We aimed to determine whether a functional link with impact on female ovarian reserve exists between FMR1 expression and expression ratios of AKT/mTOR signaling genes in human granulosa cells in vivo, as suggested from prior in vitro data. Three hundred and nine women, who were classified as normal (NOR; n = 225) and poor (POR; n = 84) responders based on their ovarian reserve, were recruited during stimulation for assisted reproductive techniques. Expressions of FMR1 and of key genes of the AKT/mTOR and AKT/FOXO1/3 signaling pathways were comparatively analyzed in their granulosa cells. FMR1 expression in granulosa cells of NOR and POR correlated significantly with AKT1, TSC2, mTOR, and S6K expression. No correlation was found between FMR1 and FOXO1 in all, and FOXO3 expression in POR, patients. AKT1 expression was significantly higher and FOXO1 expression lower in POR samples, whereas AKT1 expression was lower and FOXO1 expression was higher in NOR samples. In human native granulosa cells, FMR1 expression significantly correlated with the expression of key genes of the AKT/mTOR signaling pathway, but not with the FOXO1/3 signaling pathway. Our data point to a functional link between FMR1 expression and expression of the AKT/mTOR signaling pathway genes controlling human follicular maturation.

Fragile X-associated primary ovarian insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism and is caused by the expansion of the CGG repeat in the 5′UTR of Fragile X Mental Retardation 1 (FMR1). Approximately 20% of women carrying an FMR1 premutation (PM) allele (55–200 CGG repeat) develop FXPOI. Repeat Associated Non-AUG (RAN)-translation dependent on the variable CGG-repeat length is thought to cause FXPOI, due to the production of a polyglycine-containing FMR1 protein, FMRpolyG. Peripheral blood monocyte cells (PBMCs) and granulosa cells (GCs) were collected to detect FMRpolyG and its cell type-specific expression in FMR1 PM carriers by immunofluorescence staining (IF), Western blotting (WB), and flow cytometric analysis (FACS). For the first time, FMRpolyG aggregates were detected as ubiquitin-positive inclusions in PBMCs from PM carriers, whereas only a weak signal without inclusions was detected in the controls. The expression pattern of FMRpolyG in GCs was comparable to that in the lymphocytes. We detected FMRpolyG as a 15- to 25-kDa protein in the PBMCs from two FMR1 PM carriers, with 124 and 81 CGG repeats. Flow cytometric analysis revealed that FMRpolyG was significantly higher in the T cells from PM carriers than in those from non-PM carriers. The detection of FMRpolyG aggregates in the peripheral blood and granulosa cells of PM carriers suggests that it may have a toxic potential and an immunological role in ovarian damage in the development of FXPOI.

Fragile X mental retardation protein (FMRP) is a translational repressor encoded by FMR1. It targets bone morphogenetic protein receptor type II (BMPR2), which regulates granulosa cell (GC) function and follicle development. However, whether this interaction affects folliculogenesis remains unclear. Therefore, this study investigated the potential effect of FMRP-BMPR2 dysregulation in ovarian reserves and infertility. COV434 cells and patient-derived GCs were used to evaluate FMRP and BMPR2 expression. Similarly, FMR1, BMPR2, LIMK1, and SMAD expression were evaluated in GCs with normal (NOR) and poor (POR) ovarian responses. FMRP and BMPR2 were expressed in both cell types. They were co-localized to the nuclear membrane of COV434 cells and cytoplasm of primary GCs. FMR1 silencing increased the mRNA and protein levels of BMPR2. However, the mRNA levels of FMR1 and BMPR2 were significantly lower in the POR group. FMR1 and BMPR2 levels were strongly positively correlated in the NOR group but weakly correlated in the POR group. Additionally, SMAD9 expression was significantly reduced in the POR group. This study highlights the crucial role of FMR1/FMRP in the regulation of BMPR2 expression and its impact on ovarian function. These findings indicate that the disruption of FMRP-BMPR2 interactions may cause poor ovarian responses and infertility.

Study question: Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer: Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods: Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance: FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution: More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number: Not applicable

Is FMR1 (Fragile-X-Mental Retardation 1 Gene)-targeting miR-323a related to poor ovarian response and what impact does it have on the folliculogenesis-regulating SMAD-signaling pathway? In poor ovarian responders, miR-323a is significantly upregulated, FMRP – downregulated. Human granulosa cells, transfected with a miR-323a mimic, show an upregulation of SMAD proteins. Folliculogenesis is a complex process, regulated, among others, by FMR1 and its protein FMRP. MicroRNAs are involved in translational repression, controlling ovarian function and follicular development. miR-323a suppresses FMR1 in tumor cells, but their relationship has not been evaluated in the human ovary. miR-323a also interacts with components of SMAD pathways, which regulate cell proliferation, differentiation and apoptosis. SMADs belong to the downstream signaling of bone morphogenetic protein-receptor-2, and our previous data shows that its expression is affected by FMRP. Thus, researching miR-323a together with its target molecules may provide more insight into the pathogenesis of poor ovarian response. For ex vivo experiments, we investigated human granulosa cell (HGC) samples of poor (POR, N = 22) and normal (NOR, N = 32) ovarian responders, with an ongoing sample collection which began in 2013. Our in vitro experiments were performed on COV434 cell line, transfected with a microRNA-323a-3p mimic. RNA extraction and RT-qPCR were performed to determine the relative expression levels of the targets, with NOR serving as the control group ex vivo and non-transfected COV434 cells in vitro. 54 patients who underwent IVF/ICSI treatments were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. HGCs were collected during oocyte retrieval, and COV434 cell line was used for miRNA mimic transfection experiments. Briefly, we performed RNA extraction, cDNA synthesis and TaqMan RT-PCR, as well as protein extraction and ELISA. The collected data was analysed using the 2^–ΔΔCtmethod and Student’s t-test. We found that miR-323a was significantly upregulated in HGCs of POR (p = 0.0324). FMR1 expression in this patient group showed a slight downregulation that did not reach statistical significance (p = 0.1054). Protein expression level analysis of 22 patients from this collective (N_NOR=11, N_POR=10) demonstrated a significant downregulation of FMRP in POR (p = 0.0373), suggesting its participation in the pathogenesis of poor ovarian response. Additionally, in vitro upregulation of miR-323a resulted in altered expressions of SMAD-signaling pathway components. Namely, SMAD3 expression increased by 5.7 fold, SMAD4 – 1.6 fold, SMAD5 – 2.5 fold, and SMAD8 – 4.1 fold, compared to non-transfected COV434 cells, with SMAD3 results requiring further investigation due to an unexpected 2 fold elevation of its expression in the negative control (scramble) sample. FMR1 and SMAD1 expression levels remained stable between transfected cells and control samples. These findings show that FMR1-targeting miR-323a may play a critical role in regulating the SMAD pathways, thus affecting folliculogenesis, HGC proliferation and, ultimately, ovarian reserve, which is reduced in case of poor ovarian response. Larger patient sample size is needed to confirm the current results. An age-related influence cannot be ruled out because of the existing difference between the groups. More repeats of miR-323a-3p mimic transfection and miR-323a-3p inhibitor transfection should be carried out to support the in vitro findings. For the first time, we researched the expression levels of miR-323a in HGCs. Our results suggest that miR-323a, which, like FMRP, is downregulated in POR, is involved in the FMR1/FMRP regulatory loop, and its upregulation affects the SMAD-signaling pathway, deepening our understanding of the pathogenesis of poor ovarian response. not applicable