Several viruses have been detected in human milk, and some viruses like human immunodeficiency virus (HIV)-1 can infect the newborn.1 The presence of human papillomavirus (HPV) in breast milk has not been previously assessed. HPV is known to infect oral mucosa of infants.2 HPV might infect the epithelium of the nipple and areola.3 HPV replication in the primary human mammary ductal epithelial cells has been demonstrated (P.L. Hermonat, personal communication), and HPV types 16 and 18 can immortalize normal breast epithelium in vitro.4 Contradictory results exist on HPV detection in breast carcinomas.5
The Finnish HPV Family Study is a prospective follow-up cohort study started in 1998 to evaluate the transmission modes of HPV between newborn babies and their parents.6,7 Here we present the results of HPV-DNA testing in breast milk samples taken 3 days after delivery. The results were correlated with the earlier published data on oral and cervical HPV carriage of the spouses during a 1-year follow-up period.6,7
METHODS
Subjects.
We studied 223 mothers and 87 fathers. The mean ages of the mothers and the fathers were 25.1 years (range, 18–38 years) and 28.8 years (range, 19–46 years), respectively. At first visit after delivery, the spouses filled in questionnaires concerning their risk factors for HPV infection. Overall, 24% and 7% of the mothers and fathers reported previous genital warts and 3% and 2% had previous oral warts, respectively.
Samples.
Mothers collected the milk samples manually 3 days postpartum in the hospital into a 3 mL plastic container after washing hands with disinfectant. This procedure was done apart from feeding the infant. The container was sealed by a midwife trained for the project and the samples were immediately frozen and stored at −70°C. The milk samples were centrifuged for 20 minutes at 3000 rpm to pellet the cells, from which the DNA was extracted with the high pure polymerase chain reaction (PCR) template preparation kit (Roche Diagnostics GmbH, Penzberg, Germany), according to the manufacturer's instructions. To control the quality of extracted DNA, the human β-globin gene was first amplified with PCR from eleven samples. Oral and cervical scrapings were taken before delivery, at month 2, 6, and 12 as described earlier.6
HPV-DNA testing was done by nested PCR with MY09/MY11 and GP05+/GP06+ primers and PCR-products were hybridized with digoxigenin-labeled high-risk HPV oligoprobe cocktail as described.6 All HPV positive PCR-products were sequenced by an ABI PRISM Big Dye Terminator Cycle Sequencing Kit, and automated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, Foster City, CA). DNAs from cultured HPV negative keratinocytes were processed simultaneously with the milk samples as negative controls. Additionally, every 11th PCR sample was distilled water without any DNA. As positive controls DNA from CaSki cells was used. Each HPV positive breast milk sample was analyzed twice.
Statistical analysis was performed with SPSS computer program package (version 12.0.1 for Windows) as described.6
RESULTS
High-risk HPV-DNA was detected in 10 of the 223 milk samples (4.5%) in the cellular compartment. In sequencing 9 HPV-positive samples contained HPV-16-DNA (1 sample was inadequate for sequencing). Eight samples had identical DNA sequence of prototype HPV 16 L1. In 1 sample there was a nucleotide change at nt 6664 (G to A). HR-HPV testing of the cervical samples was done earlier and the detection rate ranged from 12% to 15%.6 HR-HPV carriage in oral samples of the mother's and fathers was detected in 20–24% and 21–26% during the 1-year follow-up, respectively.7 HPV carriage of the milk was not related to genital or oral HR-HPV-DNA status of the mother nor her demographic data, but was significant correlated with the oral HR-HPV carriage of the spouse at month 6 (odds ratio, 3.538; 95% confidence interval, 1.674–7.478; P = 0.021) and month 12 (odds ratio: 2.916; 95% confidence interval: 1.432–5.937; P = 0.022).
DISCUSSION
An interesting question is the origin of HPV-DNA in the milk. The cellular compartment of human milk contains epithelial cells as well as cells of the immune system. According to current opinion, HPV infects epithelial cells and multiplies locally at the site of entry on the skin or mucous membranes and infection does not lead to a viremia. However, contradictory to that, HPV-DNA has been found in peripheral blood leukocytes and HPV-DNA transport by bloodstream has recently been speculated.8 We found that the carriage of oral HR-HPV-DNA of the spouse at month 6 and 12 after childbirth was statistically significantly correlated with the presence of HPV carriage in breast milk, but not at the time of delivery. Oral HR-HPV infection in the male partner might be transmitted to epithelial cells of the breast of the female partner or conversely. This view is supported by a recent study suggesting that HPV can infect the epithelium of the nipple and areola.3 This supports the view of a pathogenic mechanism involving HPV transfer in a retrograde fashion via the nipple, areola, lactiferous ducts, and sinuses.3 If the spouse would have acquired oral HPV infection after delivery the amount of HPV-DNA in his oral sample could still be below the HPV detection limit at month 2. HPV-DNA was detected also at month 2 but the correlation with breast milk status was not statistically significant.
During the HPV-testing, several different steps were made to avoid contamination including DNA extraction in separate rooms and several negative controls. PCR-products were also hybridized to exclude false positive amplification. All HPV positive samples were analyzed twice with PCR. However, HPV contamination at the hospital from mother's hands and skin surrounding the nipple cannot be totally ruled out. Hand carriage of mucosal HPV DNA9 as well as contamination of environmental surfaces by genital human papillomaviruses10 has been demonstrated.
ACKNOWLEDGMENTS
The authors thank Elisa Hovinmäki for helping with sample collection and Tiia Toivonen and Tatjana Peskova for the technical assistance. Deepest gratitude belongs to Prof. Kari Syrjänen for running the statistical analysis.
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