V. I. Kulakov National Medical Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation, Moscow, Russia.
A. N. Belozersky Research Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University, Moscow, Russia.
Bull Exp Biol Med. 2022 Aug;173(4):560-568. doi: 10.1007/s10517-022-05589-x. Epub 2022 Sep 12.
We studied the effect of co-culturing of extracellular vesicles in the follicular fluid of young women and women of advanced maternal age on sperm motility. Vesicles were obtained by differential centrifugation. The sperm fraction was isolated from the seminal fluid of 18 patients (age 28-36 years). The spermatozoa were incubated with vesicles (1:2 ratio) for 60 or 120 min at 37°C in a CO2 incubator. A fraction of spermatozoa incubated without vesicles served as the control. After the incubation, the sperm samples were sedimented by centrifugation, fixed in 2.5% glutaraldehyde, and analyzed by transmission electron microscopy. RNA was isolated from the follicular fluid vesicles by column method followed by cDNA synthesis in a reaction mixture according to miScript II RT Kit protocol (Qiagen). After 60-min incubation with extracellular vesicles from the follicular fluid of women of advanced maternal age, the sperm motility and hyperactivation slightly changed in comparison with the group where incubation was performed with follicular fluid vesicles from young women and control group. Follicular fluid miRNA profiles in women of different ages varied, which suggests different functional compositions and effects of follicular fluid vesicles of different age groups on sperm characteristics. Transmission electron microscopy revealed differences in the interaction of follicular fluid vesicles from women of different age groups with spermatozoa. Further study of the effect of extracellular vesicles from the follicular fluid and analysis of their transcriptomic, proteomic, and metabolomic composition on sperm mobility and fertilizing ability will improve the effectiveness of assisted reproductive technology programs in patients with male infertility.
我们研究了年轻女性和高龄产妇的卵泡液中的细胞外囊泡共同培养对精子活力的影响。囊泡通过差速离心获得。从 18 名患者（年龄 28-36 岁）的精液中分离出精子部分。将精子与囊泡（比例为 1:2）在 37°C 的 CO2 培养箱中孵育 60 或 120 分钟。未孵育囊泡的精子部分作为对照。孵育后，将精子样本通过离心沉淀，用 2.5%戊二醛固定，并通过透射电子显微镜进行分析。通过柱法从卵泡液囊泡中分离 RNA，然后根据 miScript II RT 试剂盒（Qiagen）的方案在反应混合物中合成 cDNA。与年轻女性的卵泡液囊泡孵育 60 分钟后，与对照组相比，高龄产妇的卵泡液外囊泡对精子活力和超激活的影响略有变化。不同年龄女性的卵泡液 miRNA 图谱不同，这表明不同年龄组卵泡液囊泡的功能组成和对精子特征的影响不同。透射电子显微镜显示，不同年龄组女性的卵泡液囊泡与精子的相互作用存在差异。进一步研究卵泡液外囊泡的作用，并分析其转录组、蛋白质组和代谢组组成对精子运动和受精能力的影响，将提高男性不育症患者辅助生殖技术方案的有效性。
