一种简易的促排卵周期挽救性卵子体外成熟培养技术

A Simple Technique for Immature Oocytes Rescue by In-vitro-maturation Culture in Controlled Ovarian Hyperstimulation Cycles

【目的】探讨人类成熟卵丘细胞在未成熟卵母细胞体外成熟培养中的作用,并建立一种简易的实施技术。【方法】在控制性促排卵周期有未成熟卵母细胞时,将同周期成熟卵丘复合体切出部分卵丘细胞,用1mL注射器抽打分散细胞,贴壁培养。113个治疗周期中,298枚生发泡期卵母细胞经3种不同培养液(A、B、C)体外成熟培养(同一病人的生发泡期卵被随机分到某同一组中):第1组28个周期中73枚(A液):基础培养液+卵泡液;第2组40个周期中115枚(B液):A液+分散贴壁的卵丘细胞;第3组45个周期中110枚(C液):A液+分散贴壁的卵丘细胞+促卵泡生成激素+表皮生长因子。观察其成熟率、受精率及可用胚胎获得率等。【结果】24h成熟率:组间比较有显著性差异(A:45.2%,B:61.7%,C:78.2%,P<0.05);25～48h无显著意义。成熟卵的正常受精率在59%～67%之间,组间比较无显著差异;与第1组(54.5%,11.0%)相比,第2组(83.3%,25.2%)、第3组(90.7%,37.3%)的卵裂率和挽救率均有显著性差异(P<0.05),可用胚胎获得率组间比较依次呈现上升趋势(66.7%,82.9%,83.7%)。【结论】来自控制性促排卵周期的成熟卵丘细胞经简易吹打分散后贴壁培养,可能能协同卵泡液中或外加的生长因子,促进未成熟卵母细胞的体外成熟,而本研究技术简易有效,可用于挽救促排卵周期的未成熟卵。

[Objective] To evaluate the role of mature cumulus cells from oocyte-cumulus complex （OCC） in in-vitro maturation （IVM） and establish a new culture technique which is convenient to carry out. [Methods] The cumulus cells of OCC were cut off and dispersed by 1 mL syringe. The cumulus cells were co-cultured with the immature oocytes retrieved from the COH cycles after they adherent to the bottom of the dish. The immature oocytes were experienced IVM procedures in different culture media. They were divided into 3 groups （the oocytes at germinal vesicle stage from one woman were allotted to the same group randomly）. Group 1 （solution A）： basic culture medium＋ human follicular fluid （hFF）; Group 2（solution B）： solution A＋ cumulus cells （OCC）; Group 3（solution C）： solution A＋ OCC＋ follicle stimulating hormone （FSH） ＋ epidermal growth factor （EGF）. Then, the maturation rate, fertilization rate and formation rate of available embryo were observed. [Results] In 113 treatment cycles, 298 immature oocytes were performed IVM with solution A, B, and C. The difference for 24 hour maturation rates among 3 groups was statistically significant （A： 45.2%, B： 61.7%, C： 78.2%, P 〈 0.05）. There was no statistical difference for 25-48 hour maturation rates and normal fertilization rates of mature oocytes. The differences of cleavage rates and rescued embryo rates between group 1 and 2, group 1 and 3 were statistically significant （P 〈 0.05）. The formation rates of available embryo showed an increasing trend from group 1, 2, to 3. [ Conclusion ] After being dispersed by simply beat upon with syringe and adherent culture, the mature cumulus cells from mature OCCs in COH cycles, together with growth factors in the follicular fluid or extraneously supplemented, could promote the IVM of immature oocyte.