Sphingosine-1-phosphate protects human ovarian follicles from apoptosis in vitro

Abstract

Objective(s): We aimed to analyze if anti-apoptotic agent sphingosine-1-phosphate offers protection against in vitro follicle atresia during culture of human ovarian cortical samples. Study design: A translational research study of ex-vivo and in-vitro models of human ovarian tissue. Material and methods: Ovarian cortical tissue fragments (1 x 0.5 cm) were obtained from young patients (n = 15 mean age +/- SD: 29.4 +/- 2.5) undergoing laparoscopic excision of benign ovarian cysts. The samples were cultured for 4 days in 24-well format culture plate using conventional culture techniques. S1P was added to culture media at 200 and 400 mu M concentrations. At the end of culture period the samples were processed for both histomorphological assessment and detection of apoptosis with immunohistochemistry and western blot methods using apoptosis marker cleaved caspase-3. In vitro estradiol (E-2) and AMH productions of the samples were measured with ELISA. Follicle counts were expressed as the mean number of follicles per mm(2). Results: The mean numbers of primordial and secondary follicles were 3.2 +/- 0.4 and 0.7 +/- 0.2 respectively, in the fresh fixed uncultured samples. After four days of culture their numbers were significantly decreased to 0.8 +/- 0.2 (p < 0.01) and 0.1 +/- 0.05 (p < 0.05) respectively, in the control samples cultured without S1P compared to fresh fixed samples. SIP treatment decreased follicle atresia and significantly higher number of primordials (2.3 +/- 0.3, p < 0.01) and secondary follicles (0.5 +/- 0.1, p < 0.05) survived in the samples after 4 day culture period compared to those cultured without SIR In line with this there was dose-dependent decrease in the protein expression of cleaved caspase-3 on western blot and in the number of apoptotic follicles stained positive for cleaved caspase-3 on immunohistochemistry in the samples incubated with S113 at 200 and 400 mu M concentrations. Furthermore, those samples incubated with SlP produced significantly higher amounts of E2 (2339 +/- 321 vs. 1156 +/- 125 pg/mL respectively, p < 0.01) compared to control samples. Conclusions: These results suggest that S1P promotes follicle survival in human ovarian cortical samples in vitro. (C) 2018 Elsevier B.V. All rights reserved.