Intrauterine adhesion (IUA) is a major cause of female secondary infertility. We previously demonstrated that menstrual blood-derived stromal cell (MenSC) transplantation helped severe IUA patients have pregnancy and endometrium regeneration. We also initiated platelet-rich plasma (PRP) acted as a beneficial supplement in MenSC culturing and a potential endometrial receptivity regulator. Here, we investigated the therapeutic effect of combined transplantation of MenSCs with PRP in rat IUA models and the mechanisms of MenSCs in endometrium regeneration. Rat IUA models were established by intrauterine mechanical injured. Nine days later, all rats were randomly assigned to four groups received different treatment: placebo, MenSC transplantation, PRP transplantation, and MenSCs + PRP transplantation. The traces of MenSCs were tracked with GFP label. Endometrial morphology and pathology, tissue proliferation, inflammation, pregnancy outcomes, and mechanism of MenSCs in the regeneration of endometrium were investigated. Notably, at days 9 and 18 post-treatment, MenSC transplantation significantly improved endometrial proliferation, angiogenesis, and morphology recovery and decreased collagen fibrosis and inflammation in the uterus. MenSCs had lesion chemotaxis, colonized around the endometrial glands. Gene expression of human-derived secretory protein IGF-1, SDF-1, and TSP-1 was detected in the uterus received MenSCs at day 18. The three treatments can all improve fertility in IUA rats. Moreover, gene expressions of cell proliferation, developmental processes, and other biological processes were induced in MenSC transplantation group. Hippo signaling pathway was the most significantly changed pathway, and the downstream factors CTGF, Wnt5a, and Gdf5 were significantly regulated in treatment groups. PRP enhanced these parameters through a synergistic effect. In summary, MenSCs could effectively improve uterine proliferation, markedly accelerate endometrial damage repairment and promote fertility restoration in IUA rats, suggesting a paracrine restorative effect and Hippo signaling pathway stimulation. Our results indicate MenSCs, a valuable source of cells for transplantation in the treatment intrauterine adhesion. Combined with PRP, this cell therapy was more effective.

Background: Premature ovarian insufficiency (POI) is one of the major causes of infertility. We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) effectively improved ovarian function in a murine model of POI. Recent studies indicated that mesenchymal stem cell-derived exosomes were important components in tissue repair. In this study, we investigated the therapeutic effects of MenSCs-derived exosomes (MenSCs-Exos) in a rat model of POI and its mechanism in restoring ovulation. Methods: Ovaries of 4.5-day-old Sprague Dawley rats (SD rats) were cultured in vitro to evaluate the effects of MenSCs-Exos exposure on early follicle development. Furthermore, POI in rats was induced by intraperitoneal administration of 4-vinylcyclohexene diepoxide (VCD). Forty-eight POI rats were randomly assigned to four groups, each receiving a different treatment: PBS, MenSCs, MenSCs-Exos, and Exo-free culture supernatant of MenSCs. Estrous cyclicity, ovarian morphology, follicle dynamics, serum hormones, pregnancy outcomes, and molecular changes were investigated. Results: Exposure to MenSCs-Exos promoted the proliferation of granulosa cells in primordial and primary follicles in vitro and increased the expression of early follicle markers Deleted In Azoospermia Like (DAZL) and Forkhead Box L2 (FOXL2) while inhibiting follicle apoptosis. In vivo, MenSCs-Exos transplantation effectively promoted follicle development in the rat model of POI and restored the estrous cyclicity and serum sex hormone levels, followed by improving the live birth outcome. In addition, transplantation of MenSCs-Exos regulated the composition of the ovarian extracellular matrix and accelerated the recruitment of dormant follicles in the ovarian cortex and increased proliferation of granulosa cells in these follicles. Conclusion: MenSCs-Exos markedly promoted follicle development in vitro and in vivo and restored fertility in POI rats, suggesting a restorative effect on ovarian functions. The therapeutic effect of MenSCs-Exos transplantation was sustainable, consistent with that of MenSCs transplantation. Our results suggested that MenSCs-Exos transplantation may be a promising cell-free bioresource in the treatment of POI.

POI is characterized by “absent not abnormal” menstruation with hormonal disorders in woman younger than 40 years of age, and etiological and pathophysiological mechanisms underlying the POI development have not been clearly defined. Recently, due to advantages such as abundant sources and non-invasive methods of harvest, MenSCs have been emerging as a promising treatment strategy for the recovery of female reproductive damage. Here, we demonstrated that MenSCs graft in POI mice after CTX treatment could restore ovarian function by regulating normal follicle development and estrous cycle, reducing apoptosis in ovaries to maintain homeostasis of microenvironment and modulating serum sex hormones to a relatively normal status. Moreover, MenSCs participated in the activation of ovarian transcriptional expression in ECM-dependent FAK/AKT signaling pathway and thus restored ovarian function to a certain extent. MenSCs transplantation was proved to be an effective way to repair ovarian function with low immunogenicity, suggesting its great potential for POI treatment.

We previously reported that transplantation of menstrual blood-derived stromal cells (MenSCs) significantly improved fertility restoration in intrauterine adhesion (IUA). However, it is difficult to obtain menstrual blood samples in some severe IUA patients who have amenorrhea or oligomenorrhea. Thus, a safe and effective stem cell replacement therapy is necessary to promote endometrial regeneration. Recent studies demonstrated that the effects of MenSCs are partly mediated in a paracrine manner via small extracellular vesicles (sEVs). To explore this possibility, we performed a pre-clinical study to investigate whether concentrated MenSC-derived sEVs (MenSCs-sEVs) are sufficient to repair IUA and the mechanisms underlying their action. Rat IUA models were established by mechanical injury, followed by the administration of MenSCs or MenSCs-sEVs through intrauterine transplantation. Consistent with the efficacy of MenSCs, MenSCs-sEVs effectively recovered the morphology, promoted regeneration of the glands and angiogenesis, and reversed endometrial fibrosis in the IUA uterus. The endometrial receptivity and pregnancy outcome significantly improved after repeated MenSCs-sEVs transplantations. In addition, all rats in the MenSCs-sEVs group had no hematological or biochemical abnormalities. Three-dimensional fluorescence imaging suggested that MenSCs tended to migrate through the bloodstream, whereas MenSCs-sEVs had a better localized therapeutic effect. Moreover, MenSCs and MenSCs-sEVs inhibited the TGFβ1/SMAD3 pathway in the IUA endometrium, while promoting the phosphorylation of SMAD1/5/8 and ERK 1/2 and upregulating the expression of BMP7. Thus, MenSCs-sEVs safely and effectively enhanced endometrial restoration, suggesting a promising non-cellular therapy for endometrial regeneration and a key role in MenSC-mediated IUA treatment.

Human menstrual blood-derived stromal cells (MenSCs) are highly proliferative and show multiple differentiation capacity. The convenience and non-invasiveness make MenSC a novel cell source for regenerative medicine applications. Platelet-rich plasma (PRP) contains abundant growth factors which are beneficial to wound healing. However, the influence of PRP on MenSCs remains elusive. Here, we evaluated the role of PRP in MenSCs proliferation and assessed the effects of PRP on endometrial receptivity regulation in vitro. MenSCs cultured with 10% activated PRP were compared with those cultured with 10% fetal bovine serum (FBS). Differences in cell proliferation, differentiation, and endometrial receptivity-related gene expression were evaluated. Notably, 10% activated PRP significantly promoted MenSCs proliferation and adipogenic/osteogenic differentiation while suppressing apoptosis. Expression of the mesenchymal stem cells (MSCs) marker CD105 and the perivascular markers SUSD2 and CD146 were elevated after PRP treatment. Moreover, short-term PRP stimulation activated the phosphorylation of Akt and signal transducer and activator of transcription 3 (STAT3) pathways, upregulated expression of FoxO1, LIF, and IL1-β, and downregulated IL-6. In summary, PRP could promote MenSC proliferation, markedly accelerate cell stemness, and evaluate MenSC functions by enhancing the expression of angiogenesis and endometrial receptivity markers, suggesting its potential use as a promising supplement for MenSCs in endometrial regenerative medicine. Our results provide a theoretical basis for the clinical application of co-transplantation of PRP combined with MenSCs.