Reproductive Medical Center, Department of Obstetrics and Gynecology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai, 200025, China.
Shanghai Key Laboratory of Reproductive Medicine, Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China.
Stem Cell Res Ther. 2022 Jul 16;13(1):320. doi: 10.1186/s13287-022-03012-w.
Premature ovarian insufficiency (POI) is a refractory disease that seriously affects the reproductive health of women and is increasing in incidence and prevalence globally. There is enormous demand to improve fertility in women with POI, while there is still lack of effective therapeutic methods in clinic. Cell-free fat extract (CEFFE) has been reported to contain thousands of active proteins which possess the ability to promote tissue repair in other diseases. In our study, we aimed to observe the efficacy and biosecurity of CEFFE on the repair of ovarian function and fertility of mice with POI and further explore the underlying mechanism.
In vivo, POI mice model, established by cyclophosphamide (CTX, 120 mg/kg) and busulfan (BUS, 12 mg/kg), was treated with CEFFE via the tail vein every two days for 2 weeks. Then, the weight of ovaries, estrous cycle and follicle count by H&E staining were measured. The content of AMH, E and FSH in serum was measured by Enzyme-linked immunosorbent assay. Fertility was evaluated by the number of oocytes retrieved, the development of embryos in vitro and the litter size. Biosecurity of parent mice and their pups were examined by body mass and visceral index. The proliferation and apoptosis of cells in ovaries were examined by immunohistochemistry and transmission electron microscopy. Furthermore, the mRNA-Seq of mouse ovarian granulosa cells was performed to explore underlying mechanism of CEFFE. In vitro, KGN cell line and human primary ovarian granulosa cells (hGCs) were treated with 250 μM CTX for 48 h with/without CEFFE. The proliferative ability of cells was detected by cell counting kit-8 assay (CCK-8) and EDU test; the apoptosis of cells was detected by TUNEL and flow cytometry.
CEFFE recovered the content of AMH, E and FSH in serum, increased the number of follicles and the retrieved oocytes of POI mice (P < 0.05). CEFFE contributed to the development of embryos and improved the litter size of POI mice (P < 0.05). There was no side effect of CEFFE on parent mice and their pups. CEFFE contributed to the proliferation and inhibited the apoptosis of mouse granulosa cells in ovary, as well as in human ovarian granulosa cells (including KGN cell line and hGCs) (P < 0.05).
The treatment of CEFFE inhibited the apoptosis of granulosa cells and contributed to the recovery of ovarian function, as well as the fertility of mice with POI.
卵巢早衰（POI）是一种难治性疾病，严重影响女性的生殖健康，且全球范围内其发病率和患病率都在上升。提高 POI 患者的生育能力的需求巨大，但临床上仍缺乏有效的治疗方法。无细胞脂肪提取物（CEFFE）已被报道含有数千种具有促进其他疾病组织修复能力的活性蛋白。在我们的研究中，我们旨在观察 CEFFE 对 POI 小鼠卵巢功能和生育能力修复的疗效和生物安全性，并进一步探讨其潜在机制。
体内，采用环磷酰胺（CTX，120mg/kg）和白消安（BUS，12mg/kg）建立 POI 小鼠模型，通过尾静脉每两天给药一次，连续给药 2 周。然后，通过 H&E 染色测量卵巢重量、动情周期和卵泡计数。采用酶联免疫吸附试验（ELISA）测定血清中 AMH、E 和 FSH 的含量。通过卵母细胞回收数量、体外胚胎发育和产仔数评估生育能力。通过体质量和内脏指数检查亲代小鼠及其幼仔的生物安全性。通过免疫组织化学和透射电子显微镜检查卵巢细胞的增殖和凋亡。此外，通过对小鼠卵巢颗粒细胞的 mRNA-Seq 进行检测，以探索 CEFFE 的潜在机制。体外，用 250μM CTX 处理 KGN 细胞系和人原代卵巢颗粒细胞（hGCs）48h，并用 CEFFE 处理。通过细胞计数试剂盒-8（CCK-8）和 EDU 检测细胞增殖能力；通过 TUNEL 和流式细胞术检测细胞凋亡。
CEFFE 恢复了 POI 小鼠血清中 AMH、E 和 FSH 的含量，增加了卵泡数量和 POI 小鼠的卵母细胞回收数量（P<0.05）。CEFFE 促进了胚胎的发育，提高了 POI 小鼠的产仔数（P<0.05）。CEFFE 对亲代小鼠及其幼仔没有副作用。CEFFE 促进了小鼠和人卵巢颗粒细胞（包括 KGN 细胞系和 hGCs）中颗粒细胞的增殖，同时抑制了其凋亡（P<0.05）。
CEFFE 的治疗抑制了颗粒细胞的凋亡，有助于恢复 POI 小鼠的卵巢功能和生育能力。
