Background The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway regulates early follicular activation and follicular pool maintenance in female germline cells. Fragile X mental retardation 1 (FMR1) regulates folliculogenesis and it is variably expressed in patients with Premature Ovary Insufficiency. FMR1 expression is supposed to be linked to AKT/mTOR signaling in an ovarian response dependent manner as demonstrated in recent invitro and invivo studies in the female germline invitro and invivo. Methods We evaluated changes in the expression of AKT/mTOR signaling pathway genes by real time PCR in the peripheral blood of 74 patients with Premature Ovarian Insufficiency and 56 fertile controls and correlated their expression with FMR1 expression. Results Expression of the genes AKT1, TSC2, mTOR, and S6K was significantly more abundant in patients with POI than in the controls. For AKT1, TSC2 and mTOR, gene expression was not affected by FMR1-CGG repeat number in the 5´-untranslated region. FMR1 and S6K expression levels, however, were significantly upregulated in patients with POI and an FMR1 premutation. Independent of a premutation, expression of mTOR, S6K, and TSC2 was significantly correlated with that of FMR1 in all patients. Furthermore, when grouped according to ovarian reserve, this effect remained significant only for mTOR and S6K, with higher significance note in patients with Premature Ovarian Insufficiency than in the controls. Conclusions In Premature ovarian insufficiency patients, activation of AKT/mTOR signaling pathway is remarkable and putatively pathognomonic. Additionally, it seems to be triggered by an FMR1/mTOR/S6K linkage mechanism, most relevant in premutation carriers.

Study question 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? Summary answer 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. What is known already 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. Study design, size, duration 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. Participants/materials, setting, methods 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. Main results and the role of chance 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 (NNOR=11, NPOR=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. Limitations, reasons for caution 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. Wider implications of the 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. Trial registration number not applicable

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Study question Do FMRP (Fragile-X-Mental Retardation 1 Protein)-associated miRNA miR-125b, miR-132 and their target genes participate in the FMR1/FMRP regulatory loop and result in altered ovarian response? Summary answer miR-125b and its target gene TP53 (Tumor Protein P53) are downregulated in follicular fluid and granulosa cells of poor ovarian responders, respectively. What is known already MicroRNAs are a class of endogenous non-coding RNAs that play an important role in ovarian function and follicular development. It was found that miRNAs directly interact with FMRP and are involved in translational repression. MicroRNAs miR-125b and miR-132 have been shown to associate with FMRP in neural cells, but their relationship in the human ovary has not yet been evaluated. TP53 and LIN28A (Lin-28 Homolog A) are targets of miR-125b, the former regulating cell cycle and apoptosis, and the latter involved in germ cell formation. Investigating these molecules more thoroughly may provide insight into the pathogenesis of poor ovarian response. Study design, size, duration RNA from granulosa cells (GCs) and follicular fluid (FF) from poor (POR, nGC = 26, nFF = 12) and normal (NOR, nGC = 31, nFF = 16) ovarian responders was extracted and reverse transcription was carried out. RT-PCR was then performed to compare the relative expression of the targets, with NOR serving as the control group. Sample collection is ongoing and began in 2013. Participants/materials, setting, methods 57 patients who underwent ovarian stimulation were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. GCs (n = 57) and FF (n = 28) were collected during oocyte retrieval. RNA was extracted and specific or non-specific cDNA was synthesized depending on the target. RT-PCR was performed with TaqMan reagents. Statistical analysis was performed using the 2–ΔΔCt method, Student’s t-test and Pearson’s coefficient. Main results and the role of chance We found that miR-125b was significantly downregulated in FF of the POR group (p = 0,0190), with no similar effect observed in GCs. The miR-132 levels showed no difference between the NOR and POR groups. This finding suggests that miR-125b may play a role in the cell-to-cell communication, and its suppression may be related to poor ovarian response. We then investigated two potential miR-125b target genes, TP53 and LIN28A, and their relation to FMR1. While LIN28A is similarly expressed in both groups, TP53 is significantly downregulated in GCs of POR group (p = 0,0086). Interestingly, TP53 expression correlates moderately positively with FMR1> (Pearson’s r = 0,5) in the NOR group, while no significant correlation is evident in the POR group (Pearson’s r = 0,19). These results suggest the existence of regulatory mechanisms involving miR-125b, TP53, FMRP and FMR1 that may be disrupted in case of poor ovarian response. Limitations, reasons for caution More patient samples are needed to confirm the current results. A possible age-related influence cannot be excluded because of the existing difference between the groups. Wider implications of the findings For the first time, the role of miR-125b together with its target TP53 was investigated in the human ovary, suggesting that they are involved in the FMR1/FMRP regulatory loop and thus providing more insight into the etiology and pathogenesis of poor ovarian response. Trial registration number not applicable