS regimens was higher than with the HRT protocol in women with PCOS who undergo single-blastocyst frozen embryo transfer.

Oxidative stress markers in the follicular fluid of patients with polycystic ovary syndrome correlate with a decrease in embryo quality.

PURPOSE: Elevated oxidative stress has been proposed as an important factor in the pathogenesis of polycystic ovary syndrome (PCOS)-related infertility. Our study was aimed at simultaneously exploring local and systemic oxidative stress in PCOS individuals and its relationship with embryo quality. METHODS: We recruited 86 PCOS cases and 60 controls. Five representative oxidative stress markers, namely, total oxidant capacity (TOC), total antioxidant capacity (TAC), malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD), were measured in both follicular fluid (FF) and serum. RESULTS: Women with PCOS compared to normal controls had higher levels of TOC in both FF (10.13 ± 2.68 vs.7.03 ± 2.45, P < 0.001) and serum (11.76 ± 2.92 vs. 8.82 ± 2.57, P < 0.001). The oxidative stress index (OSI, the ratio of TOC to TAC) was also higher in PCOS cases. They were still significant after BMI adjustment (Padj<0.01). In addition, the serum OSI level was much higher than the FF OSI level in both groups. Correlation analysis showed that the FF and serum TOC were negatively correlated with the high-quality embryo rate on day 3 and the later blastocyst formation rate in the PCOS group (P < 0.05). The correlation coefficient was higher in FF. Moreover, as the regression analysis data showed, the FF MDA level was significantly associated with embryo quality indicators (P < 0.05). CONCLUSIONS: PCOS was accompanied by elevated oxidative stress in both serum and FF. Even though serum oxidative stress was severe, the study suggested that FF oxidative stress contributed more to embryo quality, to which we should give more attention in the future.

Loss of miR-23b/27b/24-1 Cluster Impairs Glucose Tolerance via Glycolysis Pathway in Mice.

Alterations in miRNAs are associated with many metabolic disorders, such as type 2 diabetes (T2DM). The miR-23b/27b/24-1 cluster contains miR-23b, miR-27b, and miR-24-1, which are located within 881 bp on chromosome 9. Studies examining the roles of miR-23b, miR-27b, and miR-24-1 have demonstrated their multifaceted functions in variable metabolic disorders. However, their joint roles in metabolism in vivo remain elusive. To investigate this subject, we constructed miR-23b/27b/24-1 cluster knockout (KO) mice. Compared with wild-type (WT) mice, the KO mice exhibited impaired glucose tolerance, which was accompanied by a reduction in the respiratory exchange rate (RER). These alterations were more noticeable after a high-fat diet (HFD) induction. Hepatic metabolomic results showed decreased expression of reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide (NAD), phosphoenolpyruvic acid (PEP), and phosphoric acid, which are involved in the glycolysis pathway. The transcriptomic results indicated that genes involved in glycolysis showed a downregulation trend. qPCR and Western blot revealed that pyruvate kinase (PKLR), the key rate-limiting enzyme in glycolysis, was significantly reduced after the deletion of the miR-23b/27b/24-1 cluster. Together, these observations suggest that the miR-23b/27b/24-1 cluster is involved in the regulation of glucose homeostasis via the glycolysis pathway.

A Recurrent Missense Mutation in ZP3 Causes Empty Follicle Syndrome and Female Infertility.

Empty follicle syndrome (EFS) is defined as the failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization. Except for some cases caused by pharmacological or iatrogenic problems, the etiology of EFS remains enigmatic. In the present study, we describe a large family with a dominant inheritance pattern of female infertility characterized by recurrent EFS. Genome-wide linkage analyses and whole-exome sequencing revealed a paternally transmitted heterozygous missense mutation of c.400 G>A (p.Ala134Thr) in zona pellucida glycoprotein 3 (ZP3). The same mutation was identified in an unrelated EFS pedigree. Haplotype analysis revealed that the disease allele of these two families came from different origins. Furthermore, in a cohort of 21 cases of EFS, two were also found to have the ZP3 c.400 G>A mutation. Immunofluorescence and histological analysis indicated that the oocytes of the EFS female had degenerated and lacked the zona pellucida (ZP). ZP3 is a major component of the ZP filament. When mutant ZP3 was co-expressed with wild-type ZP3, the interaction between wild-type ZP3 and ZP2 was markedly decreased as a result of the binding of wild-type ZP3 and mutant ZP3, via dominant negative inhibition. As a result, the assembly of ZP was impeded and the communication between cumulus cells and the oocyte was prevented, resulting in oocyte degeneration. These results identified a genetic basis for EFS and oocyte degeneration and, moreover, might pave the way for genetic diagnosis of infertile females with this phenotype.

Loss of GM130 does not impair oocyte meiosis and embryo development in mice.

Golgi matrix protein 130 (GM130), encoded by GOLGA2, is the classical marker of the Golgi apparatus. It plays important roles in various mitotic events, such as interacting with importin-alpha and liberating spindle assembly factor TPX2 to regulate mitotic spindle formation. A previous study showed that in vitro knockdown of GM130 could regulate the meiotic spindle pole assembly. In the current study, we found that knockout (KO) mice progressively died, had a small body size and were completely infertile. Furthermore, we constructed an oocyte-specific GM130 knockout mouse model (GM130-ooKO) driven by Gdf9-Cre. Through breeding assays, we found that the GM130-ooKO mice showed similar fecundity as control mice. During superovulation assays, the KO and GM130-ooKO mice had comparable numbers of ovulated eggs, oocyte maturation rates and normal polar bodies, similar to the control groups. Thus, this study indicated that deletion of GM130 might have a limited impact on the maturation and morphology of oocytes. This might due to more than one golgin sharing the same function, with others compensating for the loss of GM130.

Transmission of polycystic ovary syndrome susceptibility single-nucleotide polymorphisms and their association with phenotype changes in offspring.

STUDY QUESTION: Does the inheritance of polycystic ovary syndrome (PCOS) susceptibility single-nucleotide polymorphism affect the phenotype of offspring? SUMMARY ANSWER: Male offspring who inherit PCOS-related genetic variations from PCOS mothers were more susceptible to developing the metabolic abnormality in their later life. WHAT IS KNOWN ALREADY: Genetic factors are considered the major etiology of PCOS. Previous studies have highlighted that offspring of women with PCOS had an increased risk of the same disease or PCOS-like symptoms. STUDY DESIGN, SIZE, DURATION: The study involved 172 children born to women with PCOS and 529 children born to non-PCOS women. All offspring were conceived by assisted reproductive technologies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The offspring ranged from 1 to 8 years old. Metabolic phenotype analyses were performed in offspring aged from 2 to 8 (N = 619). Sanger sequencing, TaqMan and Sequenom MassARRAY were used to sequence the samples. MAIN RESULTS AND THE ROLE OF CHANCE: In male offspring, the fasting insulin (FINS) (P = 0.037) homeostasis model assessment of insulin resistance (HOMA-IR) (P = 0.038) and the homeostasis model assessment of pancreatic beta-cell function (HOMA-ß) (P = 0.038) levels were higher in offspring of PCOS mothers compared to controls. In female offspring, PCOS offspring had a significantly higher anti-Müllerian hormone levels (P = 0.001) compared to those from control mothers. In male offspring of PCOS mothers, subjects with a T allele at rs2349415 in the gene FSHR had higher FINS (P = 0.023), HOMA-IR (P = 0.030) and HOMA-ß levels (P = 0.013) than those in the homozygous CC group. The same increased trend in FINS, HOMA-IR and HOMA-ß levels could be found in the CC and TC group in rs2268361 located in gene FSHR compared to the TT group (P = 0.029, P = 0.030, P = 0.046, respectively). As for rs10818854 in the DENND1A gene, the AA and AG group had a higher FINS (P = 0.037) and HOMA-ß (P = 0.008) levels than the homozygous CC group. LIMITATIONS, REASONS FOR CAUTION: Firstly, the offspring may be too young to see any phenotype changes. Secondly, this study only analyzed the differences of genotype frequency using the dominant model instead of all three models due to the limited sample size of the homozygous model. The results, therefore, should be replicated and performed in a larger sample size population. Thirdly, environmental impacts cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: The findings presented in this thesis add to our understanding the changes in offspring born to PCOS women and remind us to consider early intervention to avoid more severe effects. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China 2017YFC1001000 (to Z.-J.C.), the National Natural Science Foundation of China 81430029 (to Z.-J.C.), 81622021 and 31571548 (to H.Z.), the National Natural Science Foundation of Shandong Province JQ201816 (to H.Z.) and Shandong Provincial Key Research and Development Program 2017G006036 (to L.-L.C.) and 2018YFJH0504 (to Z.-J.C.). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Verification of a ZBTB16 variant in polycystic ovary syndrome patients.

RESEARCH QUESTION: This study investigated whether rs1784692 is a risk factor for polycystic ovary syndrome (PCOS) in Han Chinese women. DESIGN: A case-control study was conducted in Han Chinese women, involving 526 PCOS patients and 522 control participants. A TaqMan MGB probe assay was used to genotype the variant rs1784692. Dominant and additive models were employed for genotype-phenotype association analysis in the PCOS and control samples. RESULTS: The minor allele C of rs1784692 is protective against PCOS (odds ratio [OR] 0.556, 95% confidence interval [CI] 0.408-0.759, P = 1.83 × 10-4), even after adjustment for body mass index (BMI) and age (ORadj 0.539, 95% CI 0.391-0.743, Padj= 1.62 × 10-4). Genotype-phenotype analysis of the dominant model showed that mean BMI in the CC+CT group was higher than in the TT group in the PCOS group (27.12 ± 5.82 versus 24.57 ± 4.52, P = 1.0 × 10-3), but not in the control groups, indicating that the minor allele C of rs1784692 associates with BMI in women with PCOS. The mean LH (luteinizing hormone) concentration in the CC+CT group was lower than in the TT group in PCOS and control participants (9.33 ± 5.08 versus 10.93 ± 5.91, P = 0.036; 4.39 ± 1.66 versus 4.89 ± 2.07, P = 0.021). Genotype-phenotype analysis of additive model showed that mean BMI in TC group was higher than in the TT group in PCOS patients compared with control participants (27.14 ± 5.81 versus 24.57 ± 4.52, P = 3.06 × 10-3). CONCLUSIONS: The SNP rs1784692 in gene ZBTB16 is protective against PCOS but is associated with increased BMI in Han Chinese women with PCOS.

Live birth after a freeze-only strategy versus fresh embryo transfer in three randomized trials considering progesterone concentration.

RESEARCH QUESTION: Is there a difference in live birth rate between a freeze-only strategy and fresh embryo transfer, and what is the effect of varying progesterone concentrations on the day of human chorionic gonadotrophin (HCG) administration? DESIGN: A secondary analysis of data from three randomized trials comparing the live birth rate after elective frozen versus fresh embryo transfer, which respectively enrolled 1508 women with polycystic ovary syndrome, 2157 ovulatory women who underwent cleavage-stage embryo transfer and 1650 ovulatory women who underwent single blastocyst transfer. Women were randomly assigned to the frozen or fresh embryo transfer group in the original trials. The primary outcome was live birth rate after the initial embryo transfer. RESULTS: The live birth rate after a freeze-only strategy was consistently higher than fresh embryo transfer at any progesterone concentration on the day of HCG administration. Nonetheless, the between-group difference in live birth rate after frozen versus fresh embryo transfer was greater in women with progesterone concentrations ≥1.14 ng/ml (52.7% versus 37.3%, odds ratio (OR) 1.88, 95% confidence interval (CI) 1.55-2.27, P = 7.89 â€¯×  10-11) than in women with progesterone concentrations <1.14 ng/ml (53.3% versus 48.1%, OR 1.23, 95% CI 1.08-1.41, P = 0.002). In women with progesterone concentration ≥1.14 ng/ml, frozen embryo transfer also resulted in higher rates of conception and clinical pregnancy than fresh embryo transfer. CONCLUSION: In women with normal or high ovarian response, a freeze-only strategy resulted in a higher live birth rate than fresh embryo transfer, irrespective of progesterone concentration. Moreover, women with progesterone concentration ≥1.14 ng/ml may benefit more from a freeze-only strategy.

Mutations in MSH5 in primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is a genetically heterogeneous disorder that occurs in familial or sporadic fashion. Through whole exome sequencing in a Chinese pedigree with POI, we identified a novel homozygous missense mutation (ENST00000375755: c.1459G > T, p.D487Y) in the MSH5 gene in two sisters with POI. The homologous mutation in mice resulted in atrophic ovaries without oocytes, and in vitro functional study revealed that mutant MSH5 impaired DNA homologous recombination repair. From sanger sequencing of MSH5 in 200 sporadic POI patients, we identified three heterozygous mutations (ENST00000375755: c.1057C > A, p.L353M; c.1459G > T, p.D487Y and c.2107 A > G, p.I703V). Considering the heterozygous p.D487Y carrier in the POI pedigree was fertile, the causality of the three heterozygous mutations in POI need more evidence. Our studies confirmed that perturbation of genes involved in DNA damage repair could lead to non-syndromic POI. The underlying mechanism-inability to repair DNA damage-will receive increasing attention with respect to POI.

MicroRNA-200b and microRNA-200c are up-regulated in PCOS granulosa cell and inhibit KGN cell proliferation via targeting PTEN.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most common endocrine metabolic disorders characterized by hyperandrogenism, polycystic ovaries and ovulatory dysfunction. Several studies have reported that the aberrant expression of miRNAs contributes a lot to disordered folliculogenesis in PCOS, though the role and underlying mechanism of microRNA-200b (miR-200b) and microRNA-200c (miR-200c) in the development of PCOS remain unclear. METHODS: The expression of miR-200b in granulosa cells (GCs) derived from 90 PCOS patients and 70 controls was analyzed by using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Granulosa-like tumor cell line (KGN) was cultured for cell counting kit-8 (CCK-8) assays after over-expression of miR-200b, miR-200c or knockdown phosphatase and tensin homolog (PTEN). TargetScan was used to identify the potential targets of miR-200b and miR-200c, which was further verified by qRT-PCR, western blot and luciferase assays. RESULTS: Significantly increased expression of miR-200b was observed in PCOS patients compared with the controls. Moreover, over-expression of miR-200b and miR-200c inhibited the proliferation of KGN cells. In addition, our results verified that miR-200b and miR-200c directly targeted PTEN, knockdown of which suppressed KGN cells proliferation. CONCLUSION: Our findings demonstrate that miR-200b and miR-200c suppress the proliferation of KGN cells by targeting PTEN, and this might provide new evidence for abnormal proliferation of GCs in PCOS.

Risk factors associated with pre-eclampsia in pregnancies conceived by ART.

RESEARCH QUESTION: What are the factors associated with the increased incidence of pre-eclampsia in pregnancies conceived through IVF using autologous oocytes? DESIGN: A nested case-control study from the combined cohort of three multicentre randomized trials comparing fresh to frozen embryo transfer, including women who achieved clinical pregnancy after the first embryo transfer. Multivariable logistic regression was used to assess the effect of baseline characteristics, ovarian response parameters, type of fertilization, type of embryo transfer, and number of gestational sacs on the risk of pre-eclampsia. RESULTS: There were 2965 clinical pregnancies and 90 women were diagnosed with pre-eclampsia. Twin gestations (odds ratio [OR] 2.34, 95% confidence interval [CI] 1.50-3.66), mean arterial pressure (OR 1.04, 95% CI 1.01-1.07), frozen embryo transfer (OR 2.06, 95% CI 1.27-3.35), body mass index (BMI) (OR 1.10, 95% CI 1.02-1.18), progesterone level on the day of human chorionic gonadotrophin trigger (OR 1.53, 95% CI 1.07-2.20), and the total dose of gonadotrophin (OR 0.999, 95% CI 0.999-1.000, P = 0.037) were associated with the risk of pre-eclampsia. When the analysis was confined to women who underwent frozen embryo transfer, twin gestations (OR 2.44, 95% CI 1.43-4.18), BMI (OR 1.13, 95% CI 1.03-1.23) and the total dose of gonadotrophin (OR 0.999, 95% CI 0.999-1.000, P = 0.014) were still related to the risk of pre-eclampsia. The embryo stage at transfer was not included in the final models. CONCLUSIONS: Frozen embryo transfer was an independent risk factor of pre-eclampsia in assisted reproductive technology. The high ovarian response may also increase the risk of pre-eclampsia. The embryo stage at transfer was not related to the risk of pre-eclampsia.

Melatonin promotes human oocyte maturation and early embryo development by enhancing clathrin-mediated endocytosis.

Embryo development potential and reproductive clinical outcomes are all deeply rooted in oocyte maturation. Melatonin has been reported to promote oocyte maturation as an antioxidant in nonprimate species. Its antioxidative functions also help reduce plasma membrane rigidity, which facilitates clathrin-mediated endocytosis (CME). Whether melatonin has effects on human oocyte maturation by regulating CME is worthy of exploration. In this study, we found that the optimal melatonin concentration for human oocyte maturation was 10-11  M, and the maturation rate of this group was 71.9% (P = .03). The metaphase II (MII) stage oocytes obtained by in vitro maturation with 10-11  M melatonin had a significantly higher fertilization rate (81.4% vs 61.4%, respectively, P = .017) and blastocyst rate (32.2% vs 15.8%, respectively, P = .039) compared to controls. During maturation, antioxidative melatonin greatly enhanced CME and decreased intra-oocyte cAMP level. The former was evidenced by the increasing numbers of coated pits and vesicles, and the upregulated expression of two major CME markers-clathrin and adaptor protein-2 (AP2). CME inhibitor dynasore increased intra-oocyte cAMP level and blocked oocyte maturation, and melatonin could partly rescue oocyte maturation and significantly elevate the expression of clathrin and AP2 in the presence of dynasore. Therefore, we conclude that melatonin could promote human oocyte maturation and early embryo development through enhancing CME.

Variants in the 5'-UTR of APELA gene in women with preeclampsia.

OBJECTIVE: To detect APELA gene variants in clinical cases with preeclampsia (PE) and evaluate the influence of the APELA variants in gene expression. METHOD: A total of 288 individuals suffering from PE and 384 unaffected individuals were chosen for case-control studies. Genomic DNA was extracted from peripheral blood. Variants screening of APELA gene was conducted, and potential influence of variants in APELA expression was evaluated with a luciferase assay. RESULTS: Two rare variants (c.-306A > G and c.-145A > G) in the 5'-UTR of APELA gene were identified exclusively in PE affected individuals. Luciferase assays in HEK293 cells and HTR-8/SVneo cells revealed that both variants impaired transcriptional activity of APELA by altering the function of promoter region. Also, a single-nucleotide polymorphism (SNP) (c.159 T > C) in exon 2 of APELA was found in both cases and controls, and there was no statistically significant difference in genotype and allele frequency between cases and controls. CONCLUSION: Variants in the 5'-UTR of APELA gene may account for variability of APELA expression among individuals with PE and may play a negative regulatory role in the pathogenesis of PE.