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Evidence of Human Papillomavirus in the Placenta

Treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). +ve: Positive sample; −ve: negative sample; M: PCR marker; PC: positive control (HeLa cell lines for HPV; Raji cell line for EBV; GMK, infected cell with HSV); NC: negative control. The oral shedding of HSV contributes to mother-child-transmission of HSV. SLPI, ubiquitously found in body fluids, is known for its an anti-microbial, anti-inflammatory and anti-protease properties. 100.PATIENTS AND METHODS: Twelve HIV-infected adults were included. While HPV-16 is associated with differentiated Bowenoid type vulvar intraepithelial neoplasia, which appears to be the most common form of early carcinoma of the vulva, the same association was not seen with respect to advanced vulvar invasive squamous cell carcinoma. HPV type 16, which is present in most HPV-associated tonsillar cancers, was the most prevalent high-risk oral HPV infection.

Controlling for subsequent circumcision status, baseline herpes simplex virus type 2 serostatus, and sexual and sociodemographic risk factors, the hazard ratio for HIV infection among men with HPV-positive glans/coronal sulcus specimens was 1.8 (95% CI, 1.1–2.9), compared with men with HPV-negative glans/coronal sulcus specimens. In vitro experiments suggested several molecular mechanisms providing plausible explanations for the association between placental HPV infection and the adverse outcome of pregnancy. Ectopic HPV-16 E5, E6, and/or E7 expression in trophoblastic cell lines has been associated with changes in viability, reduced adhesion, enhanced migration, and enhanced invasion of these cells [2, 5, 6]. Besides, the HPV-16 early promoter can be constitutively activated in several trophoblastic cell lines, which could be partly attributed to secreted progesterone, hence emphasizing the hormonal regulation of the HPV gene expression regulation during pregnancy (C. Weyn, J. Rasschaert, J.-M. Prevalence of HPV in Ocular-Surface Diseases.

Englert, and V. Fontaine, submitted). Other studies on the vertical transmission of HPVs have often been hampered by the possible contamination of the placenta with cervical cells from an infected birth canal. To circumvent this bias, we analyzed transabdominally obtained placental samples to examine strictly intrauterine HPV placental infection. From November 2008 until January 2010, 35 women gave their informed consent to use residual material from abdominal chorionic villous sampling after cytogenetical examinations for the HPV detection study by our laboratory. The gestational age of the sampling was between the 11th and the 13th gestational week. The results are disappointing in papillomavirus lesions and in chronic acyclovir-resistant herpes ulcerations, efficacy is debatable.

All samples were treated upon arrival. Individuals in Baltimore, Maryland, were recruited during 2001–2002 to participate in an oral-cancer screening program and cross-sectional study. The present analysis included men enrolled in the trial who consented to the collection of an exfoliated penile cell sample and its shipment overseas for HPV DNA testing (Figure 1). The extracted DNA from placental tissue was used in 3 different consensus PCR reactions in 2 different regions of the viral genome. The L1 region was amplified using 2 sensitive nested PCR strategies, both based on a general PGMY09/11 PCR, followed by a second nested PCR with either the GP5+/6+ or the SPF10 primers, performed as described elsewhere [8]. The pU PCR, amplifying the E6/E7 region, was performed in parallel, using 50 pmol/L pU-1-M-L forward primer, 25 pmol/L of the pU-2-R and 25 pmol/L of the pU-2-R-N reverse primers, as described elsewhere [9]. This PCR was shown to amplify HPV-16, -18, -26, -30, -31, -33, -35, -39, -45, -51, -52, -58, -59, -66, -68, -73, -85 [9,] and not HPV-34, -53, -56, -82 (our unpublished results).

Negative controls were included in every PCR reaction to rule out possible laboratory cross-contamination. HPV genotypes were determined with the INNO-LiPA Genotyping v2 kit (Innogenetics, Ghent, Belgium). This reverse hybridization line probe assay relies on the amplification of a 65 bp fragment in the L1 region with the SPF10 biotinylated primers, amplifying 23 HR- and LR-HPV types (HPV-6, -11, -16, -18, -31, -33, -35, -39, -40, -42, -43, -44, -45, -51, -52, -53, -54, -56, -58, -59, -66, -68, -70, -74), followed by streptavidin conjugation and substrate signal amplification [10]. HPV pU and GP5+/6+ PCR products were purified using a GFX column (Amersham) according to the manufacturer’s instructions. Direct DNA sequence analysis was performed using a capillary sequencer (ABI Prism 3130). The residual trophoblastic placental cells used for HPV detection were obtained from 35 placental samples of women undergoing chorionic villous sampling (CVS) for different clinical indications, mainly being patients with a high risk for chromosomal abnormalities. HPV DNA was detected in 2 out of 35 placental samples.

The first sample contained HPV-16, classified as a high-risk HPV genotype, as identified by the INNO-LiPA genotyping method. This genotype could be confirmed upon sequencing of the E6/E7 PCR product. The combination of 2 PCR reactions in different regions of the viral genome leaves no doubt about the presence of a true HPV-16 infection. Smokers were defined as individuals with ⩾1 pack-year of cigarette use, and former smokers were defined as those who had quit for >1 year. Subsequent HPV genotyping was performed by reverse line blot hybridization of PCR products, as described elsewhere [6]. Accordingly, the INNO-LiPA genotyping method after SPF10 PCR was unable to identify HPV-62, since this type is not included in the test. The results are summarized in .

It is noteworthy that HPV-62 was never detected before in our laboratory, hence further ruling out possible laboratory cross-contamination. We demonstrated the presence of HPV in 2 out of 35 transabdominally obtained placental samples, although we cannot exclude the actual HPV prevalence being higher, given that the analysis was restricted by limited amounts of placental material available, as recommended by the ethical committee. Since HPV-16 is often detected in cervical samples, we propose that placental infection might occur either through an ascending infection from the cervix or via infected sperm at fecundation. Indirect evidence supporting this first theory was postulated by Hermonat and colleagues [3] who observed a higher spontaneous abortion rate in women with cervical HPV infection compared with HPV-negative women (60% vs 20%). It is noteworthy that when retrospectively inquiring for the HPV cervical status from the 2 patients having an HPV positive CVS, the patient harboring an HPV-16 positive placenta had a HPV-negative cervical smear 9 months before and 14 months after the puncture. This information might be difficult to interpret, as the time of the puncture and of the cervical anatomo-pathological analyses did not coincide. This could, however, also suggest a placental HPV infection via infected sperm [11].

A hematogenous infection seems less likely but cannot be excluded. Our results are in concordance with previous studies suggesting vertical transmission of both α- and β-HPVs [12, 13]. On the other hand, our results are in discordance with one study reporting the absence of HPV DNA in 147 abdominally obtained placental samples, possibly due to the use of a less sensitive PCR strategy [14]. In our study, we optimized all PCR reactions in order to obtain maximum sensitivity through adaptation of annealing temperature, ramp speed, and Mg2+ concentration [8]. The etiological association between HPV placental infection and a possible adverse outcome on pregnancy was not examined in our study and could therefore not be underlined. Unknown sample quantities were derived by interpolating threshold cycle number values from a standard curve generated from logarithmic dilutions (from 50,000 to 0.5) of a diploid human cell line, CCD-18LU (ATCC CCL-205). Specimens that were indeterminate by line immunoassay were tested by PCR at Health Canada or the Fred Hutchinson Cancer Research Center (Seattle, Washington), with the PCR result deemed to be definitive.

Vertical HPV transmission could possibly be prevented by vaccination, although the clinical impact of placental HPV infection will require further study. In conclusion, HPV-16 and HPV-62 were detected in 2 out of 35 transabdominally obtained placental punctures. Additional larger studies will be needed to assess HPV type distribution and prevalence in the placenta. This study was funded by a grant from the Brussels Capital Region of Brussels, Prospective Research for Brussels, the Belgian Fund for Scientific Research (FNRS/FRSM convention 3.4568.09), ASBL Les Amis de l’Institut Pasteur de Bruxelles and the Fondation Rose et Jean Hoguet.