ABSTRACT
Little is known on the dynamics of human papillomavirus (HPV) viral load during pregnancy and on the impact of viral load on HPV vertical transmission. We described viral loads for several genotypes during pregnancy and analyse its association with vertical transmission. Data were analysed from the HERITAGE study, a cohort of pregnant women recruited between 2010 and 2016 in three centres in Canada. Vaginal samples were collected at the first and third trimesters of pregnancy, placental samples were collected at birth, and conjunctival, oral, pharyngeal, and genital samples were collected in children at birth and 3 months were tested for HPV DNA and viral load by Linear Array essay. The association between viral load and vertical transmission was measured using logistic regression. Odd ratios (ORs) and their 95% Confidence intervals (CI) were adjusted for age of the mother. We included women in the cohort infected with the 13 most common genotypes during pregnancy (n = 287). A decrease in HPV viral load was observed during pregnancy (median difference between the third and first trimester of pregnancy = −0.005 copies/cell [p < 0.05]). Women with more than 2 HPV copies/cell (compared to those with ≤ 2 copies) at first trimester had a statistically significant higher risk of vertical transmission (adjusted OR = 6.41; 95% CI: 1.10–37.34 for any genotypes and OR = 17.17; 95% CI: 1.18–250.28 for HPV‐16). Viral load values analysed continuously or categorized with different cut‐offs showed comparable results. HPV viral load varied during pregnancy and was strongly associated with HPV vertical transmission. The results provide a better understanding of risk factors associated with vertical transmission.
Keywords: human papillomavirus (HPV), pregnancy, vertical transmission, viral load
1. Introduction
Human papillomavirus (HPV) predominantly affects young adults such as women of childbearing age [1]. Several studies have documented that the prevalence of HPV among pregnant women was at least 30% [2, 3, 4]. Studies on vertical transmission of HPV estimate that the risk of HPV‐positive women transmitting HPV to their children is about 10% [5, 6]. Despite evidence of the detection of HPV in children born to HPV‐positive mothers, risk factors associated with vertical transmission are not well documented. Risk factors that have been proposed include the young age of the mothers, the persistence of HPV infection during pregnancy, vaginal delivery, and the premature rupture of the amniotic membrane [5, 7, 8, 9, 10]. Few studies have analysed the role of HPV viral load as a risk factor for HPV vertical transmission [11, 12, 13, 14]. High viral load during pregnancy is associated with an increased risk of perinatal transmission of different viruses such as human immunodeficiency virus and hepatitis [15, 16]. HPV viral load has also recently been associated with pregnancy complications, such as preterm birth and alteration of trophoblast leading to early abortion [17, 18, 19]. The objective was to describe the dynamics of HPV viral load during pregnancy and to measure its association with HPV vertical transmission.
2. Methods
Data from the HERITAGE cohort was used. The design and methods of the study have been previously published [20]. Briefly, 1050 pregnant women were recruited between 2010 and 2016 in outpatient obstetric clinics catering to the local community at three academic health centres in Montreal, Canada. Women aged 18 and above, without HIV infection, and in their first trimester of pregnancy (between 6 and 14 weeks of gestation) were eligible. All participants signed an informed consent form. The study protocol was approved by the Institutional Review Board of CHU Sainte‐Justine (protocol code: 2010‐265, date of approval: 2010‐03‐12, renewed ever year).
This study included 287 mothers who tested positive for the 13 most common HPV genotypes during pregnancy including HPV 6, 11, 16, 18, 31, 33, 39, 42, 45, 51, 52, 58, and 89. These genotypes are derived from the Family Papillomaviridae, subfamily Firstpapillomavirinae, genus Alphapapillomavirus, and species Alpha‐10 (HPV 6, HPV 11), Alpha‐9 (HPV 16, HPV 31, HPV 33, HPV 52, HPV 58), Alpha‐7 (HPV 18, HPV 39, HPV 45), Alpha‐1 (HPV 42), Alpha‐5 (HPV 51) and Alpha‐3 (HPV 89) [21]. Figure 1 described the flow chart of cohort study participants.
Figure 1.
Study flow diagram of the cohort population included in the study. HPV, Human papillomavirus; HERITAGE: Human Papillomavirus (HPV) perinatal transmission and risk of HPV persistence among children.
In the first and third trimester (32–35 weeks of gestation), a self‐collected vaginal swab (dry Dacron swabs—Copan Italia S.p.A) was provided from the mothers for HPV DNA testing. After birth, swabs were also collected for the purpose of HPV DNA testing. Four sites were sampled in children (conjunctival, oral, pharyngeal, and genital) at birth (after 24 to 48 h) and/or at 3 months of age for HPV DNA testing. Samples were obtained using a Dacron dry swab (Copan Italia S.p.A) from the genital, oral, and pharyngeal mucosa, and a soft swab (FLOQSwabs—Copan Flock Technologies) for the conjunctiva. DNA was purified for the vaginal and children's samples and subsequently stored at—70°C until testing [20]. Medical, social, and demographic characteristics were collected through questionnaires and a review of the medical records.
2.1. HPV Testing and Genotyping
HPV detection and genotyping were conducted using the Linear Array genotyping assay (LA‐HP; Roche Molecular Systems) as previously described [20]. HPV DNA and Beta‐globin were detected in each sample (2 µL). Thirty‐six HPV genotypes were detected (HPV6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, and 89).
2.2. HPV Quantification of Viral Load
HPV quantification was conducted for HPV genotypes 6, 11, 16, 18, 31, 33, 39, 42, 45, 51, 52, 58, and 89 using real‐time PCR assays as described in previous work [22, 23, 24, 25, 26, 27]. Sequences for primers and probe are described in the references above except for HPV39 (primer E6‐F 5′‐AAC CGA GGT ATA TGA ATT TG‐3′, primer E6‐R 5′‐CCG AGT CCG AGT AAT ATC‐3′ and probe FAM/CAC TAG CTG CAT GCC AAT CAT GTA‐3′, amplification protocol: 95°C × 10 min followed by 50 cycles with steps at 95°C × 15 s and 50°C × 30 s), HPV58 (primer 58‐E7‐F 5′ ATG AAA TAG GCT TGG ACG G‐3′, primer 58‐E7‐R 5′‐ GCA CAC AAT GGT ACA TGT GC‐3′, probe 58‐FAM/TGT TAC ACT TGT GGC ACC ACG GT‐3′, amplification protocol: 95°C × 10 min followed by 50 cycles with steps at 95°C × 15 s and 55°C × 30 s) and HPV89 primers (primer 89‐E7‐R 5′‐CAC GAT AGC ACA CAC CAC AC‐3′ and primer 89‐E7‐F 5′‐CTA CAA TGT GAG GAA GAA ATG C‐3′, amplification protocol: 95°C × 10 min followed by 50 cycles with steps at 95°C × 15 s and 60°C × 30 s). Briefly, HPV‐positive samples for each of the genotypes above were first assessed for the presence of PCR inhibitors by amplification of an internal control [22]. All samples were shown to be free of inhibitor activity. For each genotype individually, cycle thresholds obtained for each sample were compared to those of a titration curve obtained by serial 10‐fold dilutions of HPV DNA plasmid of the specific genotype evaluated in 75 ng of human genomic DNA (Roche Diagnostics) in 10 mM Tris‐HCl (pH 8.2). To estimate cell content, processed samples were also assessed for β‐globin DNA as reported previously [22]. Viral loads were determined by dividing the HPV DNA copies by the estimated total cell count based on β‐globin copy numbers. Results were recorded as copy numbers per cell and per sample.
2.3. Statistical Analysis
Descriptive analysis was conducted to determine the characteristics of the mothers and children using means (SD), medians, quartiles, and proportions. The description of viral load measures in mothers in terms of number of copies per cell (mean and median) was done for each HPV genotype detected in the first and third trimesters of pregnancy separately.
For women infected with persistent genotypes (HPV‐positive for the same genotype from the first to the third trimester), median differences in viral load detected at third and first trimester were estimated and compared using Wilcoxon Signed‐rank test. The viral loads for every genotype‐specific HPV episode detected in the first and third trimesters of pregnancy of each of these women were also plotted in a line graph.
The description of viral load measures was also done for each genotype detected in children during the perinatal period (birth to 3 months) (using the mean viral load for genotype presents at birth and/or 3‐month visits). Mean and median differences in viral load detected in children during the perinatal period and their respective mother during pregnancy (combining the first and the third trimester) were compared using the Wilcoxon signed‐rank test.
Logistic regression was used to estimate the crude and adjusted odd ratios (aOR) with their 95% confidence intervals (95% CI) for the association between viral load during pregnancy (measured either at first, third, or both trimesters combined) and vertical transmission. This analysis was restricted to the 253 children born alive with valid HPV testing and their mother. Perinatal transmission of HPV was defined as the detection of HPV found in children at birth and/or 3‐month visit for all sites combined. Analysis was done for all genotypes combined as well as specifically for HPV‐16. Viral load was first analysed as continuous and second as categorized based on several cut‐offs including percentiles (25% and 75%) and more than 1 or 2 copies per cell of HPV. The groups of women in the lowest category were always used as the referent in all models. Adjustment was done for the age of the mothers in all models. All tests were two‐sided, and p‐values were considered statistical at p < 0.05. Stata/SE version 14.0 (Stata‐Corp) was used for all analysis.
3. Results
From the 422 HPV‐positive women in the cohort, 287 mothers were infected with at least one of the genotypes evaluated for viral load quantification (HPV‐6, 11, 16, 18, 31, 33, 39, 42, 45, 51, 52, 58, and 89). Table 1 summarizes the differences in the characteristics of the participants who tested positive for any HPV genotype tested during pregnancy in HERITAGE cohort and those within the subgroup with viral load quantification. Overall, the two groups were similar. Most mothers in this study delivered vaginally (73.3%), were nulliparous (49.1%) and almost 50% had rupture of membranes less than 6 h before birth. Globally, among the 287 mothers of this study, 267 had HPV at first trimester and 186 at third trimester. Persistent genotype‐specific HPV infection (same genotype detected in both trimesters) was found in 166 women, transient infection was detected in the first trimester of pregnancy in 101 and new genotype at the third trimester was detected in 20 women. Among the subgroup of 253 women who had children born alive with valid HPV testing during the perinatal period, 15 (5.9%; 95% CI: 3.6%–9.6%) had offspring who also tested positive for HPV. Table 2 provides a description of the viral loads detected for these children and their mother. HPV16 was detected in 3 of these children (6.4%: 95% CI: 2.0–18.5) who were born out of the 47 mothers positive for HPV‐16 during their pregnancy. It is important to note that two children have been found with genotype that were not present in the mother during pregnancy. Viral loads for each individual genotype detected during pregnancy in the mother and in the perinatal period in children are presented in Supporting Information (Table S1).
Table 1.
Characteristics of HPV‐positive women during pregnancy in the HERITAGE study.
| Women characteristics | All HPV‐positive women at baseline N = 422 | HPV‐positive women for HPV 6, 11, 16, 18, 31, 33, 39, 42, 45, 51, 52, 58, 89 during pregnancy N = 287 | |
|---|---|---|---|
| At baseline | |||
| Age, in years | Mean (± SD) | 30.7 (± 4.8) | 30.2 (± 4.8) |
| Median (quartiles 25th and 75th) | 30 (27–34) | 30 (27–34) | |
| Range | 19–47 | 19–47 | |
| Ethnicity, n (%) | White | 329 (77.9) | 224 (78.0) |
| Native African | 37 (8.8) | 25 (8.7) | |
| African American | 3 (0.7) | 0 (0.0) | |
| East Asian | 4 (1.0) | 4 (1.4) | |
| Arab‐West Asian | 18 (4.3) | 11 (3.8) | |
| Latin American | 25 (5.9) | 18 (6.3) | |
| Native‐Aboriginal People | 1 (0.2) | 1 (0.4) | |
| Other | 5 (1.2) | 4 (1.4) | |
| Parity, n (%), | Nulliparous | 172 (50.3) | 109 (49.1) |
| Primiparous | 116 (33.9) | 75 (33.8) | |
| Multiparous | 54 (15.8) | 38 (17.1) | |
| Missing | 80 | 65 | |
| Cytology, n (%) | Normal | 376 (90.2) | 247 (87.3) |
| ASCUS | 26 (6.2) | 22 (7.7) | |
| LSIL | 7 (1.7) | 7 (2.5) | |
| HSIL | 8 (1.9) | 7 (2.5) | |
| Missing | 5 | 4 | |
| Gestational age at enrollment, in weeks | Mean (± SD) | 11.0 (± 1.5) | 11.0 (± 1.5) |
| Median (quartiles 25th and 75th) | 11 (10–12) | 11 (10–12) | |
| Range | 7–14 | 7–14 | |
| Vaccinationa, n (%) | Yes | 44 (11.2) | 34 (12.8) |
| No | 350 (88.8) | 232 (87.2) | |
| Missing | 28 | 21 | |
| During pregnancy | |||
| Pregnancy types, n (%) | Single pregnancy | 417 (98.8) | 282 (98.3) |
| Multiple pregnancy—twins | 3 (0.7) | 3 (1.0) | |
| Multiple pregnancy—triplets | 2 (0.5) | 2 (0.7) | |
| Pregnancy outcomes, n (%) | Live births | 395 (95.2) c | 270 (94.7) d |
| Spontaneous abortions | 9 (2.2) | 7 (2.5) | |
| Elective abortions | 2 (0.5) | 1 (0.4) | |
| Pregnancy termination | 7 (1.7) | 5 (1.8) | |
| Stillbirths | 2 (0.5) | 2 (0.7) | |
| Missingb | 12 | 7 | |
| Delivery mode for live births, e n (%) | Vaginal | 277 (71.9) | 192 (73.3) |
| Cesarean section | 108 (28.1) | 70 (26.7) | |
| Missing | 5 | 3 | |
Abbreviations: ASCUS, atypical squamous cells of undetermined significance; HPV, human papillomavirus; HSIL, high‐grade squamous intraepithelial lesion; LSIL, low‐grade squamous intraepithelial lesion; N, number of participants; Other, includes 1 French and 4 Haitians, SD, standard deviation.
All women were vaccinated with 4vHPV (Gardasil®4); but the vaccine type was unknown for 6 vaccinated women in each group.
Including 2 children from a triplet with missing data.
N = 395 children born alive including 3 twins and 1 triplet out of 390 pregnancies.
N = 270 children born alive including 3 twins and 1 triplet out of 265 pregnancies.
All twins and triplets born by the same delivery mode.
Table 2.
HPV viral load for the 15 children in whom HPV was detected during the perinatal period and their mothers.
| id | Mother | Children | ||||
|---|---|---|---|---|---|---|
| Viral loada (copy per cell) | HPV genotypes detected during pregnancy | Viral loada (copy per cell) | HPV genotypes detected | Sitesb | ||
| Birth 24/48 h | 3‐months | |||||
| 1 | 3.949 | 89 | 0.017 | . | 89 | O |
| 2 | 251.587 | 31, 35, 42, 51, 53, 58, 84 | 0.163 | 39, 42 | . | O‐P‐G |
| 3 | 17.148 | 31, 34, 59 | 0.011 | . | 31 | C‐O‐G |
| 4 | 0.219 | 31, 40, 45, 54, 59, 67, 81, 83, 89 | 0.000 | . | 89 | O |
| 5 | 0.005 | 6, 39 | 0.019 | 6 | . | C |
| 6 | 0.614 | 31, 39, 59, 66 | 0.006 | 31, 66 | . | C |
| 7 | 0.005 | 18 | 0.000 | 18 | . | O |
| 8 | 181.070 | 51 | 0.426 | 51 | . | C‐G |
| 9 | 6.926 | 16, 35, 42, 52 | 0.937 | 16, 35, 52 | . | G |
| 10 | 0.510 | 16, 18, 45, 53, 82 | 0.000 | 16, 53 | . | O |
| 11 | 3.537 | 18, 51 | 0.396 | 18 | . | C‐G |
| 12 | 0.073 | 35, 44, 52, 56, 89 | 0.021 | 44 | 52 | C |
| 13 | 0.049 | 53, 59, 89 | 0.172 | 51 | n.t | C |
| 14 | 15.729 | 16, 62, 81 | 0.000 | 16, 62 | n.t | C‐O‐G |
| 15 | 0.002 | 51, 84 | 0.000 | . | 51 | O |
Note: “.”: negative; “n.t”: not tested/invalid sample.
Mean viral load of all genotypes detected for multiples and persistent genotypes.
Sites where HPV was found among children: C (conjunctival), O (oral), P (pharyngeal), G (genital).
Table 3 summarizes data on HPV viral load detected in mothers during pregnancy and in children during the perinatal period. Overall, mean and median HPV viral loads were lower in the third trimester compared to the first trimester of pregnancy; the mean difference was −0.0492 copies/cell (mean difference = 1.0366−1.0858) and the median difference was −0.0001 copies/cell (median difference = 0.0007−0.0008). Among women with persistent infection, we observed a decrease in viral load detected at third trimester compared to the first trimester for most genotypes that was statistically significant for all genotypes taken together (decreases in the median viral load of −0.0002 copies/cell, p‐value: 0.0186). Interestingly, the decreases in median viral load considering all infections were mostly explained by genotype present in single infection (−0.0005 copies/cell, p‐value: 0.0024) rather than in multiple infections (> −0.0001 copies/cell, p‐value = 0.8027). A statistically significant difference was also observed specifically for HPV‐16 (median difference = −0.0100, p‐value: 0.0478). Figure 2 illustrates the variation in viral load measured throughout trimesters where each line represents a genotype‐specific HPV episode detected in each mother from which we can observe the tendency toward a reduction of viral load from the first to the third trimester.
Table 3.
HPV viral load measures in mothers during pregnancy and in children during the perinatal period.
| Genotype | Viral load (copy per cell) in mothers during pregnancy | Viral load (copy per cell) in children during the perinatal period | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| First trimester | Third trimester | Persistent infection (HPV‐positive women at both trimesters) | Both trimesters combineda | Birth to 3 monthsb | |||||||||||||||
| n | Mean | Median | n | Mean | Median | n | Mean at first trimester | Median at first trimester | Median difference (third‐first trimester) | p‐valuec | n | Mean | Median | n | Mean | Median | Median difference (children–mother) d | p‐valuea | |
| HPV 6 | 16 | 4.8220 | 0.0017 | 10 | 6.9707 | 0.0009 | 6 | 0.5491 | 0.0042 | +0.0009 | 0.7532 | 20 | 5.5178 | 0.0010 | 1 | 0.0190 | 0.0190 | +0.0146 | 0.3173 |
| HPV 11 | 0 | — | — | 1 | 0.0029 | 0.0029 | 0 | — | — | — | — | 1 | 0.0029 | 0.0029 | 0 | — | — | — | — |
| HPV 16 | 53 | 1.4985 | 0.0101 | 37 | 0.3869 | 0.0109 | 36 | 2.1844 | 0.0375 | −0.0100 | 0.0478 | 54 | 0.8750 | 0.0097 | 3 | 0.4594 | 0.0004 | −1.2745 | 0.1088 |
| HPV 18 | 23 | 2.5029 | 0.0047 | 11 | 1.8235 | 0.0044 | 10 | 5.5882 | 0.0180 | −0.0071 | 0.3329 | 24 | 1.6172 | 0.0034 | 3 | 0.3965 | 0.3965 | −2.9245 | 0.1797 |
| HPV 31 | 25 | 1.9966 | 0.0116 | 20 | 0.3711 | 0.0043 | 15 | 2.1557 | 0.0084 | −0.0026 | 0.0535 | 30 | 1.2451 | 0.0098 | 4 | 0.0130 | 0.0130 | −8.8677 | 0.1797 |
| HPV 33 | 13 | 1.3000 | 0.0002 | 7 | 0.0322 | 0.0081 | 6 | 2.8161 | 0.0132 | −0.0081 | 0.1159 | 14 | 0.6116 | 0.0001 | 0 | — | — | — | — |
| HPV 39 | 29 | 20.8464 | 0.0424 | 26 | 2.5572 | 0.0181 | 21 | 28.7559 | 0.0424 | −0.0010 | 0.5901 | 34 | 9.8758 | 0.0502 | 1 | 0.0065 | 0.0065 | — | — |
| HPV 42 | 31 | 76.5188 | 0.6357 | 22 | 92.8817 | 1.3589 | 15 | 121.1540 | 3.5673 | −0.2331 | 0.6909 | 38 | 58.21119 | 0.5271 | 3 | 0.5311 | 0.5311 | −987.8353 | 0.3173 |
| HPV 45 | 21 | 0.3248 | 0.0037 | 18 | 0.5486 | 0.0015 | 12 | 0.5624 | 0.0282 | +0.0024 | 0.4802 | 27 | 0.3092 | 0.0014 | 0 | — | — | — | — |
| HPV 51 | 42 | 9.5465 | 0.0018 | 27 | 2.5436 | 0.0039 | 20 | 19.8091 | 0.0296 | −0.0233 | 0.0228 | 49 | 4.8407 | 0.0009 | 4 | 0.3027 | 0.3345 | −90.2652 | 0.1797 |
| HPV 52 | 31 | 2.9481 | 0.0169 | 29 | 6.1703 | 0.0019 | 21 | 4.2865 | 0.0362 | −0.0105 | 0.2172 | 39 | 3.4832 | 0.0060 | 2 | 0.2593 | 0.2593 | −7.1842 | 0.1797 |
| HPV 58 | 27 | 0.0180 | 0.0008 | 14 | 0.0092 | 0.0013 | 13 | 0.0270 | 0.0052 | −0.0012 | 0.4631 | 28 | 0.0132 | 0.0015 | 0 | — | — | — | — |
| HPV 89 | 43 | 0.2717 | 0.0011 | 24 | 1.1359 | 0.0008 | 16 | 0.6322 | 0.0252 | −0.0003 | 0.9176 | 51 | 0.3852 | 0.0005 | 2 | 0.0088 | 0.0088 | −1.9659 | 0.6547 |
| All genotypes combinede | |||||||||||||||||||
| Multiple infection | 68 | 2.7420 | 0.0128 | 50 | 2.8510 | 0.0083 | 67 | 2.1689 | 0.0037 | > −0.0001 | 0.8027 | 87 | 8.2900 | 0.0547 | 10 | 0.0238 | 0.0033 | −0.0566 | 0.0593 |
| Single infection | 199 | 0.5199 | 0.0004 | 136 | 0.3696 | 0.0003 | 99 | 0.9535 | 0.0018 | −0.0005 | 0.0024 | 200 | 5.1395 | 0.0038 | 5 | 0.4826 | 0.5412 | −17.1280 | 0.0431 |
| All infection | 267 | 1.0858 | 0.0008 | 186 | 1.0366 | 0.0007 | 166 | 1.4440 | 0.0027 | −0.0002 | 0.0186 | 287f | 6.0945 | 0.0060 | 15 | 0.1767 | 0.0190 | −0.4249 | 0.0038 |
Note: HPV: human papillomavirus; n = number of HPV detected; “—”: not applicable. Bold p‐values statistically significant.
The mean viral load for the first and third trimesters combined was calculated from the mean viral load of genotypes detected at both trimesters for women with persistent genotypes, and from the viral load of genotypes detected at first trimester or third trimester only for women with transient genotypes.
The mean viral load among children (n = 15) was estimated by the mean viral load of genotypes detected at both birth and at 3‐month visits or from the viral load detected at birth or at 3 months only. If a child had the detection of a genotype detected at multiple sites at the same visit, the highest viral load was used to calculate the mean viral load.
p‐value: estimated using the Wilcoxon Signed‐Rank Test to test the difference between paired viral load at the first and third trimester in woman with persistent infection during pregnancy or to test the difference between the paired viral load detected in mothers (both trimesters combined) and in children.
Difference estimated by the median viral load of the 15 children with HPV minus the median viral load measured at both trimesters combined in their mother.
The mean viral load was estimated with the mean viral load of every genotype detected in each participant.
287 women with viral load testing at both trimesters combined (including women with transient genotypes at first trimester only (n = 101) or at the third trimester only (n = 20), or with a persistent genotype at both trimesters (n = 166).
Figure 2.
Variation in viral load of specific HPV genotypes between the first and the third trimester of pregnancy. HPV, human papillomavirus; n = number of HPV infections. (a) Includes 191 persistent genotypes specific HPV episode. (b) Includes 36 persistent HPV‐16 episode.
HPV detection in children during the perinatal period is presented in Table 3. Overall, viral load was significantly lower in the 15 children with perinatal HPV compared to their respective mothers. Considering all genotypes detected, the median difference in viral load between children and mother was of −0.4249 copies/cell (p‐value: 0.0038).
The measures of association between genotype‐specific HPV viral load during pregnancy and vertical transmission are presented in Table 4. Viral load (considered as a continuous variable) measured in the first, third or both trimesters combined, were significantly associated with vertical transmission for all genotypes, and for HPV‐16. For any genotypes and for HPV‐16 detected in the first trimester, each unit increase of viral load was associated with an increased risk of vertical transmission of 5% (aOR [95% CI]: 1.05 [1.00–1.09]) and of 14% (aOR [95% CI]: 1.14 [1.00–1.29]), respectively. The same trend was observed when considering the third and both trimesters combined. We also observed strong associations when viral load was categorized with different cut‐offs. For any genotypes and for HPV‐16 detected at first trimester, women with more than two copies per cell had a significantly higher risk of vertical transmission compared to women with two copies per cell or less (aORs = 6.41 [95% CI: 1.10–37.34] for any genotypes and 17.17 [95% CI: 1.18–250.28] for HPV‐16). Similar results were obtained for the different categorization and times when viral load was measured during pregnancy although not always significant. Moreover, when considering HPV‐16 viral load for the first and third trimesters combined, some models were not estimable because all cases of vertical transmission occurred in women in the higher categories of viral load. We also observed that the variation in viral load between third and first trimester was also associated with vertical transmission. The risk of transmission among women who had an increase in viral load between third and first trimester was higher than among those who had a decrease or similar viral load between third and first trimester (aORs = 4.51 [95% CI: 0.41–49.11] for all genotypes combined and 42.45 [95% CI: 0.89–2030.79] for HPV‐16, data not shown).
Table 4.
Association between viral load during pregnancy and vertical transmission.
| Viral load | HPV 6, 11, 16, 18, 31, 33, 39, 42, 45, 51, 52, 58, & 89 | HPV 6 only | ||||
|---|---|---|---|---|---|---|
| First trimester | First trimester | |||||
| n/N a | Crude OR (95% CI) | Adjusted OR, (95% CI) b | n/N | Crude OR (95% CI) | Adjusted OR, (95% CI) b | |
| Viral load continuous | 15/253 | 1.05 (1.00–1.09) | 1.05 (1.01–1.09) | 3/47 | 1.14 (1.01–1.29) | 1.14 (1.01–1.29) |
| Low viral load (≤ 75 percentile) | 6/192 | 1 | 1 | 1/34 | 1 | 1 |
| High viral load ( > 25 percentile) | 9/61 | 5.37 (1.83–15.76) | 5.21 (1.77–15.37) | 2/13 | 6.00 (0.49–72.77) | 6.03 (0.50–73.34) |
| Low viral load (≤ 1 copy per cell) | 10/235 | 1 | 1 | 1/38 | 1 | 1 |
| High viral load ( > 1 copy per cell) | 5/18 | 8.65 (2.58–29.03) | 8.10 (2.38–27.60) | 2/9 | 10.57 (0.84–133.07) | 10.83 (0.84–139.56) |
| Low viral load (≤ 2 copies per cell) | 13/246 | 1 | 1 | 1/40 | 1 | 1 |
| High viral load ( > 2 copies per cell) | 2/7 | 7.17 (1.27–40.53) | 6.41 (1.10–37.34) | 2/7 | 15.60 (1.19–204.78) | 17.17 (1.18–250.28) |
| Third trimester | Third trimester | |||||
| Viral load continuous | 15/253 | 1.06 (0.99–1.13) | 1.06 (0.98–1.14) | 3/47 | 2.06 (1.01–4.22) | 2.27 (0.98–5.24) |
| Low viral load (≤ 75 percentile) | 6/187 | 1 | 1 | 1/33 | 1 | 1 |
| High viral load ( > 25 percentile) | 9/66 | 4.76 (1.63–13.96) | 4.53 (1.53–13.41) | 2/14 | 5.33 (0.44–64.36) | 5.32 (0.44–64.29) |
| Low viral load (≤ 1 copy per cell) | 12/244 | 1 | 1 | 1/43 | 1 | 1 |
| High viral load ( > 1 copy per cell) | 3/9 | 9.67 (2.15–43.43) | 9.06 (1.98–41.36) | 2/4 | 6.83 (0.47–98.81) | 7.43 (0.48–116.10) |
| Low viral load (≤ 2 copies per cell) | 11/240 | 1 | 1 | 2/45 | 1 | 1 |
| High viral load ( > 2 copies per cell) | 4/13 | 9.25 (2.46–34.78) | 8.82 (2.32–33.56) | 1/2 | 21.50 (0.96–483.67) | 42.45 (0.89–2030.79) |
| First and third trimester combined | First and third trimester combined | |||||
| Viral load continuous | 15/253 | 1.06 (1.01–1.12) | 1.06 (1.00–1.12) | 3/47 | 1.35 (1.04–1.77) | 1.35 (1.04–1.76) |
| Low viral load (≤ 75 percentile) | 6/190 | 1 | 1 | 0/35 | NE | NE |
| High viral load ( > 25 percentile) | 9/63 | 5.11 (1.74–15.00) | 4.89 (1.65–14.46) | 3/12 | NE | NE |
| Low viral load (≤ 1 copy per cell) | 11/238 | 1 | 1 | 0/38 | NE | NE |
| High viral load ( > 1 copy per cell) | 4/15 | 7.50 (2.06–27.38) | 7.01 (1.89–25.98) | 3/9 | NE | NE |
| Low viral load (≤ 2 copies per cell) | 13/242 | 1 | 1 | 1/41 | 1 | 1 |
| High viral load ( > 2 copies per cell) | 2/11 | 3.91 (0.77–20.00) | 3.64 (0.70–18.89) | 2/6 | 20.00 (1.47–272.32) | 20.42 (1.48–281.87) |
Note: Bold values are statistically significant.
Abbreviations: CI, confidence interval; HPV, human papillomavirus; n, number of children with vertical transmission; N, number of HPV‐positive mothers during pregnancy, “NE”, not estimable; OR, odd ratio.
N = 253 because from 287 women with HPV detections during pregnancy we excluded 34 children with no valid HPV testing during the perinatal period.
Models were adjusted for the mother's age.
4. Discussion
This study provides a description of the dynamics of viral load of HPV during pregnancy and analysed the potential role of this viral load as a risk factor for HPV vertical transmission. Viral load of HPV decreased during pregnancy (from the first to the third trimester). Viral load detected in children was globally lower than the viral load measured in their respective mother. Importantly, viral load during pregnancy was strongly associated with vertical transmission of HPV.
We found two studies that have analysed the role of viral load during pregnancy as a risk factor for vertical transmission. In the study of Kaye et al., the mean HPV‐16 viral load observed during pregnancy in eight HPV‐16 positive mothers of the children born with HPV‐16 was 4.35 (SD ± 2.84) unit/PCR compared to 1.83 (± 1.12) unit/PCR for seven HPV‐16 positive mothers who did not transmit the virus [11]. The difference between the groups was, however, not statistically significant. Hahn et al. who have followed 72 HPV‐positive women during pregnancy, observed that the mean viral load measured in the 15 mothers who transmitted the virus to their infant was 0.30 (± 0.88) copy per cell compared to 0.35 (± 1.92) copy per cell among the 57 women who did not transmit HPV, but the difference was not statistically significant [12].
Two studies analysed the risk of vertical transmission according to HPV persistency during pregnancy. Alberico et al. who studied 23 women with HPV during pregnancy showed that the probability of vertical transmission was 57.1% among mothers with persistent infection (four children born with HPV out of the seven mothers with HPV detected at three visits during pregnancy) compared to 6.3% among those with no persistent infection (1 child out of the 16 mothers who had HPV detection at two visits or less) although they did not show whether the difference was statistically significant [13]. Castellsagué et al. who studied 66 HPV‐positive women during pregnancy observed that when the mothers had persistent HPV during pregnancy, they were more likely to transmit HPV to their children with an adjusted OR = 4.8 [95% CI: 1.4–16.9] [14]. Even if these last two studies do not directly relate to the effect of viral load, they nevertheless show a certain consistency with the results of this study since persistence could be correlated with viral load.
The literature on HPV viral load has focused on HPV‐16 or HPV‐18 among the general population of women and some have shown that viral load for these genotypes was associated with clinical outcomes such as precancerous lesions or cancer [21, 22]. Among pregnant women, viral load has been associated with higher risk of negative pregnancy outcomes [16, 17]. In a case‐control study composed of 116 women, Bruno et al. observed a higher risk of early abortion and preterm birth among women with high viral load detected in trophoblast compared to those with low levels [17]. Khayargoli et al. have also observed a strong association between HPV‐16 viral load during pregnancy and preterm birth [16] with adjusted ORs of 14 [95% CI: 1.3–153.5] and 15 [95% CI: 1.8–129.3], respectively for mothers with more than one copy per cell of HPV at first or third trimester compared to those below this threshold. We observed in this study a strong association between viral load and HPV vertical transmission. Although, the specific biological mechanisms that explain this potential association remains unknown, it is likely that higher levels of viral load could reflect higher viral replication that may predispose to a higher risk of vertical transmission.
It is also important to note that we also showed a reduction in viral load in pregnant women during pregnancy. It has been suggested that immunosuppression occurring during pregnancy in order not to reject the fetus might also lead to a greater susceptibility to viral pathogens [28]. It has also been suggested that HPV transcription in responses to growth factors and hormones, such as estrogen, progesterone and glucocorticoid, during pregnancy may increase viral load [29, 30]. The results of this study argue against these possibilities for HPV, since we observed the opposite in this cohort of pregnant women.
This study has several strengths. HERITAGE cohort is composed of a large sample of women with frequent HPV detection among mothers and children. HPV DNA genotyping and quantification were also done by real‐time PCR and the Roche Linear Array assay, known as robust methods, with high specificity and sensitivity [31]. However, this study also has limitations. Quantification of HPV viral load was obtained for 13 genotypes, representing the most common genotypes in the cohort but not all genotypes detected. Moreover, we cannot exclude the possibility that a negative HPV result rules out the presence of a latent infection in the basal epithelial cells. An HPV infection in mother may also have been missed as testing was done at only two‐time points during pregnancy. Children who had genotype detected at birth that was not detected among their mother (n = 2) are probably more likely related to an undetected/missed HPV during pregnancy and not to horizontal transmission. The precision of estimations may also be affected due to missing data (missed visits, invalid HPV testing or loss to follow‐up). The estimates regarding HPV16 particularly suffer from precision as only three cases of HPV16 vertical transmission were observed. Moreover, the ORs were adjusted for age of the mother but residual confounding bias is possible. We only adjusted for age of the mother because it is the only variable that we can think of associated with both viral load and vertical transmission. However, in a multivariable sensitivity analysis, we also included other variables possibly associated with vertical transmission only such as cytology (HSIL, LSIL, and ASCUS), type of delivery and rupture of membranes. The adjusted estimates were similar than the one obtained with the adjustment for age only (data not shown). Although residual confounding is possible, it is believed that it remains unlikely. Finally, external validity may also be affected as participants were recruited only in mostly tertiary hospital including HPV‐positive mothers at the first trimester of pregnancy.
5. Conclusion
The findings suggest that, although HPV viral load generally decreases during pregnancy, vertical transmission was strongly associated with high HPV viral load. Although, the detailed biological mechanism by which viral load is linked to vertical transmission remains to be explained, it may serve as a biomarker for the risk of vertical transmission.
Author Contributions
All authors have directly contributed to the conception and design (H.T., M.H.M., F.C., F.A., A.M.C., J.L., F.C., J.N., L.L.) or acquisition of data (J.N., L.L., H.T., M.H.M., F.C.) or analysis and interpretation (E.A.B., H.T., M.H.M., F.C.) of the study. E.A.B., H.T., and A.M.C. wrote the first draft of the manuscript. All authors have subsequently read, revised, and approved the version that is being submitted. H.T. is responsible for the overall content as the guarantor and had the full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish.
The HERITAGE Study Group
Marie‐Hélène Mayrand, François Coutlée, Patricia Monnier, Louise Laporte, Joseph Niyibizi, Monica Zahreddine, Ana Maria Carceller, Paul Brassard, Jacques Lacroix, Diane Francoeur, Marie‐Josée Bédard, Isabelle Girard, François Audibert, William Fraser, and Helen Trottier (principal investigator).
Conflicts of Interest
F.C. has received funds to evaluate novel HPV detection tests through his institution, the research centre of the CHUM, from Becton‐Dickson and Roche Molecular systems. H.T. has received occasional lecture fees from Merck and unrestricted grants form ViiV Healthcare. All other co‐authors have no conflicts of interest.
Supporting information
Supporting information.
Acknowledgments
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) (Grant MOP‐93564 and MOP‐136833) to H.T. CIHR was not involved in the study design, the collection, analysis and interpretation of the data, the writing of the report nor in the decision to submit this article for publication. H.T. held a salary award (chercheur‐boursier) for the duration of this study from the Fonds de la recherche du Québec en santé (FRQ‐S), and from CIHR (new investigator salary award). M.H.M. held a salary award (chercheur‐boursier clinicien) from the FRQ‐S until 2022. Funding for quality control of HPV testing was provided in part by the Réseau FRQS SIDA‐MI to F.C. We thank all study participants and research staff who collaborated with patients and managed specimens across all sites, and assisted with the recruitment of patients (Hasna Meddour, Myra Geoffrion, Kathleen Auclair, Véronique Prévost, Fabiola Correa Botello, Sophie Perreault, and Lise‐Angela Ouellet (Centre hospitalier universitaire (CHU) Sainte‐Justine); Sylvie Daigle, Sophie Leblanc, and Mélanie Robinson (Centre hospitalier universitaire de l'Université de Montréal (CHUM); Siham Aboulfadi (St Mary's Hospital), Josée Poirier, Audrée Janelle‐Montcalm, Isabelle Krauss, and Cindy Rousseau (CHU Sainte‐Justine) and François Beaudoin (in memorium) and Patricia Monnier (in memorium) (CHU Sainte‐Justine)). We also thank Julie Guenoun and Pierre Forest (CHUM) for their contributions to the DNA extraction and HPV testing procedures.
Contributor Information
Helen Trottier, Email: helen.trottier@umontreal.ca.
The HERITAGE study group:
Marie‐Hélène Mayrand, François Coutlée, Patricia Monnier, Louise Laporte, Joseph Niyibizi, Monica Zahreddine, Ana Maria Carceller, Paul Brassard, Jacques Lacroix, Diane Francoeur, Marie‐Josée Bédard, Isabelle Girard, François Audibert, William Fraser, and Helen Trottier
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supporting information.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.