Background Premature ovarian insufficiency (POI) is one of the leading causes of female infertility, which is caused by an abnormal ovarian reserve. Currently, there is no effective treatment to restore the fertility of POI patients. Recent studies suggested that microvesicles (MVs) released from mesenchymal stem cells (MSCs) exert therapeutic effects in various degenerative diseases. In this study, the effect of human umbilical cord MSC-derived MVs (HUCMSC-MVs) on the restoration of ovarian function in a chemotherapy-induced POI mouse model is investigated. Methods MVs were obtained from the supernatant of cultured HUCMSCs. The localization of PKH26-labeled HUCMSC-MVs in ovarian tissues was observed by confocal laser scanning microscopy. Histomorphometric analysis was performed to count the number of ovarian follicles and vessels. The ovarian sections were stained with anti-CD34 to evaluate angiogenesis. The levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent serologic assay. The mRNA expression of angiogenesis-related cytokines and the protein expression of AKT in mouse ovaries were measured by quantitative RT-PCR and western blot analysis. The parametric variables were compared by Student’s t test and analysis of variance. The non-parametric variables were compared by the Mann-Whitney U test. Categorical variables were compared by χ 2 test. P < 0.05 was considered statistically significant. Results PKH26-labeled HUCMSC-MVs were detectable within the ovaries and migrated to the ovarian follicles 24 h after transplantation. The transplantation of HUCMSC-MVs could increase the body weight and number of ovarian follicles (primordial, developing, and preovulatory follicles), induce ovarian angiogenesis, and recover the disturbed estrous cycle of POI mice. The expression levels of total AKT, p-AKT, and angiogenic cytokines (including VEGF, IGF, and angiogenin) in the ovaries of POI mice were markedly upregulated after HUCMSC-MVs transplantation, suggesting that HUCMSC-MVs transplantation might recover ovarian function by inducing angiogenesis via the PI3K/AKT signaling pathway. Conclusions This study provides valuable insight into the effects of HUCMSC-MVs on ovarian tissue angiogenesis and on the restoration of ovarian function in POI mice, which may be helpful to develop a treatment strategy for POI patients.

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It has been reported that extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (HUCMSCs) can promote the proliferative and secretive functions of granulosa cells. In vivo study further demonstrated that EVs derived from HUCMSCs can not only promote the angiogenesis of ovarian tissue but also restore the function of an ovary of chemically induced premature ovarian insufficiency (POI) mice. However, no study investigates the effects of HUCMSCs derived EVs on fertility recovery of POI mice and evaluating their offspring. This study investigates the effects of HUCMSCs derived EVs on fertility recovery and the cognitive function of their offspring. A POI model was established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS), and randomly divided into EVs-transplantation group (a single injection of 150 µg EVs proteins which suspended in 0.1 ml phosphate buffer saline [PBS] via tail vein), POI group (a single injection of 0.1 ml PBS via tail vein), and normal control group (a single injection of 0.1 ml PBS via tail vein without intraperitoneal injection of CTX and BUS). After EVs treatment, not only the ovarian function of POI mice recovered but also the fertility increased with less time to get pregnant, evaluating by in vitro fertilization and mating test. Cognitive behaviors of the offspring were similar among the three groups through the Y-maze test and novel object recognition task. An anti-apoptotic effect was identified through immunohistochemistry, real-time polymerase chain reaction and western blot. These findings indicate that HUCMSCs derived EVs can improve the fertility of POI mice without adverse effects on the cognitive behavior of their offspring, highlighting the potential value of EVs to be a cell-free therapy for patients suffering from POI.

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