Abstract and Introduction
Study Question: How does hormonal contraceptive use and menstrual cycle phase affect the female microbiome across different body sites?
Summary Answer: The menstrual cycle phase, but not hormonal contraceptive use, is associated with the vaginal and oral but not the gut microbiome composition in healthy young women.
What is Known Already: Women with low vaginal levels of Lactobacillus crispatus are at increased risk of pre-term birth, fertility treatment failure, sexually transmitted infections and gynaecological cancers. Little is known about the effect of hormonal fluctuations on other body site's microbiomes as well as the interplay between them.
Study Design, Size, Duration: This study includes a cohort of 160 healthy young Danish women using three different contraceptive regimens: non-hormonal methods (n = 54), combined oral contraceptive (COC, n = 52) or levonorgestrel intrauterine system (LNG-IUS, n = 54). Samples were collected from four body sites during the menstrual cycle (menses, follicular and luteal phases) at Copenhagen University Hospital, Rigshospitalet, Denmark.
Participants/Materials, Setting, Methods: The oral, vaginal, rectal and faecal microbiomes were characterized by shotgun sequencing. Microbial diversity and community distance measures were compared between study groups, menstrual phase timepoints and body sites. All participants answered an extensive questionnaire on current health, lifestyle and sex life. Confounding factors such as smoking, BMI and diet were analysed by PERMANOVA. Plasma oestradiol and progesterone levels are correlated with microbiome composition.
Main Results and the Role of Chance: The use of COC and LNG-IUS was not associated with the microbiome composition or diversity. However, increased diversity in the vaginal microbiome was observed during menses, followed by a subsequent expansion of Lactobacillus spp. during the follicular and luteal phases which correlated with measured serum oestradiol levels (r = 0.11, P < 0.001). During menses, 89 women (58%) had a dysbiotic vaginal microbiome with <60% Lactobacillus spp. This declined to 49 (32%) in the follicular phase (P < 0.001) and 44 (29%) in the luteal phase (P < 0.001). During menses, bacterial richness and diversity in saliva reached its lowest point while no differences were observed in the faecal microbiome. The microbiome in different body sites was on average more similar within the same individual than between individuals, despite phase or hormonal treatment. Only the vagina presented a clear cluster structure with dominance of either L. crispatus, Lactobacillus iners, Gardnerella vaginalis or Prevotella spp.
Large Scale Data: The microbiome samples analysed in this study were submitted to the European Nucleotide Archive under project number PRJEB37731, samples ERS4421369–ERS4422941.
Limitations, Reasons For Caution: The cohort is homogenous which limits extrapolation of the effects of ethnicity and socio-economic status on the microbiome. We only present three defined timepoints across the menstrual phase and miss potential important day to day fluctuations.
Wider Implications of the Findings: The use of hormonal contraception did not significantly associate with the microbiome composition in the vagina, faeces, rectum or saliva in healthy young women. This is a welcome finding considering the widespread and prolonged use of these highly efficient contraceptive methods. The menstrual cycle is, however, a major confounding factor for the vaginal microbiome. As such, the time point in the menstrual cycle should be considered when analysing the microbiome of women of reproductive age, since stratifying by vaginal dysbiosis status during menstruation could be misleading. This is the first study to confirm by direct measurements of oestradiol, a correlation with the presence of L. crispatus, adding evidence of a possible hormonal mechanism for the maintenance of this desirable microbe.
Study Funding/Competing Interest(S): This work was partly funded by the Ferring Pharmaceuticals through a research collaboration with The Centre for Translational Microbiome Research (CTMR) at the Karolinska Institutet (L.W.H., E.F., G.E. and I.S.-K.). Ferring Pharmaceuticals also funded the infrastructure to obtain the clinical samples at Copenhagen University Hospital ([#MiHSN01], M.C.K., Z.B., and H.S.N.). This work was also supported by funding from Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K.) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). M.C.K., L.W.H., E.F., Z.B., G.E., L.E., I.S.-K. and H.S.N., are partially funded by Ferring Pharmaceuticals, which also provided funds for the collection and processing of the samples analysed in this study. H.S.N.'s research is further supported by Freya Biosciences and the BioInnovation Institute. H.S.N. has received honoraria from Ferring Pharmaceuticals, Merck A/S, Astra-Zeneca, Cook Medical and Ibsa Nordic. A.N.A. reports no competing interests.
Microbial communities inhabit every inch of the human body and are thought to actively contribute to the homeostasis and health of every individual (Nelson et al., 2010; Lloyd-Price et al., 2017; Young, 2017). The implications of the microbiota in various body sites and their potential interaction and individual importance in maintaining general health are, however, inadequately understood. Most studies focus on one body site at a time and extensive work has been published documenting associations between the digestive tract microbiome and numerous health conditions, from gastrointestinal diseases to mental illnesses (Kostic et al., 2014; Meijnikman et al., 2018; Winter et al., 2018; Figuero et al., 2020; Hernández-Ceballos et al., 2021). The most studied niche in the female reproductive tract is the vaginal microbiome, which has been connected to gynaecological health (Green et al., 2015; Kroon et al., 2018). The gut and oral microbiomes have been shown to have systemic effects and the question remains whether there is an interaction between the microbiota in various body sites and how they could potentially impact women's reproductive health.
Among the most common colonizers of the vaginal tract in reproductive aged women are Lactobacillus species which have been established as the healthy vaginal microbiome linked to positive health outcomes (Ravel et al., 2011; Romero et al., 2014; Haahr et al., 2016; Kroon et al., 2018; Van Houdt et al., 2018; Brusselaers et al., 2019). Some women of reproductive age lack this Lactobacillus dominance in the vagina but have a diverse composition of other bacteria including anaerobic bacteria. This is similar to the microbial composition in bacterial vaginosis (BV), a condition characterized by thin, greyish vaginal discharge, unpleasant odour and increased vaginal pH. While common, especially among women of African descent (Zhou et al., 2010; Borgdorff et al., 2017; Gosmann et al., 2017), this microbial composition has been connected to susceptibility to sexually transmitted infections (Atashili et al., 2008; Edwards et al., 2019), difficulties achieving pregnancy after fertility treatments (Haahr et al., 2016; Koedooder et al., 2019), pre-term birth (Elovitz et al., 2019; Fettweis et al., 2019), HPV infection and gynaecological cancers (Brusselaers et al., 2019; Coudray and Kiplagat, 2019). This has led many research groups to classify a diverse vaginal microbiome with low abundance of Lactobacillus spp. as dysbiotic, even in the absence of symptoms (Forney et al., 2006; Balle et al., 2020). Interestingly, it is suspected that Lactobacillus dominance in the vagina is oestradiol- and/or progesterone-dependent as it is more common in the reproductive years of life, leaving girls and postmenopausal women with a more diverse microbiome with less abundance of Lactobacillus (Hickey et al., 2015; Muhleisen and Herbst-Kralovetz, 2016; Kaur et al., 2020).
The possible effect of hormonal contraception on the vaginal microbiome is debated. Some studies argue for a beneficial impact of the synthetic oestrogens in the combined oral contraceptive pill (COC) favouring Lactobacillus dominance (Vodstrcil et al., 2013; Brooks et al., 2017), others find no impact on the vaginal microbiome (Achilles and Hillier, 2013; Donders et al., 2017). A more diverse vaginal microbiome in levonorgestrel intrauterine systems (LNG-IUS) users have been reported (Song et al., 2020), although this alteration has also been found to be temporarily after insertion (Donders et al., 2018). Many women, especially younger women, use the monophasic COC and are continually exposed to the synthetic ethinyl oestradiol and a progestin. Both hormones work synergistically to suppress ovulation (De Leo et al., 2016), whereas most women using the progestin-only (LNG-IUS) continue to ovulate but with suppression of endometrial growth, resulting in less or absent menstrual bleeding (Grandi et al., 2018). The possible systemic effect of synthetic hormones on the microbiome composition across the body has not been extensively investigated to date, although we have demonstrated some effects for saliva (Bostanci et al., 2021).
Extensive work by Ravel's group has categorized specific clusters of microorganisms in the vagina and described the temporal dynamics of the composition in the individual woman (Ravel et al., 2011; Gajer et al., 2012). They found that some women remain stable and others shift between a Lactobacillus dominated microbiome and a more diverse community state type (CST) over time (Gajer et al., 2012). Previous studies of the vaginal microbiome have primarily used 16S rRNA gene sequencing but with time, shotgun metagenomic sequencing techniques have emerged and can provide an extensive mapping of all DNA present in the sample.
A recent study has simultaneously assessed the faecal, vaginal and cervical microbiomes of a cohort of 28 women at a single time-point, but each body site was analysed separately (Ata et al., 2019). No previous studies have investigated the longitudinal effect of the fluctuations of sex hormones during the menstrual cycle and the possible effect of hormonal contraception on the composition of the microbiome across body sites. This study compares the shotgun metagenomic profiles of four different body sites in women during a natural menstrual cycle and in women using COC or LNG-IUS to explore an impact of supressed ovulation, menstrual bleeding and shifts in sex hormones on the overall microbiome composition.
Hum Reprod. 2022;37(7):1525-1543. © 2022 Oxford University Press