Compound heterozygous mutations in TBPL2 were identified in an infertile woman with impaired ovarian folliculogenesis.

OBJECTIVE: A 32-year-old female was diagnosed with unexplained primary infertility for 10 years. She had roughly normal basal hormone levels, but her basal follicle-stimulating hormone (FSH) levels were elevated. In addition, the level of anti-Mullerian hormone was within the normal range, and she had undergone two failed oocyte collection attempts. We aimed to investigate the genetic cause of female infertility in patients with impaired ovarian folliculogenesis. METHODS: Genomic DNA was extracted from the peripheral blood of the patient and her family members. Whole-exome sequencing was performed on the patient, and TBPL2 mutations were identified and confirmed by Sanger sequencing. The Exome Aggregation Consortium (ExAC) Browser and Genome Aggregation Database (gnomAD) Browser Beta were used to search the allele frequencies of the variants in the general population. The harmfulness of the mutations was analyzed by SIFT, Mutation Taster, and CADD software. RESULT: One novel mutation, c.802C > T (p. Arg268Ter), and one known variant, c.788 + 3A > G (p. Arg233Ter), in TBPL2 were identified in the infertile family. Compound heterozygous mutations in TBPL2 may be the cause of impaired ovarian folliculogenesis, failure of superovulation, and infertility. CONCLUSIONS: We identified compound heterozygous mutations in TBPL2 that caused impaired ovarian folliculogenesis, failure of superovulation, and infertility in patients. These findings suggest an important role for compound heterozygous mutations in TBPL2 and expand the mutational spectrum of TBPL2, which might provide a new precise diagnostic marker for female infertility.

Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Ovarian Folliculogenesis and Uterine Endometrial Receptivity after Intermittent Vaginal Injection of Recombinant Human Follicle-Stimulating Hormone in Infertile Women Receiving In Vitro Fertilization and in Immature Female Rats.

The uterine first-pass effect occurs when drugs are delivered vaginally. However, the effect of vaginally administered recombinant human follicle-stimulating hormone (rhFSH) on ovarian folliculogenesis and endometrial receptivity is not well established. We aimed to compare the efficacy of rhFSH administered vaginally and abdominally in clinical in vitro fertilization (IVF) treatment, pharmacokinetic study, and animal study. In IVF treatment, the number of oocytes retrieved, endometrial thickness and uterine artery blood perfusion were not different between women who received the rhFSH either vaginally or abdominally. For serum pharmacokinetic parameters, significantly lower Tmax, clearance, and higher AUC and T1/2_elimination of rhFSH were observed in women who received rhFSH vaginally, but urine parameters were not different. Immature female rats that received daily abdominal or vaginal injections (1 IU twice daily for 4 days) or intermittent vaginal injections (4 IU every other day for two doses) of rhFSH had more total follicles than the control group. In addition, the serum progesterone and progesterone receptors in the local endometrium were significantly higher in the groups treated with intermittent abdominal or vaginal injection of rhFSH, compared with those who recieved daily injection. In summary, vaginal administration of rhFSH may provide an alternative treatment regimen in women receiving IVF.

Fundamental role of BMP15 in human ovarian folliculogenesis revealed by null and missense mutations associated with primary ovarian insufficiency.

Bone morphogenetic protein 15 (BMP15) encodes an oocyte factor with a relevant role for folliculogenesis as homodimer or cumulin heterodimer (BMP15-GDF9). Heterozygous BMP15 variants in the precursor or mature peptide had been associated with primary ovarian insufficiency (POI), but the underlying mechanism remains elusive and a double dose of BMP15 was suggested to be required for adequate ovarian reserve. We uncovered two homozygous BMP15 null variants found in two girls with POI and primary amenorrhea. Both heterozygous mothers reported physiological menopause. We then performed western blot, immunofluorescence, and reporter assays to investigate how previously reported missense variants, p.Y235C and p.R329C, located in the precursor or mature domains of BMP15, may affect protein function. The p.R329C variant demonstrates an impaired colocalization with growth/differentiation factor 9 (GDF9) at confocal images and diminished activation of the SMAD pathways at western blot and reporter assays in COV434 follicular cell line. In conclusion, BMP15 null mutations cause POI only in the homozygous state, thus discarding the possibility that isolated BMP15 haploinsufficiency can cause evident ovarian defects. Alternatively, heterozygous BMP15 missense variants may affect ovarian function by interfering with cumulin activity. Our data definitely support the fundamental role of BMP15 in human ovarian folliculogenesis.

Dynamic methylation pattern of cyp19a1a core promoter during zebrafish ovarian folliculogenesis.

In vertebrates, the aromatase coded by the cyp19a1a gene can catalyze the conversion from androgens to estrogens. Thus, the regulatory mechanisms of cyp19a1a gene expression are a critical research field in reproductive endocrinology. In this study, we use zebrafish as a model to study the dynamic methylation levels of the cyp19a1a gene core promoter during zebrafish ovarian folliculogenesis. The results show that there is an apparent fluctuation of the methylation levels of zebrafish cyp19a1a core promoter. Moreover, the methylation levels are inversely correlated with the expression levels of cyp19a1a transcripts when the ovarian follicles develop from PV into the MV stage. Also, the CpG dinucleotides which are close to the transcriptional starting site may have provided a significant blocking effect on inhibiting the transcriptional function of RNA polymerase II. Taken together, the results from the present study strongly suggest that DNA methylation was one of mechanisms that are involved in the regulation of cyp19a1a gene expression during folliculogenesis. This methylation mechanism modifying transcriptional process accompanied with zebrafish ovarian folliculogenesis might also shed new light on the regulation of cyp19a1a expression during the ovarian developmental stage in other vertebrates.

The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

Dual-specificity phosphatase 6 (DUSP6) mRNA and protein abundance is regulated by fibroblast growth factor 2 in sheep granulosa cells and inhibits c-Jun N-terminal kinase (MAPK8) phosphorylation.

Growth factors regulate ovarian follicle development and they signal through intracellular pathways including mitogen-activated protein kinase (MAPK) phosphorylation, which is negatively regulated by a subfamily of 23 dual-specificity phosphatases (DUSP). Using sheep granulosa cells as a model, we detected mRNA encoding 16 DUSPs in vivo and in vitro. Stimulation of cells in vitro with FGF2 increased (p < 0.05) abundance of DUSP1, DUSP2, DUSP5 and DUSP6 mRNA, and abundance of DUSP1 and DUSP6 proteins (p < 0.05). In contrast, neither FGF8b nor FGF18 had any major effect on DUSP mRNA abundance. Inhibition of DUSP6 action with the inhibitor BCI significantly increased (p < 0.05) MAPK8 (JNK) phosphorylation but not phosphoMAPK14 (p38) or MAPK3/1 (ERK1/2) abundance. This study suggests that FGFs stimulate DUSP protein abundance, that DUSP6 regulates MAPK8 phosphorylation in granulosa cells, and DUSPs are involved in the differential MAPK signaling of individual FGF ligands.

The Influence of Plant Isoflavones Daidzein and Equol on Female Reproductive Processes.

In this review, we explore the current literature on the influence of the plant isoflavone daidzein and its metabolite equol on animal and human physiological processes, with an emphasis on female reproduction including ovarian functions (the ovarian cycle; follicullo- and oogenesis), fundamental ovarian-cell functions (viability, proliferation, and apoptosis), the pituitary and ovarian endocrine regulators of these functions, and the possible intracellular mechanisms of daidzein action. Furthermore, we discuss the applicability of daidzein for the control of animal and human female reproductive processes, and how to make this application more efficient. The existing literature demonstrates the influence of daidzein and its metabolite equol on various nonreproductive and reproductive processes and their disorders. Daidzein and equol can both up- and downregulate the ovarian reception of gonadotropins, healthy and cancerous ovarian-cell proliferation, apoptosis, viability, ovarian growth, follicullo- and oogenesis, and follicular atresia. These effects could be mediated by daidzein and equol on hormone production and reception, reactive oxygen species, and intracellular regulators of proliferation and apoptosis. Both the stimulatory and the inhibitory effects of daidzein and equol could be useful for reproductive stimulation, the prevention and mitigation of cancer development, and the adverse effects of environmental stressors in reproductive biology and medicine.

In Vitro Folliculogenesis in Mammalian Models: A Computational Biology Study.

In vitro folliculogenesis (ivF) has been proposed as an emerging technology to support follicle growth and oocyte development. It holds a great deal of attraction from preserving human fertility to improving animal reproductive biotechnology. Despite the mice model, where live offspring have been achieved,in medium-sized mammals, ivF has not been validated yet. Thus, the employment of a network theory approach has been proposed for interpreting the large amount of ivF information collected to date in different mammalian models in order to identify the controllers of the in vitro system. The WoS-derived data generated a scale-free network, easily navigable including 641 nodes and 2089 links. A limited number of controllers (7.2%) are responsible for network robustness by preserving it against random damage. The network nodes were stratified in a coherent biological manner on three layers: the input was composed of systemic hormones and somatic-oocyte paracrine factors; the intermediate one recognized mainly key signaling molecules such as PI3K, KL, JAK-STAT, SMAD4, and cAMP; and the output layer molecules were related to functional ivF endpoints such as the FSH receptor and steroidogenesis. Notably, the phenotypes of knock-out mice previously developed for hub.BN indirectly corroborate their biological relevance in early folliculogenesis. Finally, taking advantage of the STRING analysis approach, further controllers belonging to the metabolic axis backbone were identified, such as mTOR/FOXO, FOXO3/SIRT1, and VEGF, which have been poorly considered in ivF to date. Overall, this in silico study identifies new metabolic sensor molecules controlling ivF serving as a basis for designing innovative diagnostic and treatment methods to preserve female fertility.

Follicle development as an orchestrated signaling network in a 3D organoid.

The ovarian follicle is the structural and functional unit of the ovary, composed of the female gamete (the oocyte) and supportive somatic cells. Follicles are not only the source of a female's germ cell supply, but also secrete important hormones necessary for proper endocrine function. Folliculogenesis, the growth and maturation of the follicular unit, is a complex process governed by both intrafollicular crosstalk and pituitary-secreted hormones. While the later stages of this process are gonadotropin-dependent, early folliculogenesis appears to be controlled by the ovarian microenvironment and intrafollicular paracrine and autocrine signaling. In vitro follicle culture remains challenging because of the limited knowledge of growth factors and other cytokines influencing early follicle growth. Here we discuss the current state of knowledge on paracrine and autocrine signaling influencing primary follicles as they develop into the antral stage. Given the importance of intrafollicular signaling and the ovarian microenvironment, we reviewed the current engineering approaches for in vitro follicle culture, including 3D systems using natural hydrogels such as alginate and synthetic hydrogels such as poly(ethylene glycol). Our discussion is focused on what drives the proliferation of granulosa cells, development of the thecal layer, and antrum formation-three processes integral to follicle growth up to the antral stage. Further research in this area may reveal the mechanisms behind these complex signaling relationships within the follicle, leading to more successful and physiologically-relevant in vitro culture methods that will translate well to clinical applications.

Serum Biopterin and Neopterin Levels as Predictors of Empty Follicles.

OBJECTIVE: This study measured serum and follicular fluid (FF) levels of biopterin, neopterin, vascular endothelial growth factor (VEGF), and macrophage colony-stimulating factor (M-CSF) in patients receiving mild ovarian stimulation for oocyte retrieval. PATIENTS AND METHODS: Infertile patients who underwent ovarian stimulation were divided into the following: Group 1, no oocyte retrieval (n = 12), and Group 2, retrieval of more than four oocytes (n = 13). Median total gonadotropin dose in both groups was 150 IU. Biopterin and neopterin levels were measured using high-performance liquid chromatography. VEGF and M-CSF levels were measured by enzyme-linked immunosorbent assay. RESULTS: Compared to Group 2, serum and FF levels of neopterin and VEGF and serum levels of M-CSF were significantly increased, and serum and FF levels of biopterin were significantly decreased in Group 1 (P < 0.05 each). CONCLUSION: Biopterin and neopterin levels showed similar differences in FF and serum of patients with empty follicles. Decreased biopterin and increased neopterin in serum could predict poor oocyte retrieval.

Excessive ovarian follicular development in pregnant pigtailed macaques (Macaca nemestrina).

To evaluate the status and possible control of ovarian follicular development during pregnancy, circulating levels of estrone (E1), estradiol-17ß (E2), and follicle-stimulating hormone (FSH) were measured throughout gestation in both intact and ovariectomized pregnant pigtailed monkeys (Macaca nemestrina). From an additional group of pregnant monkeys, ovaries were obtained at late gestation (on day 150 or 159 of pregnancy) for histological studies. Circulating concentrations of E1 and E2 increased on day 13 and remained elevated for about 10 days; they then declined and reached low levels on day 32 of gestation. After day 60, there were gradual but smaller increases in estrogen levels to day 140, after which both E1 and E2 levels increased significantly, reaching maximum levels (E1 = 832.2 ± 210.8 pg/ ml; E2 = 1.66 ± 0.32 ng/ml) at the end of pregnancy. Removal of ovaries on day 35 of gestation did not affect pregnancy or the pattern of estrogen secretion. Serum concentrations of FSH demonstrated only minor fluctuations during pregnancy but were similar to those found during the early follicular phase of cycling pigtailed monkeys investigated in this study. Ovarian histology revealed extensive follicular growth; in addition to the corpus luteum of pregnancy, ovaries were packed with pre-antral, small antral, and medium-sized Graafian follicles. Some of these follicles appeared to be cystic and showed various degree of atresia; their general appearance was similar to the follicles of human females with polycystic ovary syndrome. Our data suggest that FSH may initiate ovarian follicular growth during gestation. High levels of estrogens were incapable of suppressing FSH secretion but may be responsible for the induction of atresia in a large number of follicles in pregnant pigtailed monkeys.

HSP90 antibodies: a detrimental factor responsible for ovarian dysfunction.

PROBLEM: Earlier studies from our group have established that about 47% cases of autoimmune ovarian failure are due to presence of autoantibodies to Heat Shock Protein 90 (HSP90). However, there are no reports correlating pathological effects of HSP90 autoantibodies leading to ovarian failure. METHOD OF STUDY: Antibodies to HSP90 in female mouse model were generated by active immunization with an immunodominant peptide of HSP90, followed by detailed analysis of several reproductive parameters. RESULT: Estrous cyclicity remains unchanged; however, there was a significant drop in the fertility index due to an increase in pre- and post-implantation loss, associated with an increased incidence of degenerated eggs and embryos. The ovaries showed an increase in the number of empty and degenerated follicles and extensive granulosa cell deaths, which was reflected by the decrease in the levels of Nobox and Gja1 gene expression. CONCLUSION: This study underlines a critical role played by HSP90 in ovarian folliculogenesis and highlights the implications of the presence of anti-HSP90 antibodies in infertile women.