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 × 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 μM 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 (E₂) and AMH productions of the samples were measured with ELISA. Follicle counts were expressed as the mean number of follicles per mm².

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. S1P 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 S1P. 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 S1P at 200 and 400 μM concentrations. Furthermore, those samples incubated with S1P 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.

Keywords: Apoptosis; Atresia; Culture; Follicle; Human ovary; Sphingosine-1-phosphate.