In vitro maturation (IVM) of oocytes is a promising technique among assisted reproductive technologies. Although IVM has been used for many years, its efficiency is still relatively low compared to that of traditional in vitro fertilization (IVF) procedures. Therefore, we aimed to explore the hotspots and frontiers of IVM research over the past two decades and provide direction for IVM advancement.

The articles and reviews related to IVM in the Web of Science Core Collection (WoSCC) were retrieved on June 03, 2024. Three bibliometric tools, VOSviewer 1.6.18 (2010), CiteSpace 6.1. R6 (2006), and Bibliometrix R package 4.1.0 (2017), were used to generate network maps and explore knowledge frontiers and trends. To uncover the latest research advancements and frontiers in the IVM field, we conducted an analysis of the entire IVM field, including all species. Given our focus on human IVM developments, we identified the leading countries, institutions, authors, and journals driving progress in human IVM.

A total of 5150 publications about IVM and 1534 publications in the specific context of human IVM were retrieved from the WoSCC. The number of publications on both overall IVM and human IVM fields has increased steadily. In human IVM, the United States (USA) and McGill University were the most prolific country and institution, respectively. Human Reproduction was both the most published in and the most cited journal in human IVM. Seang Lin, Tan was the most productive author, and Ri-Cheng, Chian's papers were the most cited in human IVM. Furthermore, five hotspot topics were summarized, namely, culture system, supplementation, cooperation in the ovarian follicle, gene expression, and oocyte cryopreservation.

Further studies could concentrate on the following topics: (1) the mechanisms involved in oocyte maturation in vivo and in vitro, especially in energy metabolism and intercellular communications; (2) the establishment of IVM culture systems, including standardization of the biphasic IVM culture system and supplementation; (3) the genetic differences between oocytes matured in vivo and in vitro; and (4) the mechanism of cryopreservation-inflicted damage and solutions to this challenge. For human IVM, it is necessary to precisely assess the developmental stages of oocytes and adjust the IVM process accordingly to develop tailored culture media. Concurrently, clinical trials are essential for evaluating the effectiveness and safety of IVM.

Diminished ovarian reserve (DOR) is characterized by a decrease in the number and quality of oocytes, with its incidence increasing annually. Its pathogenesis remains unclear, making it one of the most challenging problems in the field of assisted reproduction. Epigenetic modification, a molecular mechanism affecting genomic activity and expression without altering the DNA sequence, has been widely studied in reproductive medicine and has attracted considerable attention regarding DOR. This review comprehensively examines the various epigenetic regulatory changes in ovarian granulosa cells (OGCs) and oocytes during DOR. DNA methylation plays a crucial role in regulating granulosa cell function, hormone production, and oocyte development, maturation, and senescence. Histone modifications are involved in regulating follicular activation, while non-coding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate granulosa cell function and oocyte development. N6-methyladenosine (m6A) modifications are associated with age-related oocyte senescence. Epigenetic clocks based on DNA methylation show potential in predicting ovarian reserve in DOR. Furthermore, it discusses the potential for utilizing epigenetic mechanisms to better diagnose and manage DOR.

Follicular fluid (FF) plays a vital role in the bidirectional communication between oocytes and granulosa cells (GCs), regulating and promoting oocyte growth and development. This fluid constitutes a complex microenvironment, rich in various molecules including hormones, growth factors, cytokines, lipids, proteins, and extracellular vesicles. Understanding the composition and metabolic profile of follicular fluid is important for investigating ovarian pathologies such as polycystic ovary syndrome (PCOS) and endometriosis. Additionally, analyzing follicular fluid can offer valuable insights into oocyte quality, aiding in optimal oocyte selection for in vitro fertilization (IVF). This review provides an overview of follicular fluid composition, classification of its components and discusses the influential components of oocyte development. It also highlights the role of follicular fluid in the pathogenesis and diagnosis of ovarian diseases, along with potential follicular fluid biomarkers for assessing oocyte quality. By understanding the intricate relationship between follicular fluid and oocyte development, we can advance fertility research and improve clinical outcomes for infertility patients.

To determine how clinical, demographic, and laboratory characteristics influence ovarian tissue oocyte quality.

Immature cumulus-oocyte complexes were isolated from removed ovaries and cultured for 48-52 h in either monophasic standard or biphasic CAPA media for fertility preservation. A total of 355 MII oocytes from 53 patients were described for intracytoplasmic and extracytoplasmic anomalies. Multiple clinical, laboratory, and demographic characteristics were analyzed. Statistically significant differences between independent groups in qualitative variables were identified using Pearson's χ2 and Fisher's exact tests. The diagnostic value of quantitative variables was assessed using the ROC curve analysis. Factors associated with the development of dysmorphism, taking patient age into account, were identified using the binary logistic regression analysis.

Dysmorphisms were observed in 245 oocytes (69.0%), with a median number of dysmorphisms of 2. Oocyte dysmorphisms were found to be 2.211 times more likely to be detected in patients with ovarian cancer, while the presence of dark-colored cytoplasm was associated with gynecologic surgery in the anamnesis (p = 0.002; OR 16.652; 95% CI, 1.977-140.237; Cramer's V 0.187). Small polar bodies developed 2.717 times more often (95% CI, 1.195-6.18) in patients older than 35. In the case of ovarian transportation on ice at 4 ℃, the chances of development of cytoplasmic granularity increased 2.569 times (95% CI, 1.301-5.179). The use of biphasic CAPA IVM media contributed to a decrease in the probability of large polar body formation (p = 0.034) compared to the standard monophasic IVM media.

Both patients' characteristics and laboratory parameters have an impact on the quality of IVM ovarian tissue oocytes.

Previous in vitro studies have suggested that SLIT ligands could play roles in regulating ovarian granulosa cell proliferation and gene expression, as well as luteolysis. However, no in vivo study of Slit gene function has been conducted to date. Here, we investigated the potential role of Slit1 in ovarian biology using a Slit1-null mouse model. Female Slit1-null mice were found to produce larger litters than their wild-type counterparts due to increased ovulation rates. Increased ovarian weights in Slit1-null animals were found to be due to the presence of greater numbers of healthy antral follicles with similar numbers of atretic ones, suggesting both an increased rate of follicle recruitment and a decreased rate of atresia. Consistent with this, treatment of cultured granulosa cells with exogenous SLIT1 induced apoptosis in presence or absence of follicle-stimulating hormone, but had no effect on cell proliferation. Although few alterations in the messenger RNA levels of follicle-stimulating hormone-responsive genes were noted in granulosa cells of Slit1-null mice, luteinizing hormone target gene mRNA levels were greatly increased. Finally, increased phospho-AKT levels were found in granulosa cells isolated from Slit1-null mice, and SLIT1 pretreatment of cultured granulosa cells inhibited the ability of both follicle-stimulating hormone and luteinizing hormone to increase AKT phosphorylation, suggesting a mechanism whereby SLIT1 could antagonize gonadotropin signaling. These findings therefore represent the first evidence for a physiological role of a SLIT ligand in the ovary, and define Slit1 as a novel autocrine/paracrine regulator of follicle development.

Follicular wave synchronization and follicular superstimulation with FSH are commonly used in OPU-IVP programs to increase oocyte developmental competence. Factors like Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), from the TGF beta superfamily, are produced by the oocyte and modulate follicular function. The aim of this study was to analyze the FSH-induced effects on (1) embryo production in dual-purpose Simmental cattle, and (2) TGF beta-mediated effects on oocyte-granulosa cell communication. Simmental heifers (n = 12, age 484 ± 62 days) underwent two OPU-IVP cycles in a cross-over design. Follicular waves were synchronized using 0.5 mg cloprostenol on Day 0, followed by 10 μg buserelin on Day 2. Subsequently, half of the heifers were randomly assigned to receive FSH/LH (four injections of 75 IU FSHp and 75IU LHp, 12 h apart on Days 4 and 5) before the first OPU, while the remaining heifers received FSH/LH before the second OPU. At the time of OPU, i.e. 7 days after the start of synchronization, granulosa cells were collected for RT-qPCR analysis. FSH treatment did not affect the number of oocytes collected (17.3 vs. 13.3, P > 0.05), but increased the percentage of quality 1 oocytes compared to controls (45.7 % vs. 22.0 %, P < 0.001). Neither cleavage (86.4 % vs. 85.7 %), nor blastocyst (42.1 % vs. 39.3 %) rate, or the number of transferable embryos produced by IVP (4.1 vs 4.8) was influenced by FSH treatment (P > 0.05 in all cases). FSH treatment increased HIF1A and FSHR levels in granulosa cells, while STAR was decreased (P = 0.008 in all cases). FSH treatment did not affect BMP15 or GDF9 mRNA expression (P > 0.05) but appeared to modulate the expression of genes involved in the BMP signaling pathway. Transcriptional levels of BMP15 receptor (BMPR1A, P = 0.016), and its downstream signaling factor SMAD1 (P = 0.008) were affected by FSH treatment. Our results demonstrated no benefit of this FSH stimulation protocol on IVP results in Simmental heifers. Further, our results suggest that the effects of FSH on bovine oocytes during acquisition of developmental competence may be mediated through BMP, but do not involve the regulation of transcriptional availability of GDF9, providing new insights into possible paracrine effects of the oocyte on granulosa cells.

To date, there is no comparative data on the effects of carbohydrates, fat, and proteins, which are macronutrients, on female reproductive functions. Therefore, in this study, we investigated the effects of diets enriched with carbohydrates, fats, and proteins on folliculogenesis and oocyte development in female rats. 21-day-old rats that were just weaned were divided into 4 groups: control, carbohydrate, fat, and protein. The control group was fed with standard chow and the carbohydrate, fat, and protein groups were fed diets enriched with 75% carbohydrate, 60% fat, and 50% protein for 11 weeks, respectively. It was found that high-fat and high-protein diets caused an increase in the estrous cycle length compared to carbohydrate group (p < 0.05). Graafian follicle number decreased in the protein group compared to the control (p < 0.05). However, the atretic follicle number was higher in the fat group compared to the control group (p < 0.05). In the carbohydrate group, Zp1 was found to be lower than the control and protein groups, Zp2 was found to be lower than the control, and Zp3 was found to be lower than the fat group (p < 0.05). While BMP15 was similar between groups (p > 0.05), GDF9 was lower in all diet groups compared to the control (p < 0.05). Foxo3a was lower in the protein group compared to carbohydrate and control (p < 0.05). GAS2 was found to be higher in the control group than the fat group, and higher in the carbohydrate group than the fat and protein groups (p < 0.05). FSH, LH, Progesterone, and E2 levels were higher in all three diet groups than in the control (p < 0.05). Also, significant differences were observed between the groups regarding adiponectin, resistin, and leptin levels. Taken together, high carbohydrate, fat, and protein intake are associated with impairment of the menstrual cycle, depletion of the developing follicle types, and altered expression of folliculogenesis-specific genes and hormones. Therefore, long-term macronutrient diets may result in shortened reproductive periods and reduced fertilization potential in females in the long run.

Concerns about inflammation-related issues affecting female reproductive health are growing. Chronic low-grade inflammation in women with polycystic ovarian syndrome (PCOS) affects follicular growth, ovulation, and androgen production. The present investigation aimed to elucidate the efficacy of flavonoid patuletin in ameliorating the letrozole-induced PCOS and associated inflammation in rats. Female Wistar rats (32 days old) were divided into five groups (n = 12): Group I, control; Group II, vehicle control; Group III, letrozole oral (1 mg/kg) for 28 days; Group IV and Group V treatment groups, patuletin i.p. (25 mg/kg) and clomiphene citrate + metformin i.p. (50 mg/kg + 300 mg/kg), respectively. Leterozole-induced PCOS and ovarian inflammation were ameliorated by patuletin, as reflected in the improved histopathology, prevention of cyst formation, significant upregulation of growth factors such as growth differentiation factor 9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) expression, and a decrease in the pro-inflammatory cytokines TNF-α, IL-6, and COX-2. Additionally, the plasma levels of reproductive hormones were restored. Upregulation of FSH-R, PR, and CYP19a1, along with downregulation of ERα, LHR, CYP17a1, CYP11a1 and HSDβ17a1, showed the regulation of gonadotropin receptors and steroid biosynthesis genes in ovarian tissues. Patuletin demonstrated a promising protective approach against the biological model of PCOS by increasing the inflammation in ovarian tissues with consequent regulation of growth factors, enzymes, and hormones, and might be used as adjuvant therapy in the treatment of problems related to female reproductive health.

Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.

Melatonin and L-carnitine are free radical scavengers with antiapoptotic and antioxidant properties that improve oocyte development.

This study aimed to find the possible effect of combining 2 antioxidant agents of melatonin and L-carnitine on oocyte morphology, maturation, apoptosis, and expression of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) genes in a mice model.

To overstimulation, 60 female NMRI mice were injected intraperitoneally using mare serum gonadotropin. On day 2 post-injection, 70 cumulus-oocyte complexes were collected from each mouse. The collected oocytes randomly were then divided into 4 groups including, the control, melatonin, L-carnitine, and melatonin + L-carnitine groups. The morphology and maturation rate of the oocytes was evaluated using a light microscope. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and the expression of BMP-15 and growth and differentiation factor GDF-9 genes was also evaluated by real-time polymerase chain reaction.

Oocyte diameter significantly was increased in combination treatment of L-carnitine and melatonin compared to other groups (p < 0.05). L-carnitine group showed the highest mean percentage of oocyte cytoplasmic pattern. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling indicated that the lowest apoptosis rate belonged to the melatonin + L-carnitine group. Moreover, the combination groups showed the highest number of oocytes and maturation rate. The BMP-15 and GDF-9 genes were significantly upregulated in all treatment groups compared to the control group.

Our results suggested a combination of melatonin + L-carnitine administration as a more effective choice for in vitro promotion of oocyte maturation.

Growth differentiation factor 9 (GDF-9) contributes to early ovarian development and oocyte survival. Higher concentrations of GDF-9 in follicular fluid (FF) are associated with oocyte nuclear maturation and optimal embryo development. In in vitro fertilization (IVF), GDF-9 affects the ability of the oocyte to fertilize and subsequent embryonic development. Bone morphogenetic protein 15 (BMP-15) is involved in the regulation of ovarian function and affects oocyte development. During IVF, BMP-15 contributes to the formation of competent blastocysts. BMP-15 may play a role in embryo implantation by affecting endometrial receptivity. Bone morphogenetic protein 4 (BMP-4) is involved in the regulation of follicle growth and development and affects granulosa cell (GC) differentiation. In relation to IVF, BMP-4 is important for embryonic development, influences cell fate and differentiation, and plays a role in facilitating embryo-endometrial interactions during the implantation process. Extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with ovulation and follicle rupture, promotes the release of mature eggs, and affects the modification of the extracellular matrix of the follicular environment. In IVF, EMMPRIN is involved in embryo implantation by modulating the adhesive properties of endometrial cells and promotes trophoblastic invasion, which is essential for pregnancy to occur. The purpose of the current article is to review the studies and recent findings of GDF-9, BMP-15, BMP-4 and EMMPRIN as fundamental factors in normal follicular development and in vitro fertilization.

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor β (Esr2/ERβ) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERβ and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERβ and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERβ and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERβ and GPER in cumulus cells.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.

Ovarian follicle culture is a powerful tool to study follicular physiology and has potential applications in clinical and commercial settings. Despite remarkable progress, recreating folliculogenesis in vitro remains challenging for many mammalian species. This study investigates the impact of platelet-rich plasma (PRP) derived from adult blood (human platelet lysate, hPL) and umbilical cord blood (Umbilical cord plasma, UCP) on murine pre-antral follicle culture and oocyte maturation. Pre-antral follicles were cultured individually for 10 days with fetal bovine serum (FBS) serving as the control and two PRP sources (hPL and UCP) and their activated forms (Ac-hPL and Ac-UCP). The results suggest that neither hPL nor UCP, regardless of activation status, improved follicle culture outcomes compared to FBS. Interestingly, activation did not significantly impact the main functional outcomes such as maturation rates, survival, and growth. Oestradiol secretion and oocyte diameter, often considered hallmarks of follicle quality, did not show significant differences between matured and non-matured oocytes across the treatment groups. However, gene expression analysis revealed a significant upregulation of Gdf-9 and Bmp-15 mRNA levels in oocytes from the Ac-UCP group, regardless of maturation stage, suggesting that the accumulation of the mRNA could be due to potential challenges in translation in the Ac-UCP group. In conclusion, this study challenges the hypothesis that PRP, as a serum source, could improve follicle culture outcomes compared to FBS, the gold standard in murine follicle culture. Further research is needed to understand the species-specific effects of PRP and explore other potential factors affecting follicle culture and oocyte quality.

The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro.

In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods.

The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions.

The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

In this study, we aimed to investigate the effect of the expression of miR-155-5p and its target genes on oocyte maturation. We analyzed the expression of miR-155-5p and its target genes in cumulus cells and oocytes using quantitative real-time reverse-transcription polymerase chain reaction. Using carboxyfluorescein, porcine cumulus cells were transfected with mimics and inhibitors of ssc-miR-155-5p to induce in vitro maturation, and subsequently, cumulus expansion, oocyte maturation, and cleavage rate were measured. We found that miR-155-5p expression in cumulus cells at the metaphase II stage was significantly higher than that at the germinal vesicle (GV) stage, whereas Cx43 expression was significantly lower than that at the GV stage (P < 0.05). Compared with those in the negative control group, the cumulus diffusion area of cumulus oocyte complexes; oocyte maturation rate; cleavage rate; HAS2, PTGS2, CD44, PTX3, and TNFAIP6 expression in cumulus cells; and GDF9, BMP15, CyclinB1, and CDK1 expression in oocytes were significantly increased in the miR-155-5p mimics group (P < 0.05), whereas the mRNA and protein expression of CX43 were significantly decreased (P < 0.05). Compared with that in the negative control group, the protein expression of CyclinB1 and p-CDK1 (Thr14, Tyr15) in the miR-155-5p mimics group was significantly increased (P < 0.05). These results suggest that miR-155-5p regulates maturation promoting factor activity by targeting Cx43, which improves the in vitro maturation and cleavage rate of porcine oocytes.

Early antral follicles (EAFs) represent the transitional stage between pre-antral and antral follicles, containing oocytes that have completed most of their growth phase. Therefore, they offer an easily exploitable reserve for producing mature oocytes and preserving genetic resources, given their higher abundance compared to antral follicles (AFs) and shorter culture period than other pre-antral follicles (PAFs). Despite these advantages, the culture of EAFs remains challenging, and the success rates of in vitro embryo production (IVEP) from EAF-derived oocytes are still far below the standard achieved with fully grown oocytes in ruminant species. The difficulty is related to developing suitable in vitro culture systems tailored with nutrients, growth factors, and other signaling molecules to support oocyte growth. In this review, we focus on the in vitro development of sheep EAFs to provide an informative reference to current research progress. We also summarize the basic aspect of folliculogenesis in sheep and the main achievements and limitations of the current methods for EAF isolation, in vitro culture systems, and medium supplementation. Finally, we highlight future perspectives and challenges for improving EAF culture outcomes.

Female reproductive efficiency is influenced by the outcomes of various processes, including folliculogenesis, apoptosis, response to gonadotropin signaling, oocyte maturation, and ovulation. The role of hormones in regulating these processes and other reproductive activities has been well established. It is becoming increasingly evident that in addition to well-characterized hormones, growth factors play vital roles in regulating some of these reproductive activities. Growth factors and their receptors are widely distributed in vertebrate ovaries at different stages of ovarian development, indicating their involvement in intraovarian reproductive functions. In the ovary, cell surface receptors allow growth factors to regulate intraovarian reproductive activities. Understanding these actions in the reproductive axis would provide a tool to target growth factors and/or their receptors to yield desirable reproductive outcomes. These include enrichment of in vitro maturation and fertilization culture media, and management of infertility. This review discusses some widely characterized growth factors belonging to the TGF, EGF, IGF, FGF, and BDNF family of peptides and their role in female reproduction in vertebrates, with a focus on mammals.

Among the natural polyphenolic compounds, resveratrol (RES) is known for reducing the effects of declining reproductive power through resisting senility, anti-oxidant and anti-inflammatory, while the molecular mechanism of RES in human ovaries is unclear. We aimed to evaluate the most likely mechanisms of RES against apoptosis induced by H₂O₂ in human ovary granulosa cells.

Ovarian granulosa cells from infertile women (≤35 years old) were collected. Those patients defined as polycystic ovary syndrome (PCOS), poor ovarian responder (POR) and Endometriosis were excluded. Then they were randomly divided into control group, model group and the treatment group. Cellular apoptosis was analyzed by flow cytometer method. The related protein and mRNA expressions were detected by western blot and RT-PCR.

Apoptosis rates of the treatment group containing RES with concentrations of 1 μM and 10 μM were significantly decreased (p < 0.001). Western blot results demonstrated that the proteins levels of transforming growth factor-β (TGF-β), Bax and Caspase 9 were decreased, and Bcl-2 was increased under RES treatment, while the protein levels of Caspase 8, Caspase 3, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) expressed no significant difference. The results by RT-PCR of follicle and ovarian development related mRNA factors were consistent with that of western blot assay.

In conclusion, the present study provides the evidence that RES may affects apoptotic factors to protect human ovarian state.

The results of G1 and G4 polymorphisms as litter-size (LS) markers of ewes remain contradictory. The aim was to evaluate the impact of G1 (c.260 G>A) and G4 (c.721 G>A) polymorphisms on the LS of sheep by synthesizing data from multiple previous studies.

Data were extracted from 14 eligible articles. The genotypes of G1 and G4 polymorphisms were homozygous wild-type (WW), heterozygous (WM), and homozygous mutant-type (MM). The standardized mean difference (SMD) method using random effect models was employed to determine the effect size of G1 and G4 polymorphisms on LS under dominant, recessive, additive, and co-dominant genetic models. Heterogeneity was analyzed with the I2 statistic index. Publication bias was depicted with funnel plots and tested by Egger's and Begg's tests.

The study showed that the correlation between G1 polymorphism and LS in sheep was not significant (p > 0.05) under all genetic models. The influence of G4 polymorphism on the LS of sheep was found significantly (p < 0.05) under dominant [SMD = 0.28, I2 = 0% (no heterogeneity)] and co-dominant [SMD = -0.14, I2 = 36% (moderate heterogeneity)] genetic models. The WM genotype of G4 polymorphism increased LS, while the MM genotype reduced LS in sheep. Publication bias among G1 and G4 polymorphism studies was absent in all genetic models.

Thus, the study revealed that G4 polymorphism could be a potential genetic marker for LS in ewes. On the contrary, G1 polymorphism has no association with the LS of ewes.

FIGLA and NOBOX are important oocyte-specific transcription factors. Both figla-/- and nobox-/- mutants showed all-male phenotype in zebrafish due to increased dominance of the male-promoting pathway. The early diversion towards males in these mutants has precluded analysis of their roles in folliculogenesis. In this study, we attenuated the male-promoting pathway by deleting dmrt1, a key male-promoting gene, in figla-/- and nobox-/- fish, which allows a sufficient display of defects in folliculogenesis. Germ cells in figla-/-;dmrt1-/- double mutant remained in cysts without forming follicles. In contrast, follicles could form well but exhibited deficient growth in nobox-/-;dmrt1-/- double mutants. Follicles in nobox-/-;dmrt1-/- ovary could progress to previtellogenic (PV) stage but failed to enter vitellogenic growth. Such arrest at PV stage suggested a possible deficiency in estrogen signaling. This was supported by lines of evidence in nobox-/-;dmrt1-/-, including reduced expression of ovarian aromatase (cyp19a1a) and level of serum estradiol (E2), regressed genital papilla (female secondary sex characteristics), and more importantly the resumption of vitellogenic growth by E2 treatment. Expression analysis suggested Nobox might regulate cyp19a1a by controlling Gdf9 and/or Bmp15. Our discoveries indicate that Figla is essential for ovarian differentiation and follicle formation whereas Nobox is important for driving subsequent follicle development.

Mammalian eggs (oocytes) are formed during fetal life and establish associations with somatic cells to form primordial follicles that create a store of germ cells (the primordial pool). The size of this pool is influenced by key events during the formation of germ cells and by factors that influence the subsequent activation of follicle growth. These regulatory pathways must ensure that the reserve of oocytes within primordial follicles in humans lasts for up to 50 years, yet only approximately 0.1% will ever be ovulated with the rest undergoing degeneration. This review outlines the mechanisms and regulatory pathways that govern the processes of oocyte and follicle formation and later growth, within the ovarian stroma, through to ovulation with particular reference to human oocytes/follicles. In addition, the effects of aging on female reproductive capacity through changes in oocyte number and quality are emphasized, with both the cellular mechanisms and clinical implications discussed. Finally, the details of current developments in culture systems that support all stages of follicle growth to generate mature oocytes in vitro and emerging prospects for making new oocytes from stem cells are outlined.

As an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by a complete arrest of follicle development at previtellogenic (PV) stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal in bmp15 mutant could be partially rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic (MV) stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-β signaling and endocytosis in the double mutant follicles. Interestingly, the expression of inhibin/activin βAa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females (bmp15-/-), returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the arrested follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. The loss of inhbaa also caused a reduction of Vtg receptor-like molecules (e.g., lrp1ab and lrp2a). In summary, the present study provided comprehensive genetic evidence that Bmp15 acts together with the activin-inhibin system in the follicle to control E2 production from the follicle, Vtg biosynthesis in the liver and its uptake by the developing oocytes.

Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors.

In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA.

Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05).

Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.

The aim of the present study was to clarify the effects of arginine vasopressin (AVP) on ovarian steroid production and its functional relationship to the ovarian bone morphogenetic protein (BMP) system. The results showed that AVP treatment significantly increased gonadotropin- and forskolin-induced progesterone synthesis by primary culture of rat granulosa cells and human granulosa cells, respectively. In contrast, estradiol production was not significantly affected by AVP. Treatment with AVP significantly increased forskolin-induced cAMP synthesis by human granulosa cells and mRNA levels of the progesterogenic enzymes CYP11A1 and HSD3B2 in the cells. On the other hand, AVP also enhanced BMP-15-induced phosphorylation of SMAD1/5/9 and ID1 transcription. It was further revealed that the expression levels of BMP receptors, including ALK3, ALK6 and BMPR2, were upregulated by AVP. Collectively, the results indicate that AVP stimulates progesterone production via the cAMP-PKA pathway with upregulation of BMP signaling that inhibits progesterone production, which may lead to fine adjustment of progesterone biosynthesis by granulosa cells.

Premature ovarian failure (POF) is an insidious cause of female infertility and a devastating condition for women. POF also has a strong familial and heterogeneous genetic background. Management of POF is complicated by the variable etiology and presentation, which are generally characterized by abnormal hormone levels, gene instability and ovarian dysgenesis. To date, abnormal regulation associated with POF has been found in a small number of genes, including autosomal and sex chromosomal genes in folliculogenesis, granulosa cells, and oocytes. Due to the complex genomic contributions, ascertaining the exact causative mechanisms has been challenging in POF, and many pathogenic genomic characteristics have yet to be elucidated. However, emerging research has provided new insights into genomic variation in POF as well as novel etiological factors, pathogenic mechanisms and therapeutic intervention approaches. Meanwhile, scattered studies of transcriptional regulation revealed that ovarian cell function also depends on specific biomarker gene expression, which can influence protein activities, thus causing POF. In this review, we summarized the latest research and issues related to the genomic basis for POF and focused on insights gained from their biological effects and pathogenic mechanisms in POF. The present integrated studies of genomic variants, gene expression and related protein abnormalities were structured to establish the role of etiological genes associated with POF. In addition, we describe the design of some ongoing clinical trials that may suggest safe, feasible and effective approaches to improve the diagnosis and therapy of POF, such as Filgrastim, goserelin, resveratrol, natural plant antitoxin, Kuntai capsule et al. Understanding the candidate genomic characteristics in POF is beneficial for the early diagnosis of POF and provides appropriate methods for prevention and drug treatment. Additional efforts to clarify the POF genetic background are necessary and are beneficial for researchers and clinicians regarding genetic counseling and clinical practice. Taken together, recent genomic explorations have shown great potential to elucidate POF management in women and are stepping from the bench to the bedside.

Mammalian female reproduction requires a functional ovary. Competence of the ovary is determined by the quality of its basic unit-ovarian follicles. A normal follicle consists of an oocyte enclosed within ovarian follicular cells. In humans and mice, the ovarian follicles are formed at the foetal and the early neonatal stage respectively, and their renewal at the adult stage is controversial. Extensive research emerges recently to produce ovarian follicles in-vitro from different species. Previous reports demonstrated the differentiation of mouse and human pluripotent stem cells into germline cells, termed primordial germ cell-like cells (PGCLCs). The germ cell-specific gene expressions and epigenetic features including global DNA demethylation and histone modifications of the pluripotent stem cells-derived PGCLCs were extensively characterized. The PGCLCs hold potential for forming ovarian follicles or organoids upon cocultured with ovarian somatic cells. Intriguingly, the oocytes isolated from the organoids could be fertilized in-vitro. Based on the knowledge of in-vivo derived pre-granulosa cells, the generation of these cells from pluripotent stem cells termed foetal ovarian somatic cell-like cells was also reported recently. Despite successful in-vitro folliculogenesis from pluripotent stem cells, the efficiency remains low, mainly due to the lack of information on the interaction between PGCLCs and pre-granulosa cells. The establishment of in-vitro pluripotent stem cell-based models paves the way for understanding the critical signalling pathways and molecules during folliculogenesis. This article aims to review the developmental events during in-vivo follicular development and discuss the current progress of generation of PGCLCs, pre-granulosa and theca cells in-vitro.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Today, fertility preservation is receiving more attention than ever. Cryopreservation, which preserves ovarian tissue to preserve fertility in young women and reduce the risk of infertility, is currently the most widely practiced. Transplantation, however, is less feasible for women with blood-borne leukemia or cancers with a high risk of ovarian metastasis because of the risk of cancer recurrence. In addition to cryopreservation and re-implantation of embryos, in vitro ovarian organ reconstruction techniques have been considered as an alternative strategy for fertility preservation. In vitro culture of oocytes in vitro Culture, female germ cells induction from pluripotent stem cells (PSC) in vitro, artificial ovary construction, and ovaria-related organoids construction have provided new solutions for fertility preservation, which will therefore maximize the potential for all patients undergoing fertility preservation. In this review, we discussed and thought about the latest ovarian organ function reconstruction techniques in vitro to provide new ideas for future ovarian disease research and fertility preservation of patients with cancer and premature ovarian failure.

Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9-/-) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9-/- and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9-/-), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9-/- gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.

To analyze the level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in follicle fluid (FF) and granulosa cells (GCs) derived from young patients with low prognosis for in vitro fertilization and embryo transfer (IVF-ET) treatment.

A prospective cohort study was carried out by enrolling 52 young patients with low prognosis according to the POSEIDON classification group 3 (low prognosis group) and 51 young patients with normal ovarian reserve (control group). The concentration of the GDF9 and BMP15 proteins in FF was determined by enzyme-linked immunosorbent assay. The mRNA level of the GDF9 and BMP15 in the GCs was measured by quantitative real-time PCR.

The concentration of GDF9 (1026.72 ± 159.12 pg/mL vs. 1298.06 ± 185.41 pg/mL) and BMP15 (685.23 ± 143.91 pg/mL vs. 794.37 ± 81.79 pg/mL) in FF and the mRNA level of GDF9 and BMP15 in the GCs and the live birth rate per treatment cycle started (30.77% vs. 50.98%) and oocytes retrieved (4.25 ± 1.91 vs.12.04 ± 4.24) were significantly lower, whereas the canceled cycle rate was significantly higher (9.62% vs. 0) in the low prognosis group compared with the control group (P < 0.05). The expression of GDF9 and BMP15 in the ovary was positively correlated with live birth (P < 0.05).

The expression of GDF9 and BMP15 in the ovary was decreased in young patients with low prognosis accompanied by a poorer outcome of IVF-ET treatment.

ChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. ( http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4 ).

Growth differentiation factor 9 (GDF9) is an oocyte-derived protein with fundamental functions in folliculogenesis. While the crucial contributions of GDF9 in follicular survival have been revealed, crystallographic studies of GDF9 structure have not yet been carried out, essentially due to the insoluble expression of GDF9 in E. coli and lack of appropriate source for structural studies. Therefore, in this study, we investigated the impact of different expression rate of bacterial thioredoxin (TrxA) using bicistronic expression constructs to induce the soluble expression of mature human GDF9 (hGDF9) driven by T7 promoter in E. coli. Our findings revealed that in BL21(DE3), the high rate of TrxA co-expression at 30 °C was sufficiently potent for the soluble expression of hGDF9 and reduction of inclusion body formation by 4 fold. We also successfully confirmed the bioactivity of the purified soluble hGDF9 protein by evaluation of follicle-stimulating hormone receptor gene expression in bovine cumulus cells derived from small follicles. This study is the first to present an effective approach for expression of bioactive form of hGDF9 using TrxA co-expression in E. coli, which may unravel the current issues regarding structural analysis of hGDF9 protein and consequently provide a better insight into hGDF9 functions and interactions.

Kisspeptin is a neuropeptide that has an important role in the female reproductive cycle which is indicated by its role in regulating the hypothalamic-pituitary-gonadal axis.

To analyze the correlation between serum kisspeptin levels, ovarian kisspeptin expression, and ovarian Bone Morphogenic Protein-15 (BMP15) expression in polycystic ovary syndrome (PCOS) model rats.

The research was accurate experimental research with a post-test design-only control group and was carried out from August to October 2022 at the Faculty of Veterinary Medicine Universitas Airlangga. 32 Rattus novergicus rats were divided into a control group and a PCOS model group. Blood serum and ovaries were obtained from all groups. In addition, blood serum was examined for kisspeptin levels by ELISA technique, and kisspeptin expression and BMP15 Ovaries were examined immunohistochemically.

Serum kisspeptin levels and ovarian kisspeptin expression of the PCOS model group were not significantly higher than those of the control group (p > 0.05, p > 0.05). The ovarian BMP15 expression of the PCOS model group was not significantly lower (p > 0.05) than that of the control group. Ovarian kisspeptin expression and ovarian BMP15 expression did not significantly correlate with serum kisspeptin levels (p > 0.05). In contrast, there was a significant correlation (p < 0.05) between ovarian kisspeptin expression and ovarian BMP15 expression.

Serum kisspeptin levels and ovarian kisspeptin expression of the PCOS model group were not higher than those of the control group, and the ovarian BMP15 expression of the PCOS model group was not lower than that of the control group. There was no correlation between serum kisspeptin levels with ovarian kisspeptin expression and ovarian BMP15 expression. However, a significant correlation was found between ovarian kisspeptin expression and ovarian BMP15 expression.

Our study aims to investigate the efficacy and clinical significance of the Zuogui pill (ZGP) on premature ovarian failure (POF) via the GDF-9/Smad2 pathway. Changes in clinical symptoms in the control group (treated with Femoston alone) and the treatment group (treated with ZGP combined with Femoston) were assessed before and after treatment. Sex hormone levels, serum inflammatory cytokine levels, and ultrasound parameters were measured before and after treatment. POF rat models were established using cyclophosphamide and the POF rats were treated with Femoston, or ZGP combined with Femoston. GDF-9 and Smad2 expression levels were determined by RT-qPCR. The follicle-stimulating hormone (FSH), luteinizing hormone (LH), interleukin (IL)-6, and IL-21 levels, and the pulsatility index (PI) and resistance index (RI) values were decreased, while the estradiol (E2) and anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), ovarian volume (OV), mean ovarian diameter (MOD), and peak systolic velocity (PSV) values were increased in the treatment group compared to the control group. After treatment with ZGP combined with Femoston, GDF-9 and Smad2 expression in the ovarian tissues of POF rats increased. ZGP has a therapeutic effect on POF via modulation of the GDF-9/Smad2 pathway.

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.

Growth differentiation factor 9 (GDF9) was the first oocyte-specific growth factor identified; however, most information about GDF9 functions comes from studies in the mouse model. In this study, we created a mutant for Gdf9 gene (gdf9-/-) in zebrafish using TALEN approach. The loss of Gdf9 caused a complete arrest of follicle development at primary growth (PG) stage. These follicles eventually degenerated, and all mutant females gradually changed to males through sex reversal, which could be prevented by mutation of the male-promoting gene dmrt1. Interestingly, the phenotypes of gdf9-/- could be rescued by simultaneous mutation of inhibin α (inha-/-) but not estradiol treatment, suggesting a potential role for the activin-inhibin system or its signaling pathway in Gdf9 actions. In gdf9-null follicles, the expression of activin βAa (inhbaa), but not βAb (inhbab) and βB (inhbb), decreased dramatically; however, its expression rebounded in the double mutant (gdf9-/-;inha-/-). These results indicate clearly that the activation of PG follicles to enter the secondary growth (SG) requires intrinsic factors from the oocyte, such as Gdf9, which in turn works on the neighboring follicle cells to trigger follicle activation, probably involving activins. In addition, our data also support the view that estrogens are not involved in follicle activation as recently reported.

Premature ovarian failure (POF) is characterized by hormonal disorders, amenorrhea, and premature loss of fertility potential in women of reproductive age. Several studies have been conducted on the effectiveness of traditional Chinese medicine (TCM) in treating POF. TCM relied primarily on apoptosis, immunity, and aging to treat POF based on the studies of domestic and foreign literature. Zuogui pills inhibited mitochondrial-dependent apoptosis in the treatment of POF. Huyang Yangkun formula regulated the downstream of the Bcl-2 family to resist apoptosis through the aquaporin-1 protein. Modified Bazhen decoction regulated apoptosis in POF by regulating X-linked inhibitors of apoptosis protein. Bushen Tianjing recipe was effective in treating POF by promoting angiogenesis and preventing apoptosis. As for immunity, Bushen Jianpi prescription and Er-Xian decoction cured autoimmunity POF models and increased follicular development-related protein expression. Bushen Huoxue Tang improved ovarian function and reduced ovarian inflammation by regulating the Nrf2/Keap1 signaling pathway and T lymphocytes. Taohong Siwu decoction promoted the proliferation and differentiation of granulosa cells of POF mice by regulating the TGF-β1/Smads signaling pathway. In addition, ginsenoside Rg1 and Jiajian Guisheng formula treated POF by regulating cell aging-related mechanisms. Si Wu Tang treated POF by activating the angiogenesis-related proteins. The goal of this review is to serve as a reference for in-depth research into the treatment of POF with TCM and provide inspiration for new diagnostic methods and treatment options.

The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo's morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.

Premature ovarian insufficiency (POI) occurs in women before the age of 40 years, accompanied by amenorrhea, hypoestrogenism, hypergonadotropinism, and infertility. The pathology of POI is complex and the molecular genetic mechanisms are poorly understood. Bone morphogenetic protein 15 (BMP15) plays a crucial role in oocyte maturation and follicular development through the activation of granulosa cells. Dysfunction of BMP15 causes ovarian dysgenesis and is related to POI. Identifying pathogenic variants contributes to revealing genetic mechanisms and making clinical diagnoses of POI.

The study involved two sisters diagnosed with POI. Whole-exome sequencing (WES) was performed to identify causative genes. Sanger sequencing was used to validate the mutations in patients with POI and members of the family with no clinical signs or symptoms. The effect of the novel mutations on the BMP15 structure was analyzed by PSIPRED. By over-expressing wild-type (WT) or mutant BMP15 plasmids in vitro, a functional study of the BMP15 mutant was conducted by real-time qPCR and western blotting. Through cocultivation with HEK293T cells, the effects of secreted BMP15 WT and variants on granulosa cell proliferation and apoptosis were detected through a cell counting kit-8 assay and flow cytometric analysis.

We identified biallelic variants in BMP15, c.791G > A (p. R264Q) and c.1076C > T (p. P359L), in two siblings with POI. Both sisters carried the same biallelic variants, while the other female members of their family carried only one of them. Structural prediction showed that the variants have not affected the secondary structure of BMP15 but may change the conformation of water molecules around protein surfaces and thermal stability of BMP15. Real-time qPCR showed no significant difference in mRNA levels among WT and the two variants. Western blotting indicated a reduction in BMP15 expression with the c.791G > A and c.1076C > T variants compared to WT. Moreover, mutants 791G > A and 1076C > T impaired the function of secreted BMP15 in promoting granulosa cell proliferation and suppressing cell apoptosis caused by reactive oxygen species.

This study identified novel biallelic variants, c.791G > A and c.1076C > T, of BMP15 in two siblings with POI. Both missense variants reduced the level of the BMP15 protein and impaired the function of BMP15 in promoting granulosa cell proliferation in vitro. Taken together, our findings provide a novel molecular genetic basis and potential pathogenesis of BMP15 variants in POI.

The suggested effects of the oocyte secreted GDF9 and BMP15 growth factors on oocyte maturation are currently based on recombinant proteins, and little is known about native GDF9 and BMP15 in humans.

Human immature cumulus-oocyte complexes (COCs) obtained in connection with ovarian tissue cryopreservation (OTC) underwent in vitro maturation (IVM). Oocyte-produced GDF9 and BMP15 were detected in COCs using immunofluorescence, and in fresh GV oocytes and in GV and MII oocytes after IVM by western blot. Concentrations of GDF9, BMP15 homodimers, and GDF9/BMP15 heterodimer in spent media after IVM were measured by ELISA. The relative expression of seven genes from the GDF9 and BMP15 signaling pathways (BMPR2, ALK5, ALK6, SMAD1, SMAD2, SMAD3, and SMAD5) was evaluated in fresh cumulus cells (before IVM) and in cumulus cells from GV and MII oocytes after IVM by RT-qPCR.

We detected native pro-mature GDF9 and BMP15 in human oocytes with molecular weights (Mw) of 47 kDa and 43 kDa, respectively. Concentrations of GDF9 and BMP15 in spent media after IVM were detected in 99% and 64% of the samples, respectively. The GDF9/BMP15 heterodimer was detected in 76% of the samples. Overall, the concentration of GDF9 was approximately 10-times higher than BMP15. The concentrations of both GDF9 and BMP15 were significantly lower in spent medium from MII oocytes than in media from oocytes that remained at the GV stage. Concentrations of the GDF9/BMP15 heterodimer did not differ between GV and MII oocytes. Furthermore, BMPR2, SMAD3, and SMAD5 were significantly upregulated in cumulus cells from MII oocytes, indicating that both GDF9 and BMP15 signaling were active during oocyte meiotic resumption in vitro.

These data suggest that the driving mechanisms for oocyte nuclear maturation may involve both GDF9 and BMP15 homodimers, while the role of the GDF9/BMP15 heterodimer is questionable.