Response of stem cells isolated from menstrual blood to metformin administration and its relationship with infertility

Introduction and Aim: Unexplained infertility is one of the most important research topics today because it is a condition in which the cause of infertility is not clear and the causes of female or male infertility cannot be determined. Metformin used for diabetes is known to have an effect on this mechanism. In this study, endometrial-derived stem cells isolated from menstrual blood were chosen as a source to study the mechanisms that may cause infertility as in patients with unexplained infertility. Accordingly, it was aimed to obtain and culture stem cells from menstrual blood samples obtained from infertile and fertile patients and to examine the changes after 30 days of metformin administration by immunocytochemical analysis. Methods: Stem cells (MenSC) were derived from menstrual blood samples obtained from fertile and infertile patients by collagenase I and ficoll protocols. After the cells were cultured in culture medium (DMEMF12, 5% Fetal bovine serum, 1% Penicillin-streptomycin), stem cell characterization was performed by CD34 and CD44 indirect immunopercosidase staining and differentiation potential to adipogenic, chondrogenic and osteogenic lineages was performed by Oil Red, Alcian Blue and Alizarin Res S staining, respectively. MenSCs obtained from fertile and infertile patients were divided into control (group 1 and group 2, respectively) and metformin treated group (group 3 and group 4, respectively). Metformin was administered at the same therapeutic dose (800 mg/day) and duration of administration (30 days) as used in humans (9 x 10-7g/ml). OCT2, OCT3/4, MATE1, αVβ3 integrin, MUC1, LIF and IGF-1 distributions in all groups were evaluated by indirect immunocytochemical analysis. Results: After the Ficoll and Collagenase I protocol, it was decided that Collagenase I protocol was appropriate for obtaining MenSCs since the cells obtained with collagenase I were 85-90% confluent in the 1st week of culture and maintained their fibroblast-like properties with increasing passage number. CD44 immunoreactivity was moderately positive and CD34 immunoreactivity was negative in MenSC. In the differentiation of MenSCs lasting 21 days, positive staining with Oil Red was detected on the 7th day in MenSCs obtained with Collagenase I protocol, while chondrogenic and osteogenic differentiation staining was less on the 14th day, but was detected on the 14th and 21st days. MATE1 immunoreactivity was strongly (+++) positive in group 1 (fertile, control) and group 4 (infertile, metformin treated), while it was moderate (++) positive in group 2 (infertile, control) and group 3 (fertile, metformin treated). OCT-2 immunoreactivity was moderate (++) in group 1 and group 3 and strong (+++) in group 2 and group 4. OCT3/4 immunoreactivity was negative (-) in group 1, group 2 and group 4 and weak (+) in group 3. IGF1 immunoreactivity was negative (-) in group 1 and group 3, moderate (++) in group 2 and weak (+) in group 4. LIF immunoreactivity was negative (-) in group 1 and group 2, but weak (+) in group 3 and group 4. Conclusion: The fact that the cells obtained from fertile and infertile patient samples with the Collagenase I protocol were more stable and morphologically more consistent than the cells obtained with ficoll indicates that it is the appropriate protocol for obtaining MenSCs. It was concluded that MenSCs can be used both as a source of mesenchymal stem cells and as a cell source that can be used in trial studies with drugs used in infertile patients. Key words: Menstrual blood stem cell, infertility, fertility, metformin, collagenase I