Objective To investigate the effects of ovarian tissue cryopreservation by needle immersed vitrification (NIV) method and subsequently orthotopic transplantation on ovarian function reconstruction in chemotherapy-induced ovary damage rat model. Methods A total of 52 matured virginal female Wistar rats at age of 8-9 weeks housed in specific-pathogen-free facilities, weighing 250-300 g. Vaginal smears were obtained daily, 50 rats having at least 2 consecutive normal estrous cycles were included in the experiment. Ten rats were selected as donors randomly, and NIV method was used for cryopreserving ovarian tissues. The remaining 40 rats were divided into 3 groups according to different treatments: cyclophosphamide group (C group, n=14), cyclophosphamide/transplantation group (C/T group, n=12), and control group (NS group, n=14). In C group and C/T group, the rats received peritoneal injection of cyclophosphamide every day for 21 days to establish the chemotherapy-induced ovary damage models; and then the frozen-thawed ovarian tissues orthotopically transplanted into the left ovarian bursae in C/ T group. The rats received peritoneal injections of 0.9% saline solution every day for 21 days in NS group. Estrous cycle recovery time, ovary weight, morphology change of ovarian tissues, and follicle count were compared among 3 groups. Results One rat died at 2 days after transplantation in C/T group; the other rats survived to the completion of the experiment. At 4 weeks after the end of injection, no significant difference in body weight was found among 3 groups (P gt; 0.05). The rats of NS group had regular estrous cycle, but cyclic changes in vaginal smears were observed in C group and C/T group during cyclophosphamide treatment. The median estrous cycle recovery was 9 days (95%CI: 7.9-10.1 days) in C group, and was 6 days (95%CI: 4.9-7.1 days) in C/ T group, showing significant difference (χ2=6.571, P=0.010). The ovarian weight showed an obvious downtrend in C group at 4 weeks after cyclophosphamide treatment, and an upward trend was observed in C/T group. The ovarian grafts survived and grew well in C/T group. Primordium follicles and primary follicles in C/T group and NS group were significantly more than those in C group (P lt; 0.05), but no significant difference was found between NS group and C/T group (P gt; 0.05). There was no significant difference in secondary follicles and antral follicles among the 3 groups (P gt; 0.05). Conclusion The method of ovarian tissue cryopreservation by NIV and subsequently orthotopic transplantation can significantly shorten the estrous cycle recovery time in chemotherapy-induced ovary damage rat model. Ovarian grafts grow well, follicle count is similar to normal level. So it has the potential ability of ovarian endocrine and fertility reconstruction after chemotherapy.
目的 探讨新型玻璃化冷冻法对人卵巢组织超微结构的保存效果，尤其是对卵巢间质细胞的保存效果。 方法 收集2007年6月－2009年1月在我院行妇科手术的患者卵巢组织共8例，使用新型玻璃化冷冻法和慢速冷冻法保存，比较两种冷冻方法对于始基卵泡卵母细胞、颗粒细胞、卵泡周围间质细胞超微结构保存效果。 结果 对于始基卵泡卵母细胞和颗粒细胞，发现两种冷冻方法之间无显著差异（P＞0.05）；在间质细胞保存中，新型玻璃化冷冻法与慢速冷冻法相比较，正常间质细胞百分率增加（P＜0.05）。 结论 新型玻璃化冷冻法与慢速冷冻法相比能更好地保存卵泡周围间质细胞，在深低温保存人卵巢组织中具有较广阔的应用前景。
ObjectiveTo compare the effect of cryoprotective vitrification agent with different concentrations on protecting human ovarian tissue. MethodsHuman ovarian biopsy tissues were obtained from nine patients between August 2013 and May 2014. The cortical tissue pieces obtained from each patient were cryopreserved using direct cover vitrification (DCV) with two different concentrations. The vitrification solutions were divided into two groups: high concentration [15% (V/V) dimethyl sulphoxide (DMSO)+15% (V/V) ethylene glycol (EG)+0.5 mol/L sucrose] and low concentration group (12% DMSO+12% EG+0.5 mol/L sucrose). The preservation effects in the two different concentration groups were compared by histologic evaluation using light microscope and apoptosis assessed by terminal dexoynucleotidyl transferase-mediated nick end labeling staining. ResultsThere was no significant difference in the proportion of morphologically normal primordial follicles between high concentration group and low concentration group (P ＞ 0.05) . The proportion of apoptotic primordial follicles in the low concentration group was 29.7% (58/195) , and was 42.1% (69/164) in the high concentration group, with a significant difference between the two groups (P ＜ 0.05) . The proportion of apoptotic stromal cells in the low and high concentration group was 30.2% (162/537) and 41.9% (206/492) respectively with a significant difference (P ＜ 0.05) . ConclusionsVitrification solutions with lower concentration can improve the preservation of the primordial follicles and stromal cells in human ovarian tissue. It is a method worth trying in order to improve the protective effect of vitrification by decreasing the toxicity of vitrification solutions.
ObjectiveTo determine if the anti-apoptosis agent can improve the protective effect of human ovarian tissue with novel vitrification method. MethodsHuman ovarian cortical tissues were collected from ten patients treated between October 2012 and August 2013. The samples obtained from each patient were divided into two groups:control (the novel immersed vitrification) and experimental group (the novel immersed vitrification supplemented with vitamin C). The preservation effects in the two groups were compared by ultrastructure using electronic microscopy, and apoptosis was assessed by terminal deoxynucleotidyl transferase-medicated dUTP nick-end labeling (TUNEL) staining. ResultsThe proportion of normal ultrastructure of oocytes and granulosa cells in the experimental group was higher than that in the control group (P<0.05). The proportion of TUNEL-positive primordial follicles in the experimental group was 21.6% (49/227) and the proportion of TUNEL-positive primordial follicles in the control group was 38.6% (91/236), with a significant difference between the two groups (P<0.001). The proportion of TUNEL-positive stromal cells in the experimental group was (21.33±3.41)% and the proportion of TUNEL-positive stromal cells in the control group was (33.46±3.09)%, also with a significant difference between the two groups (P<0.001). ConclusionAnti-apoptosis agents can improve the preservation of primordial follicles and stromal cells by inhibiting of apoptosis. It may be a method worthy of trying in order to improve the protective effect of human ovarian tissue vitrification.