Naumenko Victor A, Tyulenev Yurii A, Yakovenko Sergei A, Kurilo Lubov' F, Shileyko Ludmila V, Segal Aleksander S, Zavalishina Larisa E, Klimova Regina R, Tsibizov Anton S, Alkhovskii Sergei V, Kushch Alla A
The D, I, Ivanovsky Institute of Virology, Ministry of Health and Social Development of the Russian Federation, 123098 Gamaleya str, 16, Moscow, Russia.
Herpesviridae. 2011 Jun 28;2(1):7. doi: 10.1186/2042-4280-2-7.
The presence of human cytomegalovirus (HCMV) in male genital tract suggests its vertical transmission with spermatozoa and the development of a potentially dangerous fetal infection. The objective of the present study was to evaluate the possibility of intracellular HCMV localization in male germ cells and to examine the effect of the virus on human spermatogenesis.
Semen samples from 91 infertile and 47 fertile men were analyzed. HCMV was detected by real time PCR, rapid culture method and PCR in situ. Human testis organotypic culture and quantitative karyological analysis were used to investigate viral effects on spermatogenesis. Localization of HCMV in immature germ cells and spermatozoa was studied by immunostaining with monoclonal antibodies and ultrastructural analysis of infected organotypic culture.
Viral DNA was detected in 12.3% samples of motile spermatozoa, while infectious activity only in 2.9% infertile and fertile men without statistically significant intergroup difference. According to PCR in situ, the mean percentage of infected cell in both groups was 1.5% (0.25%-15%), which can serve as a criterion for evaluating the risk of HCMV transmission. In HCMV-infected organotypic culture viral antigens were identified in spermatides on day 4, in spermatogonia and spermatocytes on day 8, and in spermatozoa on day 14. Empty and full capsides and virions were visualized in germ cells by electron microscopy. The number of cells before introduction in culture was taken for 100%. On day 14 infected culture contained 36.8% spermatogonia, 18.7% spermatocytes, 27.6% round spermatides and 42.5% elongated spermatides; in comparison with 82.2%, 51.5%, 70.4% and 65.7% in uninfected culture, respectively (all p < 0.05). There were no changes in the number and viability of spermatozoa.
HCMV was detected in male germ cells, both in sperm samples and in testis organotypic culture. The virus may infect immature germ cells which develop to mature HCMV-carrying spermatozoa. A considerable decrease in the number of immature germ cells indicates that HCMV produces a direct gametotoxic effect and can contribute to male infertility.
男性生殖道中存在人巨细胞病毒（HCMV）提示其可通过精子垂直传播，并引发潜在危险的胎儿感染。本研究的目的是评估HCMV在男性生殖细胞内定位的可能性，并研究该病毒对人类精子发生的影响。
分析了91例不育男性和47例生育男性的精液样本。通过实时PCR、快速培养法和原位PCR检测HCMV。采用人睾丸组织块培养和定量核型分析来研究病毒对精子发生的影响。通过单克隆抗体免疫染色和对感染的组织块培养进行超微结构分析，研究HCMV在未成熟生殖细胞和精子中的定位。
在12.3%的活动精子样本中检测到病毒DNA，而在不育和生育男性中仅2.9%检测到感染活性，组间差异无统计学意义。根据原位PCR，两组中感染细胞的平均百分比为1.5%（0.25%-15%），这可作为评估HCMV传播风险的标准。在HCMV感染的组织块培养中，第4天在精子细胞中鉴定出病毒抗原，第8天在精原细胞和精母细胞中鉴定出病毒抗原，第14天在精子中鉴定出病毒抗原。通过电子显微镜在生殖细胞中观察到空衣壳和完整衣壳以及病毒粒子。将接种到培养物中的细胞数量视为100%。第14天，感染培养物中含有36.8%的精原细胞、18.7%的精母细胞、27.6%的圆形精子细胞和42.5%的伸长精子细胞；相比之下，未感染培养物中分别为82.2%、51.5%、70.4%和65.7%（所有p<0.05）。精子的数量和活力没有变化。
在男性生殖细胞中检测到HCMV，无论是在精子样本中还是在睾丸组织块培养中。该病毒可能感染未成熟生殖细胞，这些细胞发育为携带HCMV的成熟精子。未成熟生殖细胞数量的显著减少表明HCMV产生直接的配子毒性作用，并可能导致男性不育。
