Association between the vaginal microbiome and high-risk human papillomavirus infection in pregnant Chinese women

Human papillomavirus (HPV), a type of DNA virus, is associated with cervical intraepithelial neoplasia (CIN) and cervical adenocarcinoma. Indeed, HPV infection is closely related with cervical cancer and it is also present in other anogenital, head and neck cancers. According to their oncogenic potential, more than 100 types of HPV were classified as high-risk, probable high-risk, and low-risk types [1]. More than 40 types are sexually transmitted and infect the anus and genitals. Compared to warts caused by low-risk HPV types, high-risk HPV types, which account for 99% of cervical neoplasias, are usually asymptomatic [1], and as a result, these latter types are more difficult to detect. Many factors have been proven to increase the risk of genital HPV infection, e.g., sexual behaviors, number of sexual partners, early onset of sexual activity and coinfection with other sexually transmitted infections [2‐4].

A large number of microorganisms inhabit the female genital tract, and hundreds of studies have revealed that a dominance of Lactobacillus lowers bacterial richness and diversity, indicating a healthy vaginal status. A healthy microbial biofilm may prevent or hinder many urogenital diseases, such as Candida infection [5, 6], sexually transmitted diseases [7], urinary tract infections [8, 9] and human immunodeficiency virus (HIV) infection [10]. Some studies to date have also reported that changes in the vaginal microbial structure have a close connection with HPV infection [11‐14] and CIN progression [15‐17].

Pregnancy is a unique physiological state, and the composition of the vaginal microbiota changes when women become pregnant due to fluctuations in hormone levels [18, 19]. It has been claimed that a vaginal bacterial composition dominated by one or two species of Lactobacillus is especially present during pregnancy [18‐20]. However, dysbiosis of the vaginal microbiota during pregnancy has been reported to be associated with many complications of pregnancy, such as an increased risk of miscarriage, preterm birth and endometritis [18, 21, 22], yet few studies have investigated the relationship between HPV infection and the vaginal microbiome in pregnant women. It has been reported that children born to HPV-positive mothers have a significantly higher risk for developing infantile anal and genital warts [23], though the occurrence rate of cervical precancerous lesions during pregnancy is comparable to that of nonpregnant women [24]. Overall, the prevalence of HPV in pregnant women remains controversial in the literature, varying from 16.8 to 34.2% [25‐28]. Hence, it is still under debate whether pregnant women are predisposed toward HPV infection, and the factors that may influence susceptibility are unclear. Because the vaginal microbiome plays an important role in HPV infection in nonpregnant women, we hypothesized that a different vaginal microbial environment during pregnancy might facilitate HPV infection.

Therefore, the aim of this study is to investigate the association between the vaginal microbial composition and high-risk HPV infection in pregnant women. We attempt to distinguish a different microbial profile of HPV-infected pregnant women from that of nonpregnant women.

Our study addressed an undetermined topic about the association between the vaginal microbiota and hrHPV infection in pregnant women. We observed that both pregnancy and hrHPV infection were accompanied by increased vaginal bacterial richness and diversity. Lactobacillus was still the most abundant genus in all groups; however, both hrHPV infection and pregnancy had a negative influence on its abundance. Pregnancy and hrHPV infection were also accompanied by an increased proportion of CST I (dominated by Lactobacillus crispatus), as opposed to CST III. The abundances of various genera were differentially influenced by hrHPV infection and pregnancy. Overall, more anaerobic bacteria were associated with hrHPV infection and pregnancy.

According to our results, pregnancy increased vaginal bacterial richness and diversity, though some previous studies have reported lower vaginal microbial diversity during pregnancy [19, 20, 37]. A possible reason for the different findings might be that higher estrogen concentrations during pregnancy resulting in an accumulation of glycogen metabolized to lactic acid by Lactobacillus spp. on the upper layer of the vaginal epithelium, and leading to an increased abundance of Lactobacillus spp. in the vagina, thus decreasing overall bacterial richness and diversity. In this study, we observed a reduced abundance of Lactobacillus during pregnancy, which might lead to the growth of other bacteria, increasing bacterial diversity and richness in pregnant women. A similar result was reported in another study of a Chinese cohort, whereby Huang et al. found higher vaginal bacterial diversity in pregnant women, especially in the first trimester [38]. A reasonable explanation might be that fluctuation in the vaginal microbiota differs between Asian populations and Western populations and is heavily influenced by ethnicity. Regardless, the intrinsic causes need to be further studied by investigations of larger scope.

HPV infection can increase vaginal bacterial richness and diversity and lower the percentage of Lactobacillus [11, 12], and our results are in agreement with these previous studies. HPV infection is thought to alter the acidic environment of the vagina, which might promote outbreaks of bacteria [12]. In addition, the mucosal immunity and inflammation induced by HPV infection, including induction of pro-inflammatory cytokines, production of reactive oxygen species and activation of immune cells, might lead to changes in the vaginal microbiota [39]. However, other studies have indicated that hrHPV is not necessarily sufficient to induce changes in the cervicovaginal microbiota [17, 40], even though enrichment of certain anaerobic bacteria was found in patients with CIN lesions [17]. As we did not conduct cervical biopsy for high-risk HPV-infected women in our study, the presence of CIN was not assessed. Hence, we cannot exclude the possibility that the vaginal microbiota was also influenced by CIN lesions [17]. Interestingly, we found that the influence of pregnancy on bacterial richness was greater than that of HPV infection. To date, Tuominen H. et al. has also reported an altered microbial composition in cervix and placenta of HPV positive pregnant women [14]. However, it remains uncertain whether the changes result from HPV or other factors. Although both pregnancy and hrHPV infection reduced the abundance of Lactobacillus, which may contribute to increased bacterial richness and diversity, it is clear that the underlying mechanisms responsible are different. Changes in physiological hormones and the immunosuppressive state that occurs during pregnancy might lead to a fragile balance of the vaginal microbiome, which should be further explored in future studies.

Regarding community state types, the most abundant CST among nonpregnant reproductive-age women (NPN) was CST III, which was in accordance with the results from an Asian population in the study by Ravel et al. [34]. The most dominant CST in the hrHPV infection and pregnancy groups (PN, PHR and NPHR) was CST I. The predominant CSTs during pregnancy reported in previous studies varied from CST III to CST I [19, 37, 41]. Several previous studies have shown that L. iners and L. crispatus are the two most abundant Lactobacillus species found in pregnant women [18, 19, 37, 38, 41‐43]. In the vagina, a bacterial community change from CST III to CST IV is commonly observed, thus indicating that a colonization of anaerobes is more frequent in an L. iners-dominant VMB (vaginal microbiome). Conversely, an L. crispatus-dominant VMB has been associated with a low-stress environment and an adequate level of autophagy by vaginal epithelial cells to remove harmful cytoplasmic components, as well as bacteria, in pregnant women [41]. Similarly, the predominant CSTs in cases of HPV infection are controversial. Lee et al. reported that the prevalence of HPV infection did not differ between CST III and CST I [12], whereas another study found a higher HPV infection rate for an L. iners-dominant VMB [13]. It should be noted that these studies were performed using cohorts of different ethnicities, which might be one of the reasons for the differences. We also observed HPV infection to be associated with an increased proportion of CST IV in both nonpregnant and pregnant women. In two longitudinal studies, CST IV dominated by anaerobic bacteria comprised the greatest proportion of HPV-positive samples, and CST IV was associated with an increased risk of transitioning to an HPV-positive state [13, 44].

HPV infection and pregnancy equally influenced the vaginal microbial composition, but different specific genera were enriched in hrHPV-infected or pregnant women. Bifidobacterium, Bacillus, Megasphaera, Sneathia, Prevotella, Gardnerella, Fastidiosipila and Dialister were identified as significant taxa in nonpregnant hrHPV-infected populations; in previous studies, anaerobic bacteria such as Bacillus, Megasphaera, Sneathia, Prevotella, Gardnerella and Dialister have been associated with HPV infection [12, 13, 45, 46]. In general, a microenvironment with a high proportion of anaerobic bacteria and a lower proportion of Lactobacillus spp. is more susceptible to HPV infection. A surprising finding was that Bifidobacterium, a type of lactic acid-producing probiotic [47], was enriched in HPV-positive women. Although participants who used probiotics in the preceding 2 weeks were excluded from the study, we cannot rule out the influence of probiotics taken prior to that time, and they might have some persistent effects on the vaginal microbiota [48]. It has been hypothesized that Bifidobacterium might be able to guarantee a healthy vaginal balance by the production of lactic acid, in the lack of Lactobacillus. However, there are several cases reporting Bifidobacterium species as pathogens in various infectious conditions [49, 50], and a high level of stress inducers has been detected in vaginal epithelial cells when Bifidobacterium predominated the VMB [38]. Hence, the role of members of this genus should be further investigated. However, as both HPV infection and pregnancy are able to influence the vaginal microbiome, an inconsistent profiling of significant bacteria was found in hrHPV-infected pregnant women compared to nonpregnant women. Anaerobic bacteria (Megasphaera and Bacillus) and lactic acid-producing bacteria (Bifidobacterium and Lactococcus) were identified in these samples. Moreover, we found novel vaginal bacterial taxa, i.e., Acidovorax and Oceanobacillus, which were also associated with HPV infection in pregnant women. Nevertheless, the roles of these microorganisms are not clear and should be investigated further. These findings support our hypothesis that hrHPV infection and pregnancy have a dual effect on increasing vaginal bacterial diversity, leading to a differentially altered vaginal microbiota in HPV-infected pregnant women compared to nonpregnant HPV-infected women.

The strength of this study is that it addressed still-unresolved issues regarding the association between the VMB and HPV infection in pregnant women. We found high diversity and richness for the vaginal microbiome during pregnancy in a Chinese cohort and uncovered that pregnancy and HPV infection have a probable synergistic effect on altering the VMB, which caused a more complicated vaginal bacterial environment in pregnant women with hrHPV infection. The limitations of this study were that it was a small, cross-sectional study, and thus we could not determine any causation between the VMB and HPV infection or pregnancy. Further studies with longitudinal sampling are needed to assess correlations between the dynamics of the VMB microbiome and the transition of HPV at early, mild, and late gestational stages as well as postpartum and to investigate its relationship with different subtypes of HPV.

This work uncovered a probable synergistic effect of hrHPV infection and pregnancy on the vaginal microbial composition. Despite not having measured hormone level in our patients, pregnancy status seems to be a bigger driver of the vaginal diversity in our cohort, as other studies demonstrated that HPV alone does not lead to changes in it [17, 40]. HPV infection in pregnant women was associated with a more complex and diverse microbial environment.