目的 通过对不同受孕能力精子外显子的分析,寻找并验证特异性外显子作为精子受孕能力的生物标记物。 方法 基于二代测序数据进行生物信息学分析,寻找特异性外显子并设计引物。各取8份高、低受孕能力精液标本,提取精子RNA反转录后进行real time q-PCR验证外显子的表达效率,筛选表达差异恒定的精子外显子作为生物标记物。各取10份高、低受孕能力精子标本,用筛选后的外显子引物进行real time q-PCR验证。 结果 生物信息学分析得到31个候选精子外显子,从31个候选外显子中筛选出9个表达差异恒定的精子外显子GAPDHS、HSF2BP、HSPA1L、ADAM21、SPEM1、WBP2NL、DDX20、TSGA10、PGK2;real time q-PCR验证结果显示,在高、低受孕能力精液标本中这9种精子外显子表达差异明显。 结论 初步确定,差异表达恒定的九种外显子可作为评估精子质量的生物标记物。

Objective To find and verify specific exons as biomarkers of sperm fertility by analyzing sperm exons with different fertility ability.Methods Based on the second generation sequencing data, bioinformatics analysis was conducted to find specific exons and design primers. We obtained 16 semen samples, 8 of high and the other 8 of low fertilizing ability, after the sperm RNAs were extracted and reverse-transcribed, real time q-PCR was performed to verify the expression efficiency of exons, and the sperm exons with constant expression difference were selected as biomarkers. 10 high and 10 lowfertility ability sperm samples were taken for real time q-PCR verification with screened exon primers. Results Thirty-one sperm exons were obtained by bioinformatics analysis, and 9 sperm exons with constant expression differences were selected from the 31 candidate exons, including GAPDHS, HSF2BP, HSPA1L, ADAM21, SPEM1, WBP2NL, DDX20, TSGA10 and PGK2. The results of real time q-PCR verification showed that the exons of these 9 sperm were significantly different in the semen samples with high and low fertility ability. Conclusion Nine exons with constant differential expression can be used as biomarkers to evaluate sperm quality.

目的 本研究以ICSI后未成熟卵母细胞为研究对象,分析比较卵母细胞不同发育阶段冷冻对其后续效果的影响,评估ICSI后未成熟卵母细胞的利用价值。方法 未成熟卵母细胞直接成熟培养(新鲜组)与玻璃化冷冻后成熟培养(冷冻组)的成熟率,并利用孤雌激活的方法比较卵母细胞的发育潜力。结果 发现新鲜组与冷冻组体外培养卵母细胞成熟率、受精率、卵裂率、优质胚胎率和囊胚率均无差异(P>0.05)。但两组的GV期卵母细胞成熟率低于MI期(P<0.05),且冷冻组的GV期卵母细胞受精率低于MI期(P<0.05)。不过裸卵体外成熟培养效果欠佳,特别是对GV期卵母细胞,体外成熟培养后的卵母细胞发育潜力低下,无囊胚形成。结论 ICSI后未成熟卵母细胞的冷冻对卵母细胞的发育潜力没有明显影响,但体外成熟培养的卵母细胞发育潜力低下,有待进一步提高体外成熟培养技术。

Objective In this study, the immature oocytes after ICSI were used to analyze the effects of freezing on the subsequent development of oocytes at different developmental stages, and to evaluate the utilization value of immature oocytes after ICSI(intracytoplasmic sperm injection). Methods The immature oocytes was directly cultured (fresh group) and matured after vitrification (frozen group), and the development potential of the oocytes was compared by parthenogenetic activation. Results There was no significant difference in the oocyte maturation rate, fertilization rate, embryo cleavage rate, high quality embryo rate and blastocyst rate between the fresh and frozen groups (P>0.05). However, the maturation rate of GV oocytes in the two groups was lower than MI oocytes (P<0.05), and the fertilization rate of GV oocytes in the frozen group was lower than MI oocytes (P<0.05). However, the in vitro maturation of naked oocytes was not effective, especially for GV oocytes, the oocyte development potential after in vitro maturation was low, there was no blastocyst formation. Conclusion The freezing of immature oocytes after ICSI has no significant effect on the development potential, but the development potential of naked oocytes matured in vitro was low, and the in vitro maturation culture technology of naked oocyte needs to be further improved.