Novel Insight into the mechanism of di (2-ethylhexyl) phthalate (DEHP) impairing early follicle development.

Di (2-ethylhexyl) phthalate (DEHP), an artificially synthetic plasticizer, is a widespread environmental endocrine disruptor, which has raised substantial concern among the public about its potential reproductive toxicity effects. Taking large amounts of DEHP disrupts the normal functioning of the ovaries, however, the toxicological effects and the mechanisms by which DEHP impairs fetal folliculogenesis remain poorly understood. Our research aims to elucidate the associations between utero exposure to DEHP and fetal folliculogenesis in offspring. In this research, we monitored the spatiotemporal and expression levels of GDF9-Hedgehog (Hh) pathway-related genes during postnatal days 3-14, confirming initially the potential associations between defects in theca cell development and the downregulation of GDF9-Hh signaling. Moreover, utilizing an ovarian organ in vitro culture model, rescue validation experiments demonstrated that the addition of recombinant GDF9 protein effectively alleviate the theca cell damage caused by DEHP, thus supporting the aforementioned associations. In conclusion, our findings validate the significant role of the GDF9-Hh pathway in the enduring reproductive toxicity resulting from prenatal exposure to DEHP.

The Roles of GDF-9, BMP-15, BMP-4 and EMMPRIN in Folliculogenesis and In Vitro Fertilization.

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.

Revisiting GDF9 variants in primary ovarian insufficiency: A shift from dominant to recessive pathogenicity?

BACKGROUND: Primary ovarian insufficiency (POI) affects around 2-4% of women before the age of 40. Genetic factors play an important role in POI. The GDF9 gene has been identified as a significant genetic contributor of POI. However, the pathogenicity and penetrance of GDF9 variants remain uncertain. METHODS: A next-generation sequencing approach was employed to investigate the entire coding region of the GDF9 gene in a cohort of 1281 patients with POI or diminished ovarian reserve (DOR). The frequency of each identified GDF9 variant was then compared with that of the general population, taking into account the ethnicity of each individual. RESULTS: By screening the entire coding region of the GDF9 gene, we identified 19 different variants, including 1 pathogenic frameshift variant. In total, 36 patients with POI/DOR (2.8%) carried at least one GDF9 variant. With regard to missense variants, no significant overrepresentation of the most common variants was observed in our POI/DOR cohort in comparison to the general or specific ethnic subgroups. Only one homozygous subject had a frameshift loss of function variant. CONCLUSION: This epidemiological study suggests that the vast majority of heterozygous missense variants could be considered as variants of uncertain significance and the homozygous loss-of-function variant could be considered as a pathogenic variant. The identification of a novel case of a homozygous POI patient with a heterozygous mother carrying the same variant with normal ovarian function strongly suggests that GDF9 syndrome is an autosomal recessive disorder.

Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype.

Background: Polycystic ovary syndrome (PCOS) is main cause of anovulatory infertility in women with gestational age. There are currently four distinct phenotypes associated with individualized endocrinology and metabolism. Growth differentiation factor 9 (GDF9) is a candidate as potential biomarker for the assessment of oocyte competence. The effect on oocyte capacity has not been evaluated and analyzed in PCOS phenotypes. Objective: We aimed to screen the expression levels of GDF9 in mature follicles of women with controlled ovarian hyperstimulation (COS) with different PCOS phenotypes. To determine the correlation between the expression level of GDF9 and oocyte development ability. Methods: In Part 1, we conducted a retrospective study comparing the clinical outcomes and endocrine characteristics of patients with PCOS according to different subgroups (depending on the presence or absence of the main features of polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)) and non-PCOS control group. We stratified PCOS as phenotype A (n = 29), phenotype B (n = 18) and phenotype D (n = 24). In Part 2, the expression of GDF9 in follicular fluid (FF) and cumulus cells (CCs) were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Results: In Part 1, the baseline clinical, hormonal, and ultrasonographic characteristics of the study population were matched with the presence or absence of the cardinal features of each PCOS phenotypes showed a clear difference. Phenotypes A and D had statistically significant associations with blastocyst formation and clinical pregnancy compared with phenotypes B (p < 0.001). In Part 2, the levels of GDF9 in FF and CCs for phenotype A and B were significantly were higher than those of phenotype D (P = 0.019, P = 0.0015, respectively). Multivariate logistic regression analysis showed that GDF9 was an important independent predictor of blastocyst formation (P＜0.001). The blastocyst formation rate of phenotype A was higher than that of phenotype B and D (P＜0.001). Combining the results of the two parts, GDF9 appears to play a powerful role in the development of embryos into blastocysts. Conclusions: GDF9 expression varies with different PCOS phenotypes. Phenotype A had higher GDF9 levels and blastocyst formation ability.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Detection of Booroola Polymorphism of Bone Morphogenetic Protein Receptor 1b and Embrapa Polymorphism of Growth Differentiation Factor 9 in Sheep in Thailand.

This study aimed to investigate the appearance and frequencies of the Booroola polymorphism of the bone morphogenetic protein receptor 1b (BMPR1B) gene (FecB) and the Embrapa polymorphism of the growth differentiation factor 9 (GDF9) gene (FecGE) in sheep in Thailand. A total of 454 crossbred sheep blood samples were collected from four provinces in Thailand during August 2022 to July 2023. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the FecB and FecGE genotypes. The history of ewe birth types was collected from the owners to analyze the association between fecundity (Fec) genotypes and the history of birth types. The genotypic frequencies of FecB for homozygous genotype (B/B), heterozygous genotype (+/B), and wildtype (+/+) were 0.22%, 1.54%, and 98.24%, respectively. Meanwhile, the genotypic frequencies of FecGE for homozygous genotype (E/E), heterozygous genotype (+/E), and wildtype (+/+) were 0.00%, 2.42%, and 97.58%, respectively. Furthermore, three ewes exhibited both FecB and FecGE genotypes. Fisher's exact test revealed that possession of the FecB genotype was associated with multiple births (p < 0.01). Both FecB and FecGE mutations were identified in crossbred sheep in Thailand. Sheep containing FecB allele could be alternative candidates to be selected to improve the prolificacy of crossbred sheep in Thailand.

Implication of Novel BMP15 and GDF9 Variants in Unexpected Poor Ovarian Response.

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.

A new technology for the treatment of premature ovarian insufficiency: Cell sheet.

Premature ovarian insufficiency (POI) is defined as the development of hypergonadotropic hypogonadism before the age of 40 with definitive treatment being absent. In the current study, we aim to compare the efficacy of the cell sheet method with an intravenous (IV) application of adipose-derived mesenchymal stem cells (AdMSCs) to the POI with an animal model. In the current prospective study, 6-to-8-week-old Sprague Dawley rats were generated four groups: (i) a control group in which only PBS was administered; (ii) an only-POI group generated by cyclophosphamide; (iii) a POI group treated by way of IV AdMSCs; and (iv) a POI group treated by way of the cell sheet method. Twenty-eight days after an oophorectomy was performed, intracardiac blood was taken. Follicle count, immunohistochemical examination for GDF9, BMP15, and TUNEL were conducted, gene expressions of GDF9 and BMP15 were examined, and E2 was measured in the serum samples. With hematoxylin-eosin, in the third group, multi oocytes follicles were the most remarkable finding. In the fourth group, most of the follicles presented normal morphology. GDF9 involvement was similar between the first and fourth groups. BMP-15 immunoreactivity, in contrast to fourth group, was weak in all stages in the second and third groups. The current attempt represents a pioneer study in the literature in which a cell sheet method is used for the first time in a POI model. These results suggest that the cell sheet method may be a feasible and efficient method for the stem cell treatment of models with POI and could be a new treatment approach in POI.

Vitrified turkey ovarian tissue cultured and assessed through gene expression patterns: A potential screening tool.

Biobanking of turkey ovarian tissue has the potential to play a crucial part in preserving female genetics. To date, ovarian tissue has only been vitrified using a standard protocol, with immediate analyses after warming, therefore, long-term cryoinjury is unknown. Long-term cryoinjury was investigated here by in-ovo culturing, fresh (non-vitrified), a purposefully suboptimal poor vitrification (PV), and the standard vitrified (StV) protocol. Assessments were performed via cellular morphological changes and mRNA gene expression differences, immediately (day 0) or after 2, 4, or 6 days of in-ovo culturing. On day 0, the mRNA levels of heat-shock protein A2 (HSPA2) were lowest in the fresh tissue, and increased 5-fold in the StV treatment, and 18-fold in the PV treatment. Whereas, by day 6, growth determining factor 9 (GDF9) mRNA levels within the fresh tissue were over 3-fold and 21-fold higher than StV and PV treatments, respectively. After 6 days of in-ovo culture the follicle density was highest in the fresh ovarian tissue (4701 ± 950 #/mm3), followed by the StV (1601 ± 300 #/mm3), with PV having the lowest density (172 ± 145 #/mm3). This shows that although the density of follicles was higher in StV versus PV, a considerable number (∼65 %) were lost compared to the fresh treatment. Additionally, the HSPA2 expression could be an early screening tool, whereas GDF9 expression could be a late screening tool, used to assess turkey ovarian tissue vitrification protocols. We conclude that the StV protocol should be further optimized to try and improve follicle numbers post-warming.

DNA methylation of GDF-9 and GHR genes as epigenetic regulator of milk production in Egyptian Zaraibi goat.

BACKGROUND: DNA methylation is an epigenetic mechanism that takes place at gene promoters and a potent epigenetic marker to regulate gene expression. OBJECTIVE: The study aimed to improve the milk production of Zaraibi goats by addressing the methylation pattern of two milk production-related genes: the growth hormone receptor (GHR) and the growth differentiation factor-9 (GDF-9). METHODS: 54 and 46 samples of low and high milk yield groups, respectively, were collected. Detection of methylation was assessed in two CpG islands in the GDF-9 promoter via methylation-specific primer assay (MSP) and in one CpG island across the GHR promoter using combined bisulfite restriction analysis (COBRA). RESULTS: A positive correlation between the methylation pattern of GDF-9 and GHR and their expression levels was reported. Breeding season was significantly effective on both peak milk yield (PMY) and total milk yield (TMY), where March reported a higher significant difference in PMY than November. Whereas single birth was highly significant on TMY than multiple births. The 3rd and 4th parities reported the highest significant difference in PMY, while the 4th parity was the most effective one on TMY. CONCLUSION: These results may help improve the farm animals' milk productive efficiency and develop prospective epigenetic markers to improve milk yield by epigenetic marker-assisted selection (eMAS) in goat breeding programs.