The N6-methyladenosine (m6A) modification plays a central role in epigenetic regulation of the mammalian transcriptome. m6A can be demethylated by the fat mass- and obesity-associated (FTO) protein and the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. Much less is known about that whether m6A content is involved in POI (premature ovarian insufficiency) disease. In this case-controlled study, 69 POI and 53 tubal occlusion patients were recruited from the reproduction centers in our hospital. For the POI animal model experiment, ovarian tissue was obtained from ten POI and nine healthy mice. An m6A test kit was developed to determine the m6A content in the RNA, and qPCR and western blot were used to examine the mRNA and protein expression levels of FTO and ALKBH5. FACS was used to measure the levels of proliferation and apoptosis, and siRNA was used to establish FTO and ALKBH5 knockdown cell lines. Our results showed that the m6A content in the RNA from POI patients and POI mice was significantly higher than control groups and that POI was characterized by the content of m6A. The mRNA and protein expression levels of FTO were significantly lower in the POI patients than control group and were associated with a risk of POI. These data suggest that the decreased mRNA and protein expression levels of FTO may be responsible for the increase in m6A in POI, which may further increase the risk of complications of POI. High m6A should be investigated further as a novel potential biomarker of POI.

Premature ovarian failure (POF) is one of the most common causes of infertility in women. In our present study, we established cyclophosphamide- (CTX-) induced POF rat model and elucidated its effect on ovarian function. We detected the serum estrogen, follicle stimulating hormone, and anti-Müllerian hormone of mice models by ELISA and evaluated their folliculogenesis by histopathology examination. Our study revealed that CTX administration could severely disturb hormone secretion and influence folliculogenesis in rat. This study also detected ovarian cells apoptosis by deoxy-UTP-digoxigenin nick end labeling (TUNEL) and demonstrated marked ovarian cells apoptosis in rat models following CTX administration. In order to explore the potential of human umbilical cord mesenchymal stem cells (UCMSCs) in POF treatment, the above indexes were used to evaluate ovarian function. We found that human UCMSCs transplantation recovered disturbed hormone secretion and folliculogenesis in POF rat, in addition to reduced ovarian cell apoptosis. We also tracked transplanted UCMSCs in ovaries by fluorescence in situ hybridization (FISH). The results manifested that the transplanted human UCMSCs could reside in ovarian tissues and could survive for a comparatively long time without obvious proliferation. Our present study provides new insights into the great clinical potential of human UCMSCs in POF treatment.

BackgroundAlthough many reports show that various kinds of stem cells have the ability to recover the function of premature ovarian insufficiency (POI), few studies are associated with the mechanism of stem cell treatment of POI. We designed this experimental study to investigate whether human adipose stem cell-derived exosomes (hADSC-Exos) retain the ability to restore ovarian function and how hADSC-Exos work in this process.MethodsA POI mouse model was established and human ovarian granule cells (hGCs) collected from individuals with POI were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing POI. The hematoxylin and eosin assay method was employed to assess the number of follicles. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of sex hormones. The proliferation rate and marker expression levels of hGCs were measured by flow cytometry (fluorescence-activated cell sorting). Real-time PCR and western blot assays were used to determine the mRNA and protein expression levels of SMAD2, SMAD3, and SMAD5. Western blot assays were used to test the protein expression levels of apoptosis genes (Fas, FasL, caspase-3, and caspase-8).ResultsAfter the hADSC-Exos were transplanted into the POI mice model, they exerted better therapeutic activity on mouse ovarian function, improving follicle numbers during four stages. ELISA results showed that hADSC-Exos elevated the hormone levels to the normal levels. In addition, after hADSC-Exos were cocultured with POI hGCs, our results showed that hADSC-Exos significantly promoted the proliferation rate and inhibited the apoptosis rate. Furthermore, hADSC-Exos also increased the marker expression of hGCs to the normal level. Besides, mRNA and protein assays demonstrated that hADSC-Exos downregulated the expression of SMAD2, SMAD3, and SMAD5 in vivo and in vitro. Western blot assay demonstrated that hADSC-Exos inhibited expression of the apoptosis genes in POI hGCs, and SMAD knockdown increased the protein expression of apoptosis genes.ConclusionsThese findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which exosomes derived from hADSCs improved ovarian function of POI disease via regulation of the SMAD signaling pathway.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0953-7) contains supplementary material, which is available to authorized users.