关键词： 乳头状瘤病毒，人；乳多空病毒感染；肿瘤病毒感染；聚合酶链反应

【摘要】目的探讨孕期、产褥期妇女及其新生儿的人乳头状瘤病毒（HPV）亚临床感染状况。方法采用聚合酶链反应（PCR）技术检测103例孕产妇（观察组）宫颈及阴道分泌物、外周静脉血标本中HPV-6、11、16、18型DNA，其中孕早期30例、孕中期42例、孕晚期31例，孕晚期妇女监测到产褥期；以同期门诊要求带宫内节育器妇女30例为对照组，同时检测孕晚期妇女分娩的新生儿咽部分泌物标本。结果孕早、中、晚期、产褥期、对照组妇女的宫颈及阴道分泌物HPV总阳性率分别为：16.7%、28.6% 、74.2%、25.8%、26.7%；外周静脉血HPV总阳性率分别为23.3%、26.2%、58.1%、22.6%、20.0%；各组均以HPV-16/18型感染为主，其次为HPV-6/11+16/18型，而无单独的HPV-6/11型阳性。动态检测31例妇女在孕晚期、分娩前、产后6周宫颈及阴道分泌物中HPV 阳性例数分别为17例、21例、8例，其中有6例在孕期的检测中有波动；外周静脉血的HPV阳性例数分别为14例、13例、7例，其中有7例在孕期的检测中有波动；动态检测31例新生儿在出生时、出生后48～72 h、出生后6周咽部分泌物中HPV阳性例数分别为13例、6例、1例。结论整个孕期以孕晚期HPV感染明显，而孕早期、孕中期、产褥期与非孕期的HPV感染率差异无显著性；部分HPV感染的孕妇，产后可自然转为阴性，且孕期HPV表达有一自然波动；新生儿随生后时间的延长，咽部HPV感染率呈下降趋势。

Detection of the Asymptomatic Infection by Human Papillomavirus in Pregnant Women and Neonates

PENG Ping

（Department of Obstetrics and Gynecology, General Hospital, Guangzhou Military Area, Guangzhou 510010, China）

WENG Xiayun, GU Zhiyuan, et al.

【Abstract】ObjectiveTo investigate the state of human papillomavirus (HPV) infection in similar average-aged pregnant women of different gestational periods, in the puerperium and neonates. MethodsPolymerase chain reaction (PCR) assay was employed to detect HPV-6, 11, 16 and 18 DNA in 30 pregnant women in the first trimester, 42 in the second and 31 in the third (who were followed up to their puerperium), and 30 non-pregnant women asking for intrauterine device in our out-patient clinic were taken as controls. Average age in the four groups showed no significant difference (P＞0.05). Samples from cervical, vaginal exfoliated cells, maternal peripheral blood and nasopharyngeal secretion of the newborns were examined respectively. Results (1) In the first trimester, HPV-DNA was detected in the cervical, vaginal exfoliated cells of 5 cases and in the maternal peripheral blood of 7 cases. (2) In the second trimester, HPV-DNA was detected in the cervical, vaginal exfoliated cells of 12 cases and in the maternal peripheral blood of 11 cases. (3) In the third trimester, HPV-DNA was detected in the cervical, vaginal exfoliated cells of 23 cases and in the maternal peripheral blood of 18 cases. (4) In the puerperium, HPV-DNA was detected in samples of cervical, vaginal exfoliated cells of 8 cases and maternal peripheral blood of 7 cases. (5) In the control group, HPV-DNA was detected in the cervical, vaginal exfoliated cells of 8 cases and in the maternal peripheral blood of 6 cases. (6) Consecutive examinations were carried out in 31 pregnant women from the third trimester, through labor to 6 weeks of postpartum. HPV-DNA was positive in the cervical, vaginal samples of 17, 21 and 8 cases, respectively, according to the perinatal periods, and in the maternal peripheral blood of 14, 13 and 7 cases, respectively. The result through the above gestational stages was fluctuated in the cervical, vaginal samples of 6 cases and in the maternal peripheral blood of 7 cases. (7) Successive examinations in infants at time of birth, 48～72 h and 6 weeks after birth showed positive HPV-DNA in the nasopharyngeal secretion of 13, 6 cases and 1 case with respect to the examining periods. (8) The positive cases were mainly infected by HPV-16, 18. Conclusions(1) Infective rate of HPV is statistically significant in the third trimester, but no significant difference exists among the first trimester, the second trimester, the puerperium or the non-pregnancies. (2) Examining consecutively, the HPV positive rate is found to be decreased after delivery, the positive expression of HPV during the gestational periods exhibited fluctuation. (3) Infective rate of HPV in the neonatal nasopharyngeal specimens tends to decrease with time after delivery.

【Key words】Papillomavirus, human; Papovaviridae infections; Tumor virus infections; Polymerase chain reaction

人乳头状瘤病毒(human papillomavirus， HPV)是双链DNA病毒，目前已发现70多型，在生殖道上皮的病灶中至少已发现13型，其中最常见的是HPV-6、11、16、18型。近年来，育龄妇女生殖道 HPV感染率逐年上升，尤其是亚临床感染明显；而妊娠期妇女HPV感染可通过母婴传播，引起婴幼儿肛门及生殖器部位的先天性尖锐湿疣、结膜乳头状瘤及青少年喉乳头状瘤病(juvenile laryngeal papillomatosis, JLP)等相关性疾病。我们采用聚合酶链反应(PCR)技术，对平均年龄相近的不同孕期、产褥期及非孕期妇女共133例的宫颈及阴道分泌物、外周静脉血进行HPV-6、11、16、18型检测，并动态观察孕晚期、分娩前、产褥期妇女的HPV感染情况及孕晚期孕妇分娩的新生儿在出生时、出生后48～72 h、出生后6周咽部分泌物的HPV感染情况，旨在对 HPV的母婴亚临床感染状况进行直接、动态的观察，现将结果报道如下。

资料与方法

一、研究对象

1997年5月～1997年11月期间，按来院就诊日期随机选取103例孕妇(观察组)，其中孕早期30例（孕6～12周；平均年龄26.4±5.4岁）、孕中期42例（孕13～28周；平均年龄26.0±5.2岁）、孕晚期31例（孕28～41周；平均年龄27.4±4.6岁；孕晚期与产褥期为同一组病人）、非孕妇女30例为对照组（有过1次以上妊娠史，要求带宫内节育器者；平均年龄28.3±4.2岁），无内、外科疾病，无梅毒、淋病、尖锐湿疣病史，无外阴瘙痒，白带常规检查无异常。

二、方法

1.标本采集： (1)5 ml生理盐水冲洗宫颈外口及阴道穹窿，留取冲洗液，孕晚期妇女分别于孕28～32周、分娩前（破膜前）及产后6周各取1次；出生时吸取新生儿咽部分泌物，出生后 48～72 h及出生后 6周均于停喂奶4～6 h后用消毒棉签擦拭咽部，于5 ml生理盐水中反复涮洗；上述两种标本于4℃下离心（1 200 r/min, 10 min），收取沉淀细胞，混悬于1 ml 磷酸盐缓冲液（PBS,0.01mol, pH 7.2）中。(2)抽取观察组及对照组妇女的外周静脉血1～2 ml，肝素抗凝，孕晚期妇女分别于孕28～32周、分娩前及产后6周各取1次。以上标本均置-20℃冰箱保存。

2. DNA提取：(1)宫颈阴道冲洗液、新生儿咽部抽吸物用 50～100 μl裂解液（蛋白酶K终浓度为100 μg/ml）混悬细胞沉淀，于 55℃水浴消化1 h；95℃处理10 min，4℃下离心（11 000 r/min, 5 min），取上清液作为PCR扩增的模板。(2)全血标本：先反复用TE液除去红细胞成分，其余步骤同(1)。

3. PCR扩增：引物设计、扩增程序、扩增产物见参考文献［1］。每次扩增均同时设阳性和阴性对照，阳性对照为解放军总医院分子生物实验室提取的HPV-6、11、16、18型质粒DNA，阴性对照为蒸馏水。经2%琼脂糖凝胶电泳判定结果。

三、统计学处理

应用SAS软件，计数资料比较采用χ² 检验；如频数有特别小值时，两组资料采用Fisher精确检验；多组资料采用拟然比χ²检验。

结果

一、妊娠妇女HPV亚临床感染情况

不同孕期及产褥期妇女宫颈及阴道分泌物、外周静脉血的HPV感染率比较，差异均有极显著性(P均＜0.01），见表1；以孕晚期宫颈及阴道分泌物、外周静脉血的HPV感染率最高，其与孕早期、孕中期、产褥期及对照组分别比较，差异均有极显著性（P＜0.01）；而孕早期、孕中期、产褥期及对照组分别比较，差异均无显著性（P＞0.05）。所检测的各组标本中，主要为HPV16/18型阳性，约为HPV6/11+16/18型阳性的3～5倍。

动态检测31例孕妇在孕晚期（28～32周）、分娩前（37～41周）及产后6周的HPV感染情况，发现在宫颈及阴道分泌物中，不同时期共有23例HPV阳性，分娩前HPV感染率（67.7%）较孕晚期（54.8%）稍高，但差异无显著性（P＞0.05 ），其中经重复实验，排除实验误差，有5例在孕晚期为阴性，而在分娩前转为阳性，有1例在孕晚期为阳性，而在分娩前转为阴<