Sphingosine-1-phosphate reduces atresia of primordial follicles occurring during slow-freezing and thawing of human ovarian cortical strips

Abstract

We aimed in this study to explore if sphingosine-1-phosphate (S1P) reduces apoptosis of primordial follicles during cryopreservation of human ovarian cortical samples. Ovarian cortical tissue fragments obtained from young patients who underwent laparoscopic excision of benign ovarian cysts were used for the experiments. The samples were slow-frozen and thawed with and without S1P at 200 and 400 mu M, cultured for 1 day, and then were fixed and processed for both histomorphological assessment and detection of apoptosis with immunohistochemistry using apoptosis marker cleaved caspase-3. Follicle counts were expressed as the mean number of follicles per mm(2). The mean number of primordial follicles and in vitro estradiol (E2) and anti-mullerian hormone (AMH) production of the slow-frozen and thawed samples were significantly reduced compared with fresh unfrozen samples. S1P treatment at 400 mu M but not 200 mu M concentration resulted in a significant increase in the number of surviving primordial follicles and in vitro E2 and AMH productions of the samples compared with their counterparts slow-frozen without S1P. We found that that there was a significant decrease in the number of primordial follicles with their oocytes stained positive for cleaved caspase-3 in the slow-frozen samples S1P 400 mu M in comparison with the samples slow-frozen without S1P. These results suggest that S1P may ameliorate follicle atresia occurring in human ovarian cortical samples during cryopreservation.