From May 2019 to June 2020 we studied the chorionic villi of the placenta of 64 women who asked for TOP. The gestational age of women was between the 7th and 12th week. The decision on whether or not to participate in the study was at the patient’s discretion and the patient was not disadvantaged in any other way for refusing to participate in the study. Subjects were excluded if they had evidence of immunosuppression (HIV infection, transplantation, malignant tumor) or had been vaccinated against HPV. The women were invited to participate regardless of whether they had a history of HPV genital infection. The viral status (cervical HPV DNA) of the mother was not reported deliberately, first, because the aim of the research is to demonstrate the presence of HPV DNA in the placenta, second, because some data are incomplete and the risk is to reduce the number of women of the study group, already small. The prospective observational study was approved by the local institutional review committee (n. 154/2019/PO) and written informed consent was obtained from all study participants. Participants responded to a questionnaire with demographic and clinical data in order to characterize the study group (supplementary file).
Since other studies on vertical HPV transmission were often hampered by the possible contamination of the placenta with vaginal cells from an infected birth canal, other authors used the transabdominal pathway, whereas we analyzed placental samples obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina.
Women were administred prostaglandins (geneprost 1 mg) by vaginal pessaries to dilate the cervix to such an extent that a cannula (Karman’s cannula) could be introduced in the uterus, which was connected to an aspirator to aspirate ovular material. In order to avoid contamination of the ovular material with HPV coming from the maternal vagina, we resorted to a particular method. We inserted a 10 mm Karman cannula into the already dilated cervix. This cannula acts as a shirt, through which a 6 mm Karman cannula reaches the uterine cavity and aspirates the ovular material, without contaminating it. Furthermore, the cervical canal lined with cylindrical cells, is hardly contaminated by papillomavirus as it has a particular tropism for the squamous epithelial cells that line the vagina and the uterine portio; this is even more so in pregnant women whose cervix is characterized by a physiological eversion of the endocervical mucosa on the uterine portio. Moreover, the cervix represents not only an anatomical barrier but also an immunological barrier against ascending infection through mucus production, inflammatory cytokines and antimicrobial peptides. All samples of chorionic villi were manually selected from the aborted material and subjected to research for HPV DNA.
Samples and isolation of nucleic acid from chorionic villi
We have chosen to work on fresh, non-paraffinized placental tissue: in order to make the sample as homogeneous as possible. The placental tissue was processed in a sterile hood and, using a petri dish as a support, reduced to very small pieces using a scalpel from which 3 small sample sections from 3 different points were taken and placed into an Eppendorf type test tube. Following this, the treatment continued with cellular lysis by adding the lysis buffer (ATL buffer) and proteinase K with overnight incubation at 55 °C.
The day after the extraction continued using the commercial HIGH Pure PCR Template (ROCHE) kit which is based on the ability of DNA to bind to inert media or filters contained in columns; subsequently the nucleic acids bound to the filter were eluted through elution buffer and the eluate was stored at − 80 °C until the moment of use.
For the amplification of HPV DNA the Ampliquality kit HPV-TYPE EXPRESS v 3.0 (AB ANALITICA srl, Padova, Italy) was used. The kit is based on a rapid system for the identification of human Papillomavirus by single step PCR: in the preparation of the PCR session, in addition to the samples, a positive amplification and typing control (HPV 61 DNA) as well as a negative control (sterile distilled water) were also used; in addition, the kit can evaluate the suitability of the DNA extracted by the amplification of the TST gene (thiosulfate sulfurtransferase rhodanese).
The PCR mix was divided into a sufficient number of test tubes for the analysis of samples and controls, as follows:
HPV-TYPE EXPRESS MIX 20ul.
Extracted DNA 5ul.
The test tubes were then transferred to a thermocycler with the previously programmed amplification profile.
DNA genotyping using reverse line blot
The subsequent genotyping of the amplified product was carried out by an allele-specific hybridization technique; it is a technique that includes a solid phase support in which specific probes (Reverse Line Blot) have been used and includes:
Denaturing: the marked amplified product is denatured by chemical or thermal denaturation (incubation at 95 °C);
Hybridization: the strip is incubated, under specific temperature conditions, agitation and phj with the solution containing the marked and denatured amplified product;
Washes: to remove the excess marked amplified product and then to define the “astringency” of the hybridization;
Detection: Displays the hybrid that was formed based on the type of marked probe (NBT/BCIP coloring solution).
The kit used for genotyping was Ampliquality HPV-TYPE EXPRESS, able to determine the presence of 40 types of Papillomavirus, in particular: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, 67, 6, 11, 40, 42, 43, 61, 69, 70, 44, 54, 55, 62, 64, 71, 72, 81, 83, 84, 87, 89, 90.
The statistical analysis of the data was carried out with the software package SPSS 15.0 (SPSS Inc.; Chicago, IL, USA). Descriptive statistics were expressed by frequency, arithmetic mean and percentages.