Investigation on Spontaneous Abortion and Human Papillomavirus Infection

Abstract

Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA (n = 80, cases) and women who underwent a voluntary interruption of pregnancy (VI; n = 80, controls) by qualitative PCR and quantitative droplet digital PCR (ddPCR). Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p > 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p >0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p > 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.

Keywords: 9-valent HPV vaccine; 9vHPV vaccine; ELISA; HPV; PBMC; antibody; chorionic villi; droplet digital PCR; human papillomavirus; infection; quadrivalent HPV vaccine; spontaneous abortion; voluntary interruption of pregnancy.

Figure 1

Mean HPV DNA load detected by ddPCR. Mean HPV DNA load (viral DNA copy/cell) was determined in HPV-positive chorionic villi specimens from SA (n = 3) and VI (n = 4) women. Error bars represent standard error of the mean (SEM). The difference in viral load between the SA and VI groups was not statistically significant (p > 0.05).

Figure 2

Serologic profiles of human serum antibody (IgG) reactivity to human papillomavirus type 16 (HPV-16) L1 capsid protein. Immunologic data are from sera of females who had spontaneous abortion (SA, n = 80) and females who underwent voluntary interruption of pregnancy (VI, n = 80). Results are presented as mean SA and VI OD sample/OD negative sample ratios. In this scatter dot plotting, each plot represents the dispersion of OD ratios to a mean level, indicated by the line inside the box with SEM (Standard Error of Mean) for the two groups of females analyzed. The difference of OD sample/OD negative sample ratio for reactivity with HPV16 L1 capsid protein is statistically significantly higher in sera from SA women compared to sera from VI women (* p < 0.05). Data were analyzed with Mann–Whitney U test; * p < 0.05.