成熟卵母细胞玻璃化冷冻时机及解冻方法对辅助生殖结局的影响

目的 探讨玻璃化冷冻成熟卵母细胞过程中不同冷冻时机及解冻方法对辅助生殖结局的影响.方法 回顾性分析郑州大学第一附属医院生殖医学中心2007年5月-2009年5月实施辅助生殖周期中因不同原因接受成熟卵母细胞冷冻及解冻的不孕症患者30例的临床资料,其中女方双侧输卵管梗阻21例,男方无精症9例.将30例患者按照成熟卵母细胞的冷冻时机和解冻方法不同分为3组:A组共5例,取卵后4～5 h冷冻且采用常规方法解冻;B组共9例,取卵后2 h内冷冻且采用常规方法解冻;C组共16例,取卵后2 h内冷冻且采用改良法解冻.所有卵母细胞均采用玻璃化冷冻.冻存2～12个月解冻,存活的卵母细胞行卵母细胞胞质内单精子注射(ICSI)后进行胚胎移植.观察并比较3组患者的辅助生殖结局及孕期随访情况.结果 (1)B组和C组患者的卵母细胞存活率(65±33)%、(72±23)%]、移植周期率(9/9、16/16)均明显高于A组(16±17)%、1/5],差异均有统计学意义(P=0.001、0.021);B组与C组分别比较,差异均无统计学意义(P>0.05).C组的平均胚胎种植率(33±38)%]、临床妊娠率(9/16)均明显高于B组(4±11)%、1/9],差异均有统计学意义(P=0.033、0.040).(2)C组内采用自身胚胎移植、赠卵胚胎移植及供精胚胎移植者的平均年龄分别为(28.6±2.1)、(28.0±4.6)、(28.1±3.4)岁]、卵母细胞存活率分别为(73±25)%、(88±10)%、(66±25)%]、受精率分别为(84.6±0.9)%、(79.3±2.0)%、(82.8±15.0)%]、胚胎种植率分别为(20.0±44.7)%、(33.0±0.1)%、(41.6±41.7)%]及临床妊娠率(分别为1/5、3/3、5/8)之间比较,差异均无统计学意义(P>0.05).(3)A组1例患者行胚胎移植,但未妊娠;B组1例临床妊娠,2个月后胚胎停止发育;C组9例临床妊娠,其中1例妊娠4个月流产,8例已顺利分娩5男婴、4女婴,新生儿染色体及发育均正常,每解冻卵母细胞活产率为5.9%(8/135),平均孕周为(39.4±0.9)周,平均新生儿出生体质量为(3574±569)g.结论 取卵后2 h内玻璃化冷冻及改良法解冻成熟卵母细胞,可以提高胚胎质量及改善临床妊娠结局.

Effects of cryopreservation time and thawing method of human oocyte vitrification on the outcome of assisted reproduction

Objective To evaluate the effects on pregnancy outcome of freezing time from oocyte retrieval and thawing method for metaphase Ⅱ human oocytes vitrification. Methods From Mar 2007 to Mar 2009, the clinical outcome of 30 infertile women undergoing vitrified-thawing oocytes of in vitro fertilizationembryo transfer(IVF-ET) in the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University was studied retrospectively, including 21 women with double fallopian tube obstruction and 9 women's husband azoospermia. All infertile women were divided into three groups, including 5 cases in group A (freezing between 4 and 5 hours from oocyte retrieval and conventional thawing method), 9 cases in group B (freezing within 2 hours from retrieval and conventional thawing method) and 16 cases in group C (freezing within 2 hours from retrieval and improved thawing method). The vitrified oocytes were preserved for 2 months to I year and thawed for Intracytoplasmic sperm injection (ICSI) and embryo transfer. The outcome of IVF and pregnancy were recorded. Results (1) The rates of oocyte survival was (65±33) % in group B and (72±23)% in group C and the rate of transfer cycle was 9/9 in group B and 16/16 in group C, which were all significantly higher than (16±17) % of oocyte survival and 1/5 of transfer cycle in group A (P = 0. 001,0. 021). However, the rate of oocyte survival and transfer cycle between group B and group C did not reach statistical difference (P > 0. 05). The rate of implantation and clinical pregnancy of (33±38) % and 9/16 in group C were significantly higher (4±11)% and 1/9 in group B (P =0. 033,0. 040).(2)The mean age of women in group C were (28.6±2.1) in oneself oocyte, (28.0±4.6) in donor oocyte and (28.1±3.4) in donor sperm. The rate of oocyte survival was (73±25) %, (88±10) % and (66±25) %. The rate of fertilization rate was (84. 6±0. 9) %, (79. 3±2. 0) % and (82. 8±15.0) %. The rate of implantation was (20. 0±44. 7) %, (33. 0±0. 1) % , (41.6±41.7) %. The rate of clinical pregnancy was 1/5 in oneself cycles,3/3 in donor oocyte cycles, 5/8 banked donor sperm cycles in group C. All above clinical parameters were not statistically different (P >0. 05). (3) In group A, one women underwent IVFET and no clinical pregnancy was observed. One women pregnancy was terminated at two months in group B.The clinical pregnancies rate of group C was 9/16, late abortion occurred in 1 woman, the other 8 women underwent term pregnancy, including 5 male infants and 4 female infants. All of infants showed normal Karyotype. Live-birth rates per warmed oocyte was 5.9% (8/135). The mean gestational weeks and birth weight of the infants were (39. 4±0. 9) weeks and (3574±569) g, respectively. Conclusions Embryo quality and clinical outcome of thawing cycles could be significantly improved when oocyte vitrification was performed within 2 hours from oocyte retrieval and improved thawing method.