Metabolic profiles of children aged 2-5 years born after frozen and fresh embryo transfer: A Chinese cohort study.

BACKGROUND: Frozen embryo transfer (FET) has become a widely employed assisted reproductive technology technique. There have historically been concerns regarding the long-term metabolic safety of FET technology in offspring due to pregnancy-induced hypertension and large for gestational age, both of which are well-recognized factors for metabolic dysfunction of children. Therefore, we aimed to compare the metabolic profiles of children born after frozen versus fresh embryo transfer at 2 to 5 years of age. METHODS AND FINDINGS: This was a prospective cohort study. Using data from the "Assisted Reproductive Technology borned KIDs (ARTKID)," a birth cohort of offspring born from assisted reproductive technology at the Institute of Women, Children and Reproductive Health, Shandong University, China. We included 4,246 singletons born after FET (n = 2,181) and fresh embryo transfer (n = 2,065) enrolled between 2008 and 2019 and assessed the glucose and lipid variables until the age of 2 to 5 years. During a mean follow-up of 3.6 years, no significant differences were observed in fasting blood glucose, fasting insulin, Homeostatic Model Assessment of Insulin Resistance Index, total cholesterol, triglycerides, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol levels between offspring conceived by fresh and frozen embryo transfer in the crude model and adjusted model (adjusted for parental age, parental body mass index, parental education level, paternal smoking, parity, offspring age and sex). These results remained consistent across subgroup analyses considering offspring age, the stage of embryo transfer, and the mode of fertilization. Results from sensitivity analysis on children matched for age within the cohort remains the same. The main limitation of our study is the young age of the offspring. CONCLUSIONS: In this study, the impact of FET on glucose and lipid profiles during early childhood was comparable to fresh embryo transfer. Long-term studies are needed to evaluate the metabolic health of offspring born after FET.

Metabolic Profiles of Offspring Born From Biopsied Embryos from Toddlerhood to Preschool Age.

CONTEXT: Embryo biopsy, which is necessary for preimplantation genetic testing (PGT), has not been fully investigated regarding its potential influences and safety. Previous studies of children born from biopsied embryos (PGT children) have primarily centered around their growth and neuropsychological development, while there remains limited knowledge concerning their endocrine and metabolic parameters. OBJECTIVE: This study aims to examine the effect of trophectoderm (TE) biopsy on metabolic outcomes for PGT children. METHODS: A total of 1267 children from the Center for Reproductive Medicine, Shandong University, who were conceived through in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) with and without PGT, were analyzed in this study. Three sets of measurements pertaining to growth and metabolism were taken at each predetermined follow-up time point. The linear regression models within a generalized estimating equation were employed to examine the associations between the PGT and each outcome measure and the approach of false discovery rate was used to correct for multiple comparisons. RESULTS: After controlling for confounding factors and correcting for multiple comparisons, no statistically significant difference was identified in any of the measured variables between the PGT children and children conceived by IVF alone (IVF children) and children conceived through IVF using ICSI (ICSI children). The same is true also for age- or sex-based subgroup analyses. CONCLUSION: Between the ages of 1 and 5 years, there are no clinically adverse metabolic outcomes observed in PGT children, and their metabolic profiles are essentially identical to those of IVF children and ICSI children.

Association between endometrial thickness and birthweight of singletons from vitrified-warmed cycles: a retrospective cohort study.

RESEARCH QUESTION: What is the association between endometrial thickness (EMT) and the birthweight of singleton infants born from frozen-thawed embryo transfer cycles? DESIGN: This retrospective cohort study was conducted from January 2016 to December 2019. Participants were categorized into a natural cycle (NC, n = 8132) group and hormone replacement therapy (HRT, n = 4975) group. Only singleton deliveries were included. The primary outcomes were measures of birthweight and relevant indexes. Multivariable logistic regression and multivariable-adjusted linear regression models that incorporated restricted cubic splines were used. RESULTS: In the HRT group, the risk of delivering a small for gestational age (SGA) infant was increased in women with an EMT <8.0 mm (adjusted odds ratio [aOR] 1.85, 95% confidence interval [CI] 1.17-2.91) compared with women with an EMT of 8.0 to <12.0 mm, and increased with an EMT ≥12.0 mm (aOR 1.85, 95% CI 1.03-3.33). An inverted U-shaped relationship was found between EMT and birthweight in women with HRT. No significant differences were shown in birthweight z-score, or being SGA or large for gestational age, in singletons among the three EMT groups in the natural cycles. CONCLUSIONS: A thinner endometrium seen in women undergoing HRT cycles was associated with a lower birthweight z-score, as well as a higher risk of SGA. However, no significant association was observed between EMT and birthweight z-score or SGA in the NC group. It is noteworthy that a thicker endometrium was not associated with a higher birthweight in frozen-thawed embryo transfer (FET) cycles. Women with a thin endometrium who achieve pregnancy require specialized attention, particularly if they are undergoing FET with HRT cycles.

DENND1A desensitizes granulosa cells to FSH by arresting intracellular FSHR transportation.

Polycystic ovary syndrome (PCOS) is a complex disorder. Genome-wide association studies (GWAS) have identified several genes associated with this condition, including DENND1A. DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport. However, the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood. In this study, we investigated DENND1A expression in ovarian granulosa cells (GCs) from PCOS patients and its correlation with hormones. Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases, which was positively correlated with testosterone levels. To further explore the functional implications of DENND1A, we generated a transgenic mouse model overexpressing Dennd1a (TG mice). These TG mice exhibited subfertility, irregular estrous cycles, and increased testosterone production following PMSG stimulation. Additionally, the TG mice displayed diminished responsiveness to FSH, characterized by smaller ovary size, less well-developed follicles, and abnormal expressions of FSH-priming genes. Mechanistically, we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor (FSHR), promoting its internalization and inhibiting recycling. These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms, thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.

Excessive Exogenous Gonadotropins and Genetic and Pregnancy Outcomes After Euploidy Embryo Transfer: A Secondary Analysis of a Randomized Clinical Trial.

Importance: The safety of exogenous gonadotropin treatment, based on its effect on embryos and pregnancy outcomes, remains inconclusive. Objective: To evaluate the associations of different doses and durations of gonadotropins with embryonic genetic status and pregnancy outcomes after euploid embryo transfer in couples with infertility. Design, Setting, and Participants: This study was a post hoc analysis of a multicenter randomized clinical trial (RCT) conducted at 14 reproductive centers throughout China from July 2017 to June 2018 that evaluated the cumulative live birth rate with or without preimplantation genetic testing for aneuploidy (PGT-A) among couples with infertility and good prognosis. The PGT-A group from the original RCT was selected for secondary analysis. Patients were divided into 4 groups according to the total dosage of exogenous gonadotropins and treatment duration: group 1 (≤1500 IU and <10 days), group 2 (≤1500 IU and ≥10 days), group 3 (>1500 IU and <10 days), and group 4 (>1 500 IU and ≥10 days). Group 1 served as the control group. Data were analyzed from June through August 2023. Interventions: Blastocyst biopsy and PGT-A. Main outcomes and measures: The primary outcomes were embryonic aneuploidy, embryonic mosaicism, and cumulative live birth rates after euploid embryo transfer. Results: A total of 603 couples (mean [SD] age of prospective mothers, 29.13 [3.61] years) who underwent PGT-A were included, and 1809 embryos were screened using next-generation sequencing. The embryo mosaicism rate was significantly higher in groups 2 (44 of 339 embryos [13.0%]; adjusted odds ratio [aOR], 1.69 [95% CI, 1.09-2.64]), 3 (27 of 186 embryos [14.5%]; aOR, 1.98 [95% CI, 1.15-3.40]), and 4 (82 of 651 embryos [12.6%]; aOR, 1.60 [95% CI, 1.07-2.38]) than in group 1 (56 of 633 embryos [8.8%]). There were no associations between gonadotropin dosage or duration and the embryo aneuploidy rate. The cumulative live birth rate was significantly lower in groups 2 (83 of 113 couples [73.5%]; aOR, 0.49 [95% CI, 0.27-0.88]), 3 (42 of 62 couples [67.7%]; aOR, 0.41 [95% CI, 0.21-0.82]), and 4 (161 of 217 couples [74.2%]; aOR, 0.53 [95% CI, 0.31-0.89]) than in group 1 (180 of 211 couples [85.3%]). Conclusions and relevance: In this study, excessive exogenous gonadotropin administration was associated with increased embryonic mosaicism and decreased cumulative live birth rate after euploid embryo transfer in couples with a good prognosis. These findings suggest that consideration should be given to minimizing exogenous gonadotropin dosage and limiting treatment duration to improve embryo outcomes and increase the live birth rate. Trial Registration: ClinicalTrials.gov Identifier: NCT03118141.

High-fat and high-sucrose diet impairs female reproduction by altering ovarian transcriptomic and metabolic signatures.

BACKGROUND: Excessive energy intake in modern society has led to an epidemic surge in metabolic diseases, such as obesity and type 2 diabetes, posing profound threats to women's reproductive health. However, the precise impact and underlying pathogenesis of energy excess on female reproduction remain unclear. METHODS: We established an obese and hyperglycemic female mouse model induced by a high-fat and high-sucrose (HFHS) diet, then reproductive phenotypes of these mice were evaluated by examing sexual hormones, estrous cycles, and ovarian morphologies. Transcriptomic and precise metabolomic analyses of the ovaries were performed to compare the molecular and metabolic changes in HFHS mice. Finally, orthogonal partial least squares discriminant analysis was performed to compare the similarities of traits between HFHS mice and women with polycystic ovary syndrome (PCOS). RESULTS: The HFHS mice displayed marked reproductive dysfunctions, including elevated serum testosterone and luteinizing hormone levels, irregular estrous cycles, and impaired folliculogenesis, mimicking the clinical manifestations of women with PCOS. Precise metabolomic overview suggested that HFHS diet disrupted amino acid metabolism in the ovaries of female mice. Additionally, transcriptional profiling revealed pronounced disturbances in ovarian steroid hormone biosynthesis and glucolipid metabolism in HFHS mice. Further multi-omics analyses unveiled prominent aberration in ovarian arginine biosynthesis pathway. Notably, comparisons between HFHS mice and a cohort of PCOS patients identified analogous reproductive and metabolic signatures. CONCLUSIONS: Our results provide direct in vivo evidence for the detrimental effects of overnutrition on female reproduction and offer insights into the metabolic underpinnings of PCOS.

MALAT1 expression in granulosa cells in PCOS patients with different phenotypes.

Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine metabolic disorders. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) reportedly can regulate the reproductive system. Despite studies, the alteration of MALAT1 expression in granulosa cells (GCs) from PCOS patients was inconsistent. To evaluate MALAT1 expression in GCs in different PCOS subgroups and its association with PCOS phenotypes, we collected GCs from 110 PCOS cases and 71 controls, and examined MALAT1 expression by quantitative PCR. The results showed MALAT1 expression was upregulated in PCOS cases, especially in insulin resistant (IR) PCOS subgroup, obese PCOS subgroup and non-hyperandrogenic (NHA) PCOS subgroup. MALAT1 expression was positively correlated with BMI and several metabolic parameters in controls. Interestingly, MALAT1 expression was notably associated with some critical endocrine indexes for PCOS, including E2, FSH, LH and LH/FSH ratio. In different PCOS subgroups, we found significant positive correlations with LH/FSH ratio in IR-PCOS and PCOS with normal weight, and with serum T and LH level in NHA-PCOS subgroup. Integrated analysis with lncRNA target databases and PCOS-related databases revealed MALAT1 could participate in PCOS by influencing immune response and lipids metabolism in GCs. In conclusion, MALAT1 was differently expressed in GCs in PCOS, especially in IR, obese and NHA PCOS subgroups. MALAT1 was likely involved in metabolism and immune response in GCs in PCOS. However, more studies are necessary to establish this concept.

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.

Roles of bone morphogenetic proteins in endometrial remodeling during the human menstrual cycle and pregnancy.

BACKGROUND: During the human menstrual cycle and pregnancy, the endometrium undergoes a series of dynamic remodeling processes to adapt to physiological changes. Insufficient endometrial remodeling, characterized by inadequate endometrial proliferation, decidualization and spiral artery remodeling, is associated with infertility, endometriosis, dysfunctional uterine bleeding, and pregnancy-related complications such as preeclampsia and miscarriage. Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor-ß (TGF-ß) superfamily, are multifunctional cytokines that regulate diverse cellular activities, such as differentiation, proliferation, apoptosis, and extracellular matrix synthesis, are now understood as integral to multiple reproductive processes in women. Investigations using human biological samples have shown that BMPs are essential for regulating human endometrial remodeling processes, including endometrial proliferation and decidualization. OBJECTIVE AND RATIONALE: This review summarizes our current knowledge on the known pathophysiological roles of BMPs and their underlying molecular mechanisms in regulating human endometrial proliferation and decidualization, with the goal of promoting the development of innovative strategies for diagnosing, treating and preventing infertility and adverse pregnancy complications associated with dysregulated human endometrial remodeling. SEARCH METHODS: A literature search for original articles published up to June 2023 was conducted in the PubMed, MEDLINE, and Google Scholar databases, identifying studies on the roles of BMPs in endometrial remodeling during the human menstrual cycle and pregnancy. Articles identified were restricted to English language full-text papers. OUTCOMES: BMP ligands and receptors and their transduction molecules are expressed in the endometrium and at the maternal-fetal interface. Along with emerging technologies such as tissue microarrays, 3D organoid cultures and advanced single-cell transcriptomics, and given the clinical availability of recombinant human proteins and ongoing pharmaceutical development, it is now clear that BMPs exert multiple roles in regulating human endometrial remodeling and that these biomolecules (and their receptors) can be targeted for diagnostic and therapeutic purposes. Moreover, dysregulation of these ligands, their receptors, or signaling determinants can impact endometrial remodeling, contributing to infertility or pregnancy-related complications (e.g. preeclampsia and miscarriage). WIDER IMPLICATIONS: Although further clinical trials are needed, recent advancements in the development of recombinant BMP ligands, synthetic BMP inhibitors, receptor antagonists, BMP ligand sequestration tools, and gene therapies have underscored the BMPs as candidate diagnostic biomarkers and positioned the BMP signaling pathway as a promising therapeutic target for addressing infertility and pregnancy complications related to dysregulated human endometrial remodeling.

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.

TOX3 deficiency mitigates hyperglycemia by suppressing hepatic gluconeogenesis through FoxO1.

BACKGROUND: Excessive hepatic glucose production is a hallmark that contributes to hyperglycemia in type 2 diabetes (T2D). The regulatory network governing this process remains incompletely understood. Here, we demonstrate that TOX3, a high-mobility group family member, acts as a major transcriptional driver for hepatic glucose production. METHODS: Tox3-overexpressed and knockout mice were constructed to explore its metabolic functions. Transcriptomic and chromatin-immunoprecipitation sequencing (ChIP-seq) were used to identify downstream targets of TOX3. Both FoxO1 silencing and inhibitor approaches were used to assess the contribution of FoxO1. TOX3 expression levels were examined in the livers of mice and human subjects. Finally, Tox3 was genetically manipulated in diet-induced obese mice to evaluate its therapeutic potential. RESULTS: Hepatic Tox3 overexpression activates the gluconeogenic program, resulting in hyperglycemia and insulin resistance in mice. Hepatocyte-specific Tox3 knockout suppresses gluconeogenesis and improves insulin sensitivity. Mechanistically, integrated hepatic transcriptomic and ChIP-seq analyses identify FoxO1 as a direct target of TOX3. TOX3 stimulates FoxO1 transcription by directly binding to and activating its promoter, whereas FoxO1 silencing abrogates TOX3-induced dysglycemia in mice. In human subjects, hepatic TOX3 expression shows a significant positive correlation with blood glucose levels under normoglycemic conditions, yet is repressed by high glucose during T2D. Importantly, hepatic Tox3 deficiency markedly protects against and ameliorates the hyperglycemia and glucose intolerance in diet-induced diabetic mice. CONCLUSIONS: Our findings establish TOX3 as a driver for excessive gluconeogenesis through activating hepatic FoxO1 transcription. TOX3 could serve as a promising target for preventing and treating hyperglycemia in T2D.

Oral micronized progesterone versus vaginal progesterone for luteal phase support in fresh embryo transfer cycles: a multicenter, randomized, non-inferiority trial.

STUDY QUESTION: Does oral micronized progesterone result in a non-inferior ongoing pregnancy rate compared to vaginal progesterone gel as luteal phase support (LPS) in fresh embryo transfer cycles? SUMMARY ANSWER: The ongoing pregnancy rate in the group administered oral micronized progesterone 400 mg per day was non-inferior to that in the group administered vaginal progesterone gel 90 mg per day. WHAT IS KNOWN ALREADY: LPS is an integrated component of fresh IVF, for which an optimal treatment regimen is still lacking. The high cost and administration route of the commonly used vaginal progesterone make it less acceptable than oral micronized progesterone; however, the efficacy of oral micronized progesterone is unclear owing to concerns regarding its low bioavailability after the hepatic first pass. STUDY DESIGN, SIZE, DURATION: This non-inferiority randomized trial was conducted in eight academic fertility centers in China from November 2018 to November 2019. The follow-up was completed in April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1310 infertile women who underwent their first or second IVF cycles were enrolled. On the day of hCG administration, the patients were randomly assigned to one of three groups for LPS: oral micronized progesterone 400 mg/day (n = 430), oral micronized progesterone 600 mg/day (n = 440) or vaginal progesterone 90 mg/day (n = 440). LPS was started on the day of oocyte retrieval and continued till 11-12 weeks of gestation. The primary outcome was the rate of ongoing pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: In the intention-to-treat analysis, the rate of ongoing pregnancy in the oral micronized progesterone 400 mg/day group was non-inferior to that of the vaginal progesterone gel group [35.3% versus 38.0%, absolute difference (AD): -2.6%; 95% CI: -9.0% to 3.8%, P-value for non-inferiority test: 0.010]. There was insufficient evidence to support the non-inferiority in the rate of ongoing pregnancy between the oral micronized progesterone 600 mg/day group and the vaginal progesterone gel group (31.6% versus 38.0%, AD: -6.4%; 95% CI: -12.6% to -0.1%, P-value for non-inferiority test: 0.130). In addition, we did not observe a statistically significant difference in the rate of live births between the groups. LIMITATIONS, REASONS FOR CAUTION: The primary outcome of our trial was the ongoing pregnancy rate; however, the live birth rate may be of greater clinical interest. Although the results did not show a difference in the rate of live births, they should be confirmed by further trials with larger sample sizes. In addition, in this study, final oocyte maturation was triggered by hCG, and the findings may not be extrapolatable to cycles with gonadotropin-releasing hormone agonist triggers. WIDER IMPLICATIONS OF THE FINDINGS: Oral micronized progesterone 400 mg/day may be an alternative to vaginal progesterone gel in patients reluctant to accept the vaginal route of administration. However, whether a higher dose of oral micronized progesterone is associated with a poorer pregnancy rate or a higher rate of preterm delivery warrants further investigation. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a grant from the National Natural Science Foundation of China (82071718). None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: This trial was registered at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/) with the number ChiCTR1800015958. TRIAL REGISTRATION DATE: May 2018. DATE OF FIRST PATIENT'S ENROLMENT: November 2018.

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.

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.

Investigation of androgen receptor CAG repeats length in polycystic ovary syndrome diagnosed using the new international evidence-based guideline.

BACKGROUND: To study whether CAG repeat polymorphism of androgen receptor (AR) contributes to the risk of polycystic ovarian morphology (PCOM) with antral follicle count (AFC) ≥ 20 in the context of new international guideline of polycystic ovary syndrome (PCOS). METHODS: Blood of 109 PCOS cases and 61 controls were collected for the measurement of AR CAG repeats length by sequencing. The mean number and frequency distribution of CAG repeats length were observed. Detailed analysis was conducted by dividing PCOS cases into low AFC group (L-AFC, AFC < 20) and high AFC group (H-AFC, AFC ≥ 20) according to the new international evidence-based guideline. RESULTS: The portion of individuals with lower CAG repeats length in H-AFC group was significantly larger than those with higher CAG repeats length. Logistic model revealed individuals with lower CAG length tended to develop H-AFC. CONCLUSION: Lower CAG repeats length in the AR gene of PCOS cases increases risk of PCOM.

Effects of duration of long-acting GnRH agonist downregulation on assisted reproductive technology outcomes in patients with adenomyosis: a retrospective cohort study.

Objectives: To elucidate the relationship between long-acting GnRH agonist (GnRHa) downregulation and assisted reproductive technology (ART) outcomes and identify the optimal duration of downregulation in patients with adenomyosis. Design: Retrospective cohort study. Participants: The study was designed to evaluate ART outcomes in adenomyosis patients with and without GnRHa downregulation between January 2016 and December 2020. A total of 374 patients with adenomyosis (621 cycles) were included with 281 cycles in downregulation group versus 340 cycles in non-downregulation group. After 1:1 propensity score matching (PSM), a sample size of 272 cycles in each group was matched. The matched downregulation group was further divided into 1-month (147 cycles), 2-months (72 cycles), and ≥3 months downregulation (53 cycles) subgroups. Stratification analysis was conducted on pregnancy outcomes in 239 fresh embryo transfer (ET) cycles and 305 frozen embryo transfer (FET) cycles. Results: The downregulation group had larger mean diameter of initial uterus and higher proportion of severer dysmenorrhea compared to non-downregulation group. The pregnancy-related parameters in GnRHa downregulation group were similar to those in non-downregulation group, except for higher late miscarriage rate (MR) (13.4% vs. 3.1%, P = 0.003). The subgroup comparisons in fresh ET cycles indicated that implantation rate (75.0% vs. 39.2%, P = 0.002), biochemical pregnancy rate (91.7% vs. 56.0%, P = 0.036) and clinical pregnancy rate (83.3% vs. 47.0%, P = 0.016) could be improved by prolonged GnRHa downregulation (≥3 months), whereas late MR was difficult to be reversed (30.0% vs. 3.2%, P = 0.017). In FET cycles, higher MR (53.6% vs. 29.9%, P = 0.029; 58.8% vs. 29.9%, P = 0.026) and lower live birth rate (18.8% vs. 34.1%, P = 0.023; 17.1% vs. 34.1%, P = 0.037) were observed in the 1-month and ≥3 months downregulation group, while no differences were found in the 2-months downregulation group compared to the non-downregulation group. Conclusion: In patients with severer adenomyosis, long-acting GnRHa downregulation might be correlated with improved ART outcomes. In fresh ET cycles, prolonged downregulation (≥3 months) might be beneficial to improve live birth rate, which needed to be verified by further study with larger sample. In FET cycles, the optimal duration of downregulation was not certain and still needed further exploration.

Letrozole use in vitrified single-blastocyst transfer cycles is associated with lower risk of large for gestational age infants in patients with polycystic ovary syndrome.

PURPOSE: To evaluate the obstetric and perinatal outcomes of three routine endometrial preparation protocols in women with PCOS who underwent frozen embryo transfer (FET). METHODS: This was a retrospective study in women with PCOS who underwent FET in an academic reproductive medical center. A total of 2710 cycles were enrolled and classified into three groups according to different endometrial preparation protocols; human menopausal gonadotropin (HMG), letrozole + HMG, or hormone replacement therapy (HRT). RESULTS: The stimulation groups had reduced risks of hypertensive disorders of pregnancy (HDP), large for gestational age (LGA) infants, and cesarean delivery than the HRT group. After adjustment for different confounder combinations in the two models, the frequencies of LGA and HDP in the letrozole + HMG group and the HMG group were still significantly lower than those in the HRT group. The letrozole + HMG group exhibited a reduced risk of LGA than HMG group after adjustment of confounders. A trend toward risk reductions in HDP and LGA was observe in turns of HRT, HMG, and letrozole + HMG groups, and the trends were statistically significant (Ptrend = 0.031 and 0.001). CONCLUSION: In patients with PCOS, ovarian stimulation protocols for endometrial preparation are associated with reduced risks of HDP and LGA compared to HRT cycles. The use of letrozole could further reduce risk of LGA compared to HMG only protocol. We propose that ovarian stimulation protocols can be used widely for endometrial preparation in FET cycles in women with PCOS, especially with the use of letrozole.

Effects of ovarian stimulation protocols on outcomes of assisted reproductive technology in adenomyosis women: a retrospective cohort study.

Objective: To evaluate the effects of different ovarian stimulation protocols on in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes in infertile women with adenomyosis. Methods: We carried out a retrospective cohort study among infertile women with adenomyosis receiving IVF/ICSI treatment, including 257 fresh embryo transfer (ET) cycles and 305 frozen embryo transfer (FET) cycles. In fresh ET cycles, ultra-long, long, short, and antagonist protocols were adopted. In FET cycles, patients received long-acting GnRH agonist (GnRHa) pretreatment or not. The primary outcome was clinical pregnancy rate (CPR), and the secondary outcomes included implantation rate (IR), miscarriage rate (MR), and live birth rate (LBR). Results: In fresh ET cycles, compared with ultra-long and long protocols, IR (49.7%, 52.1% versus 28.2%, P=0.001) and CPR (64.3%, 57.4% versus 35.6%, P=0.004) significantly decreased in the short protocol. Similarly, compared with ultra-long and long protocols, a decreased inclination of IR (49.7%, 52.1% versus 33.3%) and CPR (57.4%, 64.3% versus 38.2%) existed in the antagonist protocol, although no statistical significance was detected because of strict P adjustment of Bonferroni method (Padj=0.008). Compared with long protocol, LBR in short protocol decreased obviously (48.2% versus 20.3%, P<0.001). In FET cycles, no matter which origin of embryos, there were no statistical differences in IR, CPR, and LBR. For women ≥35 years receiving fresh ET, CPR was higher in ultra-long and long protocols (52.1%, 50.0% versus 20.0%, 27.5%, P=0.031) compared to antagonist and short protocols. For women ≥35 years receiving FET, compared with ultra-long and antagonist protocols, cycles with embryos originating from long and short protocols had higher proportions of long-acting GnRHa pretreatment (30.4%,30.00 versus 63.9%, 51.4%, P=0.009). IR (61.1%, 48.6% versus 32.6%, 25.0%, P=0.020) and CPR (58.3%, 48.6% versus 30.4%, 25.0%, P=0.024) in long and short protocols were higher than rates of ultra-long and antagonist protocols, but no statistical differences were supported because of strict Bonferroni method (Padj=0.008). Conclusion: In infertile women with adenomyosis, if a fresh embryo was planned for transfer, an ultra-long or long protocol might be beneficial. If antagonist and short protocols were used, whole embryos frozen followed by FET was recommended. In FET cycles, embryos derived from different protocols had no impact on pregnancy outcomes.

Identification of novel first-trimester serum biomarkers for early prediction of preeclampsia.

BACKGROUND: Preeclampsia (PE) is a leading cause of maternal and perinatal mortality and morbidity worldwide, but effective early prediction remains a challenge due to the lack of reliable biomarkers. METHODS: Based on the extensive human biobank of our large-scale assisted reproductive cohort platform, the first-trimester serum levels of 48 cytokines, total immunoglobulins (Igs), anti-phosphatidylserine (aPS) antibodies, and several previously reported PE biomarkers [including placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and activin A] were measured in 34 women diagnosed with PE and 34 matched normotensive controls. RESULTS: The PE group has significantly higher first-trimester serum levels of interleukin (IL)-2Rα, IL-9, tumor necrosis factor-ß (TNF-ß), RANTES, hepatocyte growth factor (HGF), total IgM, and total IgG, and aPS IgG optical density (OD) value, as well as lower first-trimester serum levels of PlGF and total IgA and aPS-IgG immune complexes (IC) OD value than the control group. Combining top five first-trimester serum biomarkers (total IgM, total IgG, PlGF, aPS IgG, and total IgA) achieved superior predictive value [area under the curve (AUC) and 95% confidence interval (CI) 0.983 (0.952-1.000), with a sensitivity of 100% and a specificity of 94.1%] for PE development compared to PlGF and PlGF/sFlt-1 independently [AUC and 95% CI 0.825 (0.726-0.924) and 0.670 (0.539-0.800), respectively]. CONCLUSION: We identified novel first-trimester serum biomarkers and developed an effective first-trimester prediction model using immune-related factors and PlGF for PE, which could facilitate the development of early diagnostic strategies and provide immunological insight into the further mechanistic exploration of PE.

Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

Knowledge gaps that limit the development of therapies for polycystic ovary syndrome (PCOS) concern various environmental factors that impact clinical characteristics. Circadian dysrhythmia contributes to glycometabolic and reproductive hallmarks of PCOS. Here, we illustrated the amelioration of Limosilactobacillus reuteri (L. reuteri) on biorhythm disorder-ignited dyslipidemia of PCOS via a microbiota-metabolite-liver axis. A rat model of long-term (8 weeks) darkness treatment was used to mimic circadian dysrhythmia-induced PCOS. Hepatic transcriptomics certified by in vitro experiments demonstrated that increased hepatic galanin receptor 1 (GALR1) due to darkness exposure functioned as a critical upstream factor in the phosphoinositide 3-kinase (PI3K)/protein kinase B pathway to suppress nuclear receptors subfamily 1, group D, member 1 (NR1D1) and promoted sterol regulatory element binding protein 1 (SREBP1), inducing lipid accumulation in the liver. Further investigations figured out a restructured microbiome-metabolome network following L. reuteri administration to protect darkness rats against dyslipidemia. Notably, L. reuteri intervention resulted in the decrease of Clostridium sensu stricto 1 and Ruminococcaceae UCG-010 as well as gut microbiota-derived metabolite capric acid, which could further inhibit GALR1-NR1D1-SREBP1 pathway in the liver. In addition, GALR antagonist M40 reproduced similar ameliorative effects as L. reuteri to protect against dyslipidemia. While exogenous treatment of capric acid restrained the protective effects of L. reuteri in circadian disruption-induced PCOS through inhibiting GALR1-dependent hepatic lipid metabolism. These findings purport that L. reuteri could serve for circadian disruption-associated dyslipidemia. Manipulation of L. reuteri-capric acid-GALR1 axis paves way for clinical therapeutic strategies to prevent biorhythm disorder-ignited dyslipidemia in PCOS women.