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Background Primary ovarian insufficiency (POI) is a complex endocrine disorder with impairment of ovarian function before the age of 40 that can associate with mutations in TAp63alpha (TAp63α), a transcription factor essential for maintaining genome integrity in the female germline. Variants located in the C-terminal can constitutively self-activate TAp63α and induce oocyte apoptosis, resulting in POI. This research examined the involvement of the R643* truncating variant of TAp63α in the development of POI. Methods Experiments were performed in the human osteosarcoma-derived SAOS-2 cell line, a non-germline in vitro model selected due to its lack of endogenous TAp63α expression. This cell line cannot fully recapitulate the ovarian follicle microenvironment; however, the R643* variant was investigated alongside the previously validated R594* POI-related variant to confirm the robustness of the methodology. DNA damage was induced using Doxorubicin treatment, and subsequent analyses were performed using Western immunoblotting, luciferase assays, quantitative PCR, and flow cytometry. Results The R643* variant of the TAp63α protein was expressed at minimal RNA and protein levels in the transfected SAOS-2 cells ( p  < 0.001). The overexpression of the TAp63α mutant R643* in SAOS-2 cells resulted in a significant alteration in the expression of target genes. Specifically, BAX expression was decreased (adjusted p  = 0.0008), whereas the expression of IRF6, K14, WNT4, and MASPIN was significantly elevated (adjusted p  < 0.0001). MDM2 expression also increased (adjusted p  < 0.05). Notably, stable R643* variant expression increased cell survival following Doxorubicin treatment (adjusted p  < 0.05). Conclusions We propose a new molecular mechanism for the induction of POI. Our in vitro characterization indicated that the R643* variant of TAp63α may increase the susceptibility of germ cells to DNA damage accumulation during the extended period of meiotic arrest, likely due to a decrease in apoptosis. Concurrently, it appears to stimulate pathways and genes driving uncontrolled follicular development and proliferation. The consequence of these combined effects would be the persistence of poorly competent oocytes, which ultimately leads to premature depletion of the follicular reserve. Clinical trial number not applicable.

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.

PurposeLow mitochondrial DNA (mtDNA) content in oocytes and in cumulus cells is an indicator of poor oocyte quality. Moreover, initial evidence showed a correlation between mtDNA content in cumulus cells and mtDNA copy number in peripheral blood cells. On these bases, we deemed of interest investigating the correlation between mtDNA copy number in peripheral blood and natural fecundity.MethodsThis is a nested case–control study drawn from a prospective cohort of pregnant women referred for routine first trimester screening for aneuploidies (from 11 + 0 to 12 + 6 weeks of gestation) between January 2012 and March 2013 at the “Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico” of Milan, Italy. Cases were subfertile women who attempted to become pregnant for 12–24 months. Controls were the two subsequently age-matched women who became pregnant in less than 1 year. MtDNA was quantified using real-time PCR and normalized to nuclear DNA.ResultsOne hundred and four subfertile women and 208 controls were selected. The median (IQR) mtDNA copy number was 95 (73–124) and 145 (106–198), respectively (p < 0.001). The area under the ROC curve was 0.73 (95% CI 0.67–0.79) (p < 0.001). The Youden index was 105 mtDNA copy number. The crude OR for subfertility in women with mtDNA copy number below this threshold was 5.72 (95% CI 3.43–9.55). The accuracy of mtDNA copy number assessment in peripheral blood progressively decreased with increasing female age.ConclusionsLow mtDNA copy number in peripheral blood is associated with an increased risk of subfertility and may represent a biomarker of natural fecundity.

Abstract Context Despite a growing number of studies, the genetic etiology in many cases of ovarian dysgenesis is incompletely understood. Objectives This work aimed to study the genetic etiology causing absence of spontaneous pubertal development, hypergonadotropic hypogonadism, and primary amenorrhea in 2 sisters. Methods Whole-exome sequencing was performed on DNA extracted from peripheral lymphocytes of 2 Palestinian sisters born to consanguineous parents. Following a BMP15 variant identification, confirming genetic segregation studies were performed in family members. Three-dimensional (3D) modeling for BMP15 dimer and BMP15-GDF-9 heterodimer were followed by functional studies in human ovarian COV434 granulosa cells cotransfected with plasmid harboring either the variant or a wild-type (WT) control, and a second plasmid harboring a luciferase-reporter-gene with a BMP-responsive element. Results A novel homozygous c.G959A/p.C320Y BMP15 mutation was identified in both sisters, and segregated with the disease in the family. By 3D-structure modeling, the mutations were predicted to damage a cysteine-knot motif, disrupt BMP15 dimerization, and severely impair activation of the BMP pathway. The homologous mutation C53Y occurring and identified spontaneously in sheep results in sterility in homozygotes, mimicking the human phenotype here. A 3.8-fold decrease in BMP15 signaling was observed in vitro in cells expressing the homozygous BMP15 mutant when compared to the WT control. Conclusion The novel homozygous missense C320Y mutation is the first homozygous human BMP15 variant causing impaired signaling ability, which correlates with the predicted 3D-structural changes leading to ovarian dysgenesis. The homologous mutation in sheep mimics the human phenotype by infertility. Beyond genetic counseling, and considering ovarian preservation, the ovine model enables further elucidation and interventions in the BMP signaling.

Primary Ovarian Insufficiency (POI) is a female fertility disorder which affects 1% of women before 40 years of age and manifests with amenorrhea, elevation of serum gonadotrophins and low estrogens. POI has a strong genetic component with incomplete penetrance. Several candidate genes have been described so far, however, its etiopathogenesis is mostly unknown. In order to discover the POI-related causative mechanisms, microarray transcriptome analysis in human granulosa cells (hGCs) stimulated with recombinant human BMP15 (rhBMP15) and next generation sequencing analysis (NGS) on the identified differentially expressed genes in a selected group of patients with POI were conducted on NGS Illumina platform. In the present study, we obtained 19 differentially expressed genes upon rhBMP15 stimulation in hGCs. Results: showed that all identified genes were upregulated and associated to pluripotency, inhibition of apoptosis, cell proliferation, BMP signaling and apoptosis. Moreover, we identified nine POI patients bearing six rare variants in 5 of the BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C). The BMP15-induced transcriptome analysis in hGCs contributed the understanding of BMP15 role as transcriptional regulator, through the activation of transcriptional repressors, by inducing pathways inhibiting the ovarian follicle maturation, thus possibly maintaining an undifferentiated state of hGCs. These findings lead to the identification of novel candidate genes for POI.

Some sheep breeds are naturally prolific, and they are very informative for the studies of reproductive genetics and physiology. Major genes increasing litter size (LS) and ovulation rate (OR) were suspected in the French Grivette and the Polish Olkuska sheep populations, respectively. To identify genetic variants responsible for the highly prolific phenotype in these two breeds, genome-wide association studies (GWAS) followed by complementary genetic and functional analyses were performed. Highly prolific ewes (cases) and normal prolific ewes (controls) from each breed were genotyped using the Illumina OvineSNP50 Genotyping Beadchip. In both populations, an X chromosome region, close to the BMP15 gene, harbored clusters of markers with suggestive evidence of association at significance levels between 1E-05 and 1E-07. The BMP15 candidate gene was then sequenced, and two novel non-conservative mutations called FecXGr and FecXO were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (pFecXGr = 5.98E-06 and pFecXO = 2.55E-08). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecXGr/FecXGr, LS = 2.50±0.65 versus FecX+/FecXGr, LS = 1.93±0.42, p<1E-03 and FecXO/FecXO, OR = 3.28±0.85 versus FecX+/FecXO, OR = 2.02±0.47, p<1E-03). Both mutations are located in very well conserved motifs of the protein and altered the BMP15 signaling activity in vitro using a BMP-responsive luciferase test in COV434 granulosa cells. Thus, we have identified two novel mutations in the BMP15 gene associated with increased LS and OR. Notably, homozygous FecXGr/FecXGr Grivette and homozygous FecXO/FecXO Olkuska ewes are hyperprolific in striking contrast with the sterility exhibited by all other known homozygous BMP15 mutations. Our results bring new insights into the key role played by the BMP15 protein in ovarian function and could contribute to a better understanding of the pathogenesis of women's fertility disorders.

Bone Morphogenetic Protein 15 (BMP15) is a TGF beta -like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGF beta family member was performed. A maximum likelihood phylogenetic tree of several TGF beta /BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFs family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.

Cytokines are key regulatory mediators involved in the host response to immunological challenges, but also play a critical role in the communication between the immune and the central nervous system. For this, their expression in both systems is under a tight regulatory control. However, pathological conditions may lead to an overproduction of pro-inflammatory cytokines that may have a detrimental impact on central nervous system. In particular, they may damage neuronal structure and function leading to deficits of neuroplasticity, the ability of nervous system to perceive, respond and adapt to external or internal stimuli. In search of the mechanisms by which pro-inflammatory cytokines may affect this crucial brain capability, we will discuss one of the most interesting hypotheses: the involvement of the neurotrophin brain-derived neurotrophic factor (BDNF), which represents one of the major mediators of neuroplasticity.

Psychiatric disorders are associated with altered function of inhibitory neurotransmission within the limbic system, which may be due to the vulnerability of selective neuronal subtypes to challenging environmental conditions, such as stress. In this context, parvalbumin-positive GABAergic interneurons, which are critically involved in processing complex cognitive tasks, are particularly vulnerable to stress exposure, an effect that may be the consequence of dysregulated redox mechanisms. Adult Male Wistar rats were subjected to the chronic mild stress procedure for 7 weeks. After 2 weeks, both control and stress groups were further divided into matched subgroups to receive chronic administration of vehicle or lurasidone (3 mg/kg/d) for the subsequent 5 weeks. Using real-time RT-PCR and western blot, we investigated the expression of GABAergic interneuron markers and the levels of key mediators of the oxidative balance in the dorsal and ventral hippocampus. Chronic mild stress induced a specific decrease of parvalbumin expression in the dorsal hippocampus, an effect normalized by lurasidone treatment. Interestingly, the regulation of parvalbumin levels was correlated to the modulation of the antioxidant master regulator NRF2 and its chaperon protein KEAP1, which were also modulated by pharmacological intervention. Our findings suggest that the susceptibility of parvalbumin neurons to stress may represent a key mechanism contributing to functional and structural impairments in specific brain regions relevant for psychiatric disorders. Moreover, we provide new insights on the mechanism of action of lurasidone, demonstrating that its chronic treatment normalizes chronic mild stress-induced parvalbumin alterations, possibly by potentiating antioxidant mechanisms, which may ameliorate specific functions that are deteriorated in psychiatric patients.