Successful fertility following optimized perfusion and cryopreservation of whole ovary and allotransplantation in a premature ovarian insufficiency rat model.

BACKGROUND: Fertility preservation by whole ovary cryopreservation and transplantation (WOCP&TP) with vascular anastomosis requires successful cryopreservation. In this study, we investigated the possibility of restoring ovarian function and natural fertility after WOCP&TP in a premature ovarian insufficiency (POI) rat model. The influence of cryopreservation on the offspring of rats following WOCP&TP was also explored. METHOD: Rats aged 8-10 weeks were used as donors and recipients for allotransplantation. Fifteen rat whole ovaries were divided into three groups: the optimized group, the conventional group, and the fresh group. Different perfusion modes were used before cryopreservation and after thawing. Whole ovaries were observed by morphologic analysis, immunohistochemical staining, and transferase-mediated deoxyuridine triphosphate nick end-labeling assay. Ovarian function and fertility after WOCP&TP were then observed in 25 cyclophosphamide-induced POI rats for 8 months. Ovarian function was assessed by vaginal smears and blood hormone levels. Fertility restoration was quantified as the live birth rate after mating. The filial generation of rats was mated at 8-10 weeks of age. Offspring were observed for birth defect. RESULTS: Histological evaluation demonstrated intact morphology of follicles in all groups, with 77.6% of the total number of follicles identified as intact in the optimized group. The apoptotic rates of ovarian cells in the optimized group were significantly lower than that in the conventional group. Of the 20 live POI rats, 14 (70%) began to recover ovarian function after 2 weeks of transplantation, with normal hormone levels achieved 4 weeks after transplantation. Four of 14 rats were pregnant and delivered live offspring. One rat had a second pregnancy and delivered a second litter of live offspring. When the offspring matured, they were mated, and second and third generations of rats were born. All offspring had no abnormalities in appearance. CONCLUSIONS: High rates of restoration of ovarian function and natural fertility with multiple generations of offspring were obtained following WOCP&TP in a cyclophosphamide-induced POI rat model by utilizing optimized perfusion. Cryopreservation did not affect the viability of successive generations.

[Effects of hydroxycamptothecin on TGFb1, a-SMA and collagen I expression in rat hepatic satellite cells].

To investigate the molecular mechanism of hydroxycamptothecin (HCPT)-mediated anti-hepatic fibrosis by evaluting its effects on expression of tumor growth factor-beta 1 (TGFb1), alpha-smooth muscle actin (a-SMA) and collagen I (Col I) in hepatic satellite cells (HSCs). Cultured HSCs were treated with different concentrations of HCPT: low-dose group, 0.25 mg/L; middle-dose group, 0.5 mg/L; high-dose group, 0.75 mg/L; and control group, 0 mg/L. Cell proliferation was assessed by the MTT assay. The mRNA expressions of TGFb1, a-SMA and Col I were determined by reverse transcription-polymerase chain reaction. The protein expressions of TGFb1 and a-SMA were detected by Western blot. The content of Col I in the cultured HSCs' supernatant was measured by enzyme-linked immunosorbent assay. The MTT absorbance values of the low-dose group (0.631+/-0.074), middle-dose group (0.469+/- 0.012) and high-dose group (0.204+/- 0.001) were significantly lower than that of the control group (0.793+/-0.098; F = 82.86, P less than 0.01). Compared with the control group, the HCPT-treated groups showed significantly down-regulated gene expressions of TGFb1 (control: 0.716+/-0.064 vs. low: 0.611+/-0.040, middle: 0.510+/-0.014, high: 0.403+/-0.026), a-SMA (control: 0.696+/-0.075 vs. low: 0.579+/-0.037, middle: 0.470+/-0.024, high: 0.299+/-0.017), and Col I (control: 1.019+/-0.056 vs. low: 0.835+/-0.022, middle: 0.696+/-0.055, high: 0.322+/-0.104) (all, P less than 0.01). Meanwhile, HCPT-treated HSCs showed significantly reduced protein expressions of TGFb1 (control: 0.872+/-0.053 vs. low: 0.654+/-0.047, middle: 0.545+/-0.042, high: 0.436+/-0.039) and a-SMA (control: 0.858+/-0.050 vs. low: 0.620+/-0.045, middle: 0.525+/-0.042, high: 0.434+/-0.052) (all, P less than 0.01). The Col I levels secreted by HSCs were significantly lower in the HCPT-treated groups (low: 168.367+/-16.453 ng/ml; middle: 141.284+/-11.731 ng/ml; high: 132.910+/-10.048 ng/ml) than in the control group (188.733 +/-18.299 ng/ml) (all, P less than 0.01). The mechanism of HCPT-mediated anti-hepatic fibrosis may involve down-regulation of TGFb1 expression to inhibit HSC proliferation and activation, as well as reduction of Col I synthesis and secretion.