Since chronic inflammation is a typical feature of polycystic ovary syndrome (PCOS), both clinical and experimental studies have demonstrated that resveratrol (RES) can effectively alleviate it. However, the underlying mechanism remains unclear. To further investigate this, granulosa cells (GCs) derived from PCOS patients, lipopolysaccharide (LPS)-treated human granulosa cells (KGN), LPS-induced chronic inflammation mouse models, and dehydroepiandrosterone (DHEA)-induced PCOS mouse models were treated with RES. The expression of inflammatory cytokines, including interleukin (IL)-6, IL-1β, chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX2), as well as absent in melanoma 2 (AIM2), was examined. Additionally, ovarian morphological changes in these mouse models were assessed using hematoxylin-eosin (HE) staining. The results showed that the expression of these inflammatory cytokines and AIM2 increased significantly in GCs derived from PCOS patients and LPS-induced KGN cells, as well as in the ovaries of LPS-induced chronic inflammation and DHEA-induced PCOS mouse models. Furthermore, blocking AIM2 in LPS-treated KGN cells and mice with LPS-induced inflammation or PCOS significantly reduced the upregulation of inflammatory cytokines, similar to the results observed following RES treatment. In addition, LPS-induced phosphorylation of the JAK2/STAT3 pathway in KGN cells was completely abolished by RES treatment. Notably, LPS-induced upregulation of AIM2 and these inflammatory cytokines was completely reversed by blocking the JAK2/STAT3 pathway using AZD-1480 and SH-4-54, respectively. Further in vivo studies showed that ovarian morphological and estrous cycle disturbances in DHEA-induced PCOS mouse models were effectively ameliorated by RES and A151. In conclusion, RES alleviates chronic inflammation in PCOS by inhibiting AIM2 via blocking the JAK2/STAT3 pathway. Our findings suggest that the targeted inhibition of AIM2 could represent a novel therapeutic approach for PCOS.

Preeclampsia (PE) is a major hypertensive disorder of pregnancy and a leading cause of maternal and perinatal mortality and morbidity worldwide. Aberrant placental development, characterized by shallow trophoblast invasion and incomplete remodeling of maternal spiral arteries, remains central to PE pathogenesis. In PE, tissue transglutaminase (TGM2) is dysregulated in the placenta, contributing to vascular dysfunction, inflammation, and impaired trophoblast differentiation, yet the upstream regulatory signals remain unknown. Bone morphogenetic protein 7 (BMP7) is abundantly expressed at the maternal-fetal interface and implicated in vascular homeostasis, but its role in regulating trophoblast function, especially related to PE pathogenesis, remains undefined. Here, single-cell transcriptomic profiling of placental tissues revealed coordinated downregulation of BMP7 and upregulation of TGM2 in PE relative to normotensive controls. Functional assays in immortalized and primary human trophoblasts demonstrated that BMP7 promotes trophoblast migration and invasion by suppressing TGM2 expression through the ALK2/3-SMAD1/5/9-SMAD4 pathway. Trophoblast organoid models further demonstrated the anti-invasive role of TGM2 in trophoblast differentiation during early placentation. In vivo, an adenovirus-expressing sFlt-1 (Ad sFlt-1)-induced PE mouse model showed increased placental Tgm2 expression, while recombinant BMP7 administration ameliorated PE-like symptoms. Although the protective effects of BMP7 in vivo are likely mediated by mechanisms beyond TGM2 alone, together these findings identify the BMP7-TGM2 axis as an important trophoblast-regulatory mechanism in PE and support BMP7 as a potential therapeutic candidate.

Proportion-based laboratory outcomes in assisted reproductive technology (ART), including two-pronuclear fertilization rate, good-quality embryo rate, and blastocyst formation rate, are widely used to evaluate treatment effectiveness and laboratory performance. These outcomes are bounded between 0 and 1, frequently skewed, and arise from hierarchical data structures in which oocytes or embryos are nested within cycles and patients. Inappropriate statistical handling of such data can lead to biased inference and misinterpretation of treatment effects. This narrative review aimed to identify common methodological misuses in the analysis of proportion-based ART laboratory outcomes and to summarize appropriate statistical approaches. This narrative review examined methodological and applied ART studies published between 2000 and 2025. Statistical practices were extracted and categorized according to data reporting (numerator–denominator structure), modeling strategies, and handling of clustering and boundary values. Recommended analytical frameworks were synthesized for binary outcomes reported either as a proportion or a count. Common analytical issues included pooling embryo- or oocyte-level observations and applying chi-square tests, reporting absolute counts without denominators, describing skewed proportion outcomes using mean ± standard deviation and linear models, and failing to account for intra-patient or intra-cycle clustering. Appropriate approaches depended on data structure. Beta regression was suitable for continuous proportions within the open interval (0,1), while zero–one-inflated beta models accommodated observed boundary values. When numerator and denominator counts were available, binomial generalized linear mixed models (GLMMs) and generalized estimating equations (GEEs) preserved denominator information and accounted for hierarchical clustering. Misapplication of statistical methods to proportion-based ART laboratory outcomes remains common and may compromise reproducibility and clinical interpretation. Adoption of model–data alignment and transparent reporting practices will improve the validity of ART laboratory research and support evidence-based decision-making in reproductive medicine.

The dysregulation of growth factors is associated with defective trophoblast invasion, which leads to uteroplacental malperfusion due to the inadequate remodeling of spiral arteries. Pregnancy disorders, including preeclampsia, particularly early-onset preeclampsia, are closely related to compromised placental function caused by aberrant trophoblast invasion. Activin A, a growth factor detectable in serum that belongs to the transforming growth factor-β (TGF-β) superfamily, has been implicated in the development of preeclampsia, as evidenced by its elevated serum levels and its role in regulating trophoblast invasion. However, the existing research on its regulatory mechanisms in trophoblast invasion has focused mainly on intracellular, nonsecretory epithelial-mesenchymal transition (EMT) markers in conventional trophoblast cell lines, which limits its translational relevance to clinical applications. In this study, we performed small RNA sequencing combined with cell biology studies in primary human trophoblast and 2D human trophoblast stem cell models and revealed that the upregulation of the SOX4 (SRY-box transcription factor 4) and miR-103a-3p induced by activin A contributes to trophoblast invasion and potential extravillous differentiation. The bioinformatic analysis of proteomic and microRNA profiles from public databases revealed increased expression of the activin A protein and exosomal miR-103a-3p in maternal blood during the second trimester of pregnancy complicated by preeclampsia. Overall, our integrated approach reveals the regulatory mechanism by which activin A, SOX4, and miR-103a-3p regulate human trophoblast invasion and EVT differentiation, highlighting their potential as early diagnostic biomarkers for preeclampsia.

Background: Trial evidence of lifestyle intervention is scarce and has largely focused on women with obesity and subfertility. We evaluated the effects of a multifaceted preconception behavioural intervention within the Sino-Canadian Healthy Life Trajectories Initiative (SCHeLTI) trial. Methods: In this 1:1 parallel, open-label, cluster-randomized trial, 36 maternal and child health centers in Shanghai were randomly assigned to intervention or control (routine care). The community-family-mother-child intervention promoted healthy lifestyle behaviors targeting nutrition, physical activity, sleep, and emotional health. Healthcare providers used “healthy conversation” skills combined with an e-health app and community resources. The primary outcome was clinical pregnancy confirmed by ultrasound within 12 months, using intention-to-treat analyses. Findings: 1470 couples were assigned, 631 became pregnant within one year. Clinical pregnancy rates were 47.8% in the intervention group and 44.7% in the control group (adjusted hazard ratio [HR] 1.11, 95% CI 0.95–1.30; p=0.201). Among participants with normal baseline BMI, the intervention significantly increased clinical pregnancy (adjusted HR 1.22, 95% CI 1.02–1.48; p=0.029; p-interaction=0.068), reduced sedentary time (–0.44 h, 95% CI –0.81 to –0.07; p=0.020), and lowered systolic blood pressure (–2.93 mmHg, 95% CI –5.12 to –0.73; p=0.011). Interpretation: The SCHeLTI multifaceted preconception lifestyle intervention showed minimal overall improvement in clinical pregnancy but yielded broader reproductive, behavioral, and cardiovascular benefits among women with normal BMI. These findings support targeted implementation of preconception lifestyle strategies for normal-weight women and emphasize the need to maximize sustained engagement to optimize benefits.

Abstract:Oocyte in vitro maturation (IVM) is an evolving component of assisted reproductive technology (ART) that offers a less invasive and cost-effective alternative to conventional controlled ovarian stimulation. It is particularly beneficial for patients at risk of ovarian hyperstimulation syndrome (OHSS), those with polycystic ovary syndrome (PCOS), and individuals requiring urgent fertility preservation. Despite these advantages, clinical uptake has remained limited owing to concerns about the developmental competence and quality of IVM-derived oocytes. To address this, we conducted a comprehensive literature search of PubMed, Embase, and Web of Science for articles published between January 2000 and June 2025, using combinations of keywords related to IVM, oocyte maturation, culture protocols, oocyte quality, and clinical outcomes. Recent progress in the field has led to the development of biphasic culture systems, pre-IVM priming strategies, and the incorporation of regulatory factors such as C-type natriuretic peptide (CNP) and oocyte-secreted factors, all of which have contributed to improved oocyte maturation and embryo development. Nonetheless, variability in outcomes persists due to differences in patient selection, stimulation protocols, and laboratory practice. Continued optimisation of IVM culture systems and a deeper understanding of oocyte maturation mechanisms will be essential for enhancing clinical efficacy. Future research should prioritise standardisation, patient-tailored protocols, and systematic long-term outcome data to support wider adoption of IVM. This review provides a comprehensive overview of recent advances and ongoing challenges in human oocyte IVM, offering perspectives on future directions for clinical translation and improved ART outcomes. Lay summary:IVM is a fertility treatment that allows immature eggs to mature in the laboratory rather than within the body. This approach can be safer, simpler, and more affordable than traditional IVF, especially for women with polycystic ovary syndrome (PCOS), those at risk of ovarian hyperstimulation, or those who need to preserve their fertility quickly, such as cancer patients. Although IVM holds great promise, it is not yet widely used because its success rates are not as high as those of conventional methods. This review looks at the latest scientific progress to improve IVM, including better laboratory techniques, the use of natural hormones and growth factors, and new ways to support egg development outside the body. These advancements have helped improve the quality of eggs and embryos, but challenges remain. Differences in patient types, medications used, and lab practices can affect how well IVM works. More research is needed to make IVM more consistent and effective so it can become a routine fertility option for a broader population.

BACKGROUND:RNA modifications, collectively known as the epitranscriptome, represent the third layer of gene regulation, influencing gene expression at transcriptional, post-transcriptional, and translational levels. RNA-modifying proteins (RMPs), including writers, erasers, and readers, are responsible for depositing, removing, and recognizing chemical modifications on RNA molecules. These modifications play a crucial role in linking molecular processes to cellular functions. Over the past few decades, a growing body of laboratory evidence, alongside advances in sequencing technologies, has uncovered connections between aberrant RNA modifications and reproductive disorders, highlighting their emerging roles in female fertility. Given the rapid expansion of epitranscriptomic research in female reproduction, a comprehensive review is needed to summarize the broader impacts of various RNA modifications, rather than focusing on individual RNA modifications alone. OBJECTIVE AND RATIONALE:This review aims to elucidate the progress in understanding the role of RNA modifications in reproductive biology and how their dysregulations contribute to infertility-related conditions, such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency (POI), and endometriosis. Special focus will be given to RNA modifications in coding RNAs, particularly those linked to female fertility and supported by solid evidence. The ultimate objective is to explore how targeting the RNA-modification machinery can lead to the development of novel therapeutic interventions for restoring fertility. SEARCH METHODS:We conducted a thorough review of peer-reviewed original research articles and reviews published over the past two decades using the PubMed search engine. Keywords included terms related to RNA modifications, such as 'N6-methyladenosine (m6A)', 'N4-acetylcytidine (ac4C)', and 'adenosine-to-inosine (A-I) editing', combined with terms related to female reproduction, such as 'ovary', 'oocyte', and 'embryo'. Additional relevant search phrases were also utilized to ensure comprehensive coverage of the topic. OUTCOMES:RNA modification has emerged as a transformative area in reproductive biology, with our understanding of the epitranscriptome growing rapidly due to significant advances in high-throughput sequencing technologies. Regulatory proteins play a crucial role in the correct deposition and functional implementation of RNA modifications. Knockout animal models have identified a broad, though still incomplete, list of RNA modifications involved in mammalian reproductive processes. These include prevalent modifications in mRNA, such as m6A, as well as A-I editing, and, to a lesser extent, 5-methylcytosine (m5C) and ac4C. These regulatory mechanisms impact various reproductive functions, including folliculogenesis, oocyte maturation, fertilization, and embryo development. Dysregulation of RNA modifications may exacerbate infertility-related conditions, such as POI, PCOS, and endometriosis. Although clinical investigations are still in their early stages, RNA modifications show great promise as diagnostic biomarkers and therapeutic targets, with the potential to improve fertility and reproductive health outcomes. WIDER IMPLICATIONS:This review explores a relatively underexamined area of epitranscriptomic research in female reproduction, offering the potential to significantly advance our understanding of reproductive biology. It underscores the clinical relevance of RNA modifications in infertility-related disorders and identifies potential biomarkers, as well as RMP-targeted therapies, that could shape future clinical decision-making and personalized treatments. These insights are crucial for reproductive clinicians and embryologists, presenting new avenues for diagnosis and therapeutic interventions in reproductive medicine. REGISTRATION NUMBER:N/A.

The effective utilization of immature oocytes holds promise for improving conception rates in women with low ovarian reserve. The oocyte microenvironment, comprising the extracellular matrix and intercellular communication with somatic cells, is crucial for supporting oocyte maturation. This study hypothesizes that a three-dimensional (3D) culture system, constructed with hydrogels modified by laminin-mimetic peptides-a major component of the extracellular matrix-and granulosa cells (GCs) from mature mouse oocytes, can replicate the biomechanical microenvironment required for oocyte maturation, thereby promoting the development of immature mouse oocytes. To validate this hypothesis, we initially assessed the physical properties of hydrogels and determined the optimal concentration for in vitro oocyte culture. A total of 304 germinal vesicle (GV) phase mouse oocytes were retrieved and randomly allocated to two-dimensional (2D), 3D, and 3D-GCs culture groups. The 3D-GCs group exhibited the highest rate of first polar body extrusion (83.1 ± 5.0 %). Oxidative stress levels and mitochondrial membrane potential were evaluated using DCFH-DA and JC-1 staining, confirming that 3D culture significantly reduced oxidative stress and enhanced mitochondrial activity. Cytoplasmic maturation, assessed by cortical granule distribution and endoplasmic reticulum organization, further highlighted the superiority of the 3D-GCs system. The 3D-GCs group demonstrated 87.0 % of oocytes with grade III cortical granule distribution and 84.6 % with mature endoplasmic reticulum, significantly surpassing the other groups (p < 0.05). In conclusion, the 3D-GCs culture system effectively supports synchronized nuclear and cytoplasmic maturation in immature oocytes. This approach provides a promising platform for improving the developmental competence of oocytes, potentially benefiting women with diminished ovarian reserve.

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-beta (TGF-beta) 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. Discoveries about the functions of bone morphogenetic proteins (BMPs) in endometrial remodeling have deepened our understanding and enabled development of therapeutic technologies in reproductive medicine. ALK, activin receptor-like kinase; BMPR2, bone morphogenetic protein receptor type 2; GDF, growth differentiation factor. This schematic diagram was created with Biorender.com.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Human trophoblast stem cells (hTSCs) and related trophoblast organoids are state-of-the-art culture systems that facilitate the study of trophoblast development and human placentation. Using single-cell transcriptomics, we evaluate how organoids derived from freshly isolated first-trimester trophoblasts or from established hTSC cell lines reproduce developmental cell trajectories and transcriptional regulatory processes defined in vivo. Although organoids from primary trophoblasts and hTSCs overall model trophoblast differentiation with accuracy, specific features related to trophoblast composition, trophoblast differentiation, and transcriptional drivers of trophoblast development show levels of misalignment. This is best illustrated by the identification of an expanded progenitor state in stem cell-derived organoids that is nearly absent in vivo and transcriptionally shares both villous cytotrophoblast and extravillous trophoblast characteristics. Together, this work provides a comprehensive resource that identifies strengths and limitations of current trophoblast organoid platforms.

Neuropeptide FF (NPFF) belongs to the RFamide peptide family. NPFF regulates a variety of physiological functions by binding to a G protein-coupled receptor (GPCR), NPFFR2. Epithelial ovarian cancer (EOC) is a leading cause of death among gynecological malignancies. The pathogenesis of EOC can be regulated by many local factors, including neuropeptides, through an autocrine/paracrine manner. However, to date, the expression and/or function of NPFF/NPFFR2 in EOC is undetermined. In this study, we show that the upregulation of NPFFR2 mRNA was associated with poor overall survival in EOC. The TaqMan probe-based RT-qPCR showed that NPFF and NPFFR2 were expressed in three human EOC cells, CaOV3, OVCAR3, and SKOV3. In comparison, NPFF and NPFFR2 expression levels were higher in SKOV3 cells than in CaOV3 or OVCAR3 cells. Treatment of SKOV3 cells with NPFF did not affect cell viability and proliferation but stimulated cell invasion. NPFF treatment upregulates matrix metalloproteinase-9 (MMP-9) expression. Using the siRNA-mediated knockdown approach, we showed that the stimulatory effect of NPFF on MMP-9 expression was mediated by the NPFFR2. Our results also showed that ERK1/2 signaling was activated in SKOV3 cells in response to the NPFF treatment. In addition, blocking the activation of ERK1/2 signaling abolished the NPFF-induced MMP-9 expression and cell invasion. This study provides evidence that NPFF stimulates EOC cell invasion by upregulating MMP-9 expression through the NPFFR2-mediated ERK1/2 signaling pathway.

Placental insufficiency disorders, including preeclampsia and intrauterine growth restriction, are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes have underlying poor placental trophoblast cell invasion into uterine tissues. Placental invasion is controlled by many hormones and growth factors. Myostatin (MSTN) is a transforming growth factor‐β superfamily member recognized for its important role in muscle growth control. MSTN has also been shown to be secreted and functioning in the placenta, and its serum and/or placental levels were found to be upregulated in preeclampsia and intrauterine growth restriction. Considering that the mechanistic role of MSTN in placentation remains poorly understood, we hypothesized that MSTN uses ALK4/5‐SMAD2/3/4 signaling to increase human trophoblast invasion through a group of epithelial–mesenchymal transition genes including SERPINE2, PAI‐1, and SOX4. mRNA sequencing of control and MSTN‐treated primary human trophoblast cells (n = 5) yielded a total of 610 differentially expressed genes (false discovery rate <0.05) of which 380 genes were upregulated and 230 were downregulated. These differentially expressed genes were highly enriched in epithelial–mesenchymal transition genes, and a subset including SERPINE2, PAI‐1, and SOX4 was investigated for its role in MSTN‐induced trophoblast cell invasion. We found that MSTN induced upregulation of SERPINE2 via ALK4/5‐SMAD2/3/4 signaling; however, SMAD2 was not involved in MSTN‐induced PAI‐1 upregulation. SOX4 was involved in MSTN‐induced upregulation of SERPINE2, but not PAI‐1. Collectively, this study discovers novel molecular mechanisms of MSTN‐induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.

Recurrent pregnancy loss (RPL) remains an unsolved problem in obstetrics and gynecology, and up to 50% of RPL cases are unexplained. Unexplained RPL (uRPL) is widely considered to be related to an aberrant endometrial microenvironment. BMP2 is an important factor involved in endometrial decidualization and embryo implantation, and intercellular adhesion molecule 1 (ICAM1) is a critical inflammatory regulator in the endometrium. In this study, we found that endometrial samples obtained from Unexplained RPL patients have significantly lower BMP2 and higher ICAM1 levels than fertile controls. For further research on the relationship between BMP2 and ICAM1 and the potential molecular mechanisms in Unexplained RPL, immortalized human endometrial stromal cells (HESCs) and primary human decidual stromal cells (HDSCs) were used as study models. Our results showed that BMP2 significantly decreased ICAM1 expression by upregulating DNA-binding protein inhibitor 3 (ID3) in both HESCs and HDSCs. Using kinase receptor inhibitors (dorsomorphin homolog 1 (DMH-1) and dorsomorphin) and siRNA transfection, it has been found that the upregulation of ID3 and the following downregulation of ICAM1 induced by BMP2 is regulated through the ALK3-SMAD4 signaling pathway. This research gives a hint of a novel mechanism by which BMP2 regulates ICAM1 in the human endometrium, which provides insights into potential therapeutics for unexplained RPL.

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6β1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6β1.

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Abstract Background:Pentraxin 3 contributes to the formation of cumulus-oophorus complex,its level is considered to indicate the quality and potential of oocytes. Bone morphogenetic protein 6 is a key regulator of ovary follicular development and regulates female reproduction. It is unclear whether Pentraxin 3 is differentially expressed in granulosa cells derived from Polycystic ovary syndrome and whether Bone morphogenetic protein 6 affects Pentraxin 3 in human granulosa-lutein cells.To evaluate whether Pentraxin 3 is differentially expressed in the granulosa cells derived from women with Polycystic Ovary Syndrome and whether granulosa cell-derived Bone Morphogenetic Protein 6 can regulate the expression of Pentraxin 3 in hGL cells. Materials and methods:The expression levels of BMP6 and PTX3 in granulosa cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 and PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3. TGF-β type 1 inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways. Results:The levels of Bone Morphogenetic Protein 6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of Pentraxin 3 were positively correlated with the corresponding oocyte maturation rate in women with Polycystic Ovary Syndrome. Additionally, the in vitro cell cultured results showed Bone Morphogenetic Protein 6 significantly inhibited the expression of Pentraxin 3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in hGL cells. Conclusions:The suppressive effect of Bone Morphogenetic Protein 6 on Pentraxin 3 expression was mediated by ALK2/ALK3 type 1 receptors and BMPR2/ACVR2A type 2 receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in women with Polycystic Ovary Syndrome.Our findings provides new insights into the understanding of the pathogenesis of Polycystic Ovary Syndrome-related ovulatory disorders.

Purpose To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells. Methods The expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-β type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells. Results The levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells. Conclusions The suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.