Immediate protective effect of rUCMSC-EVs on ovarian function in a cyclophosphamide -induced premature ovarian insufficiency rats: counteracting granulosa cell apoptosis.

Abstract

Backgrounds: Premature ovarian insufficiency (POI), which is defined as the loss of ovarian activity before the age of 40 leading to amenorrhea and infertility, is often induced in female patients treated with alkylating agents such as cyclophosphamide (CTX). Currently, the standard clinical management for POI is hormone replacement therapy, which alleviates symptoms but does not restore fertility. Although multiple previous studies have demonstrated that mesenchymal stem cells and their derived extracellular vesicles can protect ovarian function and restore fertility, whether concurrent EV intervention during chemotherapy can preserve ovarian reserve remains unclear.

Methods: The rat umbilical cord mesenchymal stem cells derived extracellular vesicles (rUCMSC-EVs) in this study were isolated from the culture supernatant of rat umbilical cord mesenchymal stem cells (rUCMSC), and POI rat model was established via intraperitoneal injection of CTX and subcutaneous administration of busulfan. Concurrently with the chemotherapeutic drug injection, rUCMSC-EVs were injected into the ovaries. ELISA, histological assessment and mating experiments were used to evaluate ovarian function and reproductive outcomes. Additionally, ovarian granulosa cells were isolated and co-treated with CTX and rUCMSC-EVs to assess apoptosis and DNA repair capacity via flow cytometry and gene expression assays.

Results: Treatment with rUCMSC-EVs effectively counteracted CTX-induced ovarian damage, as evidenced by restored ovarian weight, improved follicular reserve (increased antral follicles and reduced atreic follicles), and elevated serum anti-Müllerian hormone (AMH) and estradiol (E2) levels. Consequently, EV-treated rats exhibited reduced ovarian fibrosis and apoptosis, resulting in improved pregnancy and live birth rates. Furthermore, in ovarian granulosa cells, compared to cells treated with CTX alone, co-treatment with CTX and EVs significantly reduced apoptosis and DNA damage, while upregulating the expression levels of key DNA repair molecules (including BRCA1, BRCA2, MRE11 and RAD51).

Conclusions: Our study demonstrates that the combined intervention of rUCMSC-EVs and chemotherapy drugs can alleviate the apoptosis of ovarian granulosa cells, thereby preserving the ovarian function and improving the fertility of the POI rats. The reduction in granulosa cell apoptosis may be related to the enhancement of DNA damage repair capacity.