Homogenous subpopulation of human mesenchymal stem cells and their extracellular vesicles restore function of endometrium in an experimental rat model of Asherman syndrome.

BACKGROUND: Asherman syndrome (AS), or intrauterine adhesions, is a main cause of infertility in reproductive age women after endometrial injury. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are promising candidates for therapies that repair damaged endometria. However, concerns about their efficacy are attributed to heterogeneity of the cell populations and EVs. A homogenous population of MSCs and effective EV subpopulation are needed to develop potentially promising therapeutic options in regenerative medicine. METHODS: AS model was induced by mechanical injury in adult rat uteri. Then, the animals were treated immediately with homogeneous population of human bone marrow-derived clonal MSCs (cMSCs), heterogenous parental MSCs (hMSCs), or cMSCs-derived EV subpopulations (EV20K and EV110K). The animals were sacrificed two weeks post-treatment and uterine horns were collected. The sections were taken, and hematoxylin-eosin was used to examine the repair of endometrial structure. Fibrosis was measured by Masson's trichrome staining and α-SMA and cell proliferation by Ki67 immunostaining. The function of the uteri was explored by the result of mating trial test. Expression changes of TNFα, IL-10, VEGF, and LIF were assayed by ELISA. RESULTS: Histological analysis indicated fewer glands, thinner endometria, increased fibrotic areas, and decreased proliferation of epithelial and stroma of the uteri in the treated compared with intact and sham-operated animals. However, these parameters improved after transplantation of both types of cMSCs and hMSCs and/or both cryopreserved EVs subpopulations. The cMSCs demonstrated more successful implantation of the embryos in comparison with hMSCs. The tracing of the transplanted cMSCs and EVs showed that they migrated and localized in the uteri. Protein expression analysis results demonstrated downregulation of proinflammatory factor TNFα and upregulation of anti-inflammatory cytokine IL-10, and endometrial receptivity cytokines VEGF and LIF in cMSC- and EV20K-treated animals. CONCLUSION: Transplantation of MSCs and EVs contributed to endometrial repair and restoration of reproductive function, likely by inhibition of excessive fibrosis and inflammation, enhancement of endometrial cell proliferation, and regulation of molecular markers related to endometrial receptivity. Compared to classical hMSCs, cMSCs were more efficient than hMSCs in restoration of reproductive function. Moreover, EV20K is more cost-effective and feasible for prevention of AS in comparison with conventional EVs (EV110K).

Thrombophilic mutations in Iranian patients with infertility and recurrent spontaneous abortion.

Factor V Leiden (FVL) G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T, and factor II (FII) G20210A mutations are three important causes of thrombophilia, the condition that might be related to infertility and recurrent spontaneous abortion (RSA). In this study we evaluated the presence of these three mutations in 36 female patients with unexplained infertility, 65 female patients with unexplained RSA, and 62 healthy fertile women as control group. DNA was extracted from peripheral blood samples and PCR-RFLP was performed for the molecular diagnosis of each mutation. In addition, activated protein C resistance (APC-R) was also evaluated. The frequencies of FVL, MTHFR, and FII mutations (heterozygous and homozygous) in the control group were 0.0%, 38.7%, and 3.2%, respectively. The frequency of FVL mutation in patients with infertility (30.6%) or RSA (20.0%) was significantly higher than that of the control group. A significantly higher MTHFR mutation rate was also observed in patients with RSA (63.1%) as compared to controls. However, the mutation rate of MTHFR in patients with infertility (50.0%) was not statistically different from that in controls. No significant difference was observed in the frequencies of FII mutations between the patients and controls. Decreased levels of APC-R were observed in 25.0% of infertile patients and 18.9% of patients with RSA. In conclusion, our results show a skew towards higher mutation frequencies of FVL and MTHFR in patients that may necessitate detection of such mutations in these Iranian patients.