Evidence for ease of transmission of human papillomavirus DNA from sperm to cells of the uterus and embryo
Evidence for ease of transmission of human papillomavirus DNA from sperm to cells of the uterus and embryo
复制标题
DOI：
10.1007/bf02066536
复制
发表时间：
1996-07-01
影响因子：
3.1
通讯作者：
King, A
中科院分区：
文献类型：
--
作者：
Chan, PJ;Seraj, IM;King, A
材料与方法HPV检测阴性的液化供体精液通过上游法处理。简言之，将液化的精液在Hams' F-10（GIBCO，Grand Island，NY）培养基中混合，并以300 g离心10分钟。所得精子沉淀用1.0ml培养基分层，并在37 ℃下在空气培养箱中培养30分钟，以使活动精子从沉淀中游出。孵育后，取出顶部0.5 ml（总活力超过50%;计数超过2000万/ml），并将等分试样冷冻作为未处理精子对照。剩余标本用于下文所述的HPV DNA程序。将重悬的精子细胞加入等体积的来自HPV 16和18型的两种类型的HPV DNA片段（每种约1 μ g/l）的混合物中。HPV DNA片段先前通过聚合酶链反应（PCR）合成，使用先前报道的这些HPV类型的特异性引物：16型，98 bp;和t8型，80 bp（9）。所有HPV引物均靶向E6-E7开放阅读框
MATERIALS AND METHODSLiquefied donor semen testing negative for HPV was processed through the swim-up method. Briefly, the liquefied semen was mixed in Hams' F-10 (GIBCO, Grand Island, NY) culture medium and was centrifuged for 10 rain at 300g. The resultant sperm pellet was layered over with 1.0 ml culture medium and incubated for 30 min at 37 C in an air incubator to allow the motile sperm to swim out of the pellet. After incubation, the top 0.5 ml was removed (total motility, over 50%; count, over 20 million/ml) and an aliquot was frozen as the untreated sperm control. The remainder of the specimen was used for the HPV DNA procedure described below. HPV DNA (80-98 bp) was chosen as the foreign DNA because of its availability and because of the relevance of HPV infections in the reproductive tract.The resuspended sperm cells were added to an equal volume of a mixture of two types of HPV DNA fragments (approximately I~ g/~ l each) from HPV types 16 and 18. The HPV DNA fragments were previously synthesized by polymerase chain reaction (PCR) using primers specific for these HPV types as reported previously: Type 16, 98 bp; and type t8, 80 bp (9). All HPV primers target the E6-E7 open reading frame