The healthy female microbiome across body sites: effect of hormonal contraceptives and the menstrual cycle

Abstract

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.

Keywords: hormonal contraceptives; menstrual cycle; microbiome; shotgun sequencing; womens reproductive health.

Figure 1.

Schematic representation of the sampling scheme and taxonomic profiles. Over 50 women were recruited using one of three contraceptive regimens: NHC (non-hormonal contraceptives), COC (combined oral contraceptives) and LNG-IUS (levonorgestrel intrauterine system). Included women were sampled at the hospital during the menstrual phase (cycle days 1–3), follicular phase (cycle days 8–12) and luteal phase (cycle days 18–22). Blood samples, saliva and rectal swabs were collected at the hospital; vaginal swabs and faeces were collected by the women at home. Women on LNG-IUS with oligo-/amenorrhoea started their sampling at a random day. The average taxonomic profile observed at each body site is depicted as circle charts. Only the vaginal swabs are divided by contraceptive and sampling point in this figure, since they are the only ones with clear visible differences.

Figure 2.

Taxonomic profiles across body sites, contraceptive groups and phase of the menstrual cycle. Vaginal samples are dominated by Lactobacillus crispatus, Lactobacillus iners and Gardnerella vaginalis, as well as various Prevotella spp. Women are separated by contraceptive and sorted after their dominant group in the menstrual phase and are in the same order in each panel, so each individual’s progression through the menstrual cycle can be followed directly. For some women, the microbiome remains fairly consistent across all three time-points, while other women exhibit sharp changes. Saliva samples are dominated by Veillonella spp., Neisseria spp., Haemophilus spp. and Prevotella spp., while faecal and rectal samples are dominated by Faecalibacterium prausnitzii, Bacteroides spp., Roseburia spp. and Prevotella spp., the latter being more abundant in the rectum than in faeces. Samples are remarkably stable over time. COC, combined oral contraceptives; LNG-IUS, levonorgestrel intrauterine system; NHC, non-hormonal contraceptives.

Figure 3.

Only vaginal swabs have significant differences in diversity across the menstrual cycle. Violin plots of bacterial diversity (Simpson’s inverted index) for vaginal, saliva, faecal and rectal samples. In each panel, samples from women using NHC are depicted in pink, COC in yellow and LNG-IUS in blue. The phase of the menstrual cycle is given in the x-axes. COC, combined oral contraceptives; LNG-IUS, levonorgestrel intrauterine system; NHC, non-hormonal contraceptives.

Figure 4.

Different body sites have a unique signature, but there are points of contact. (a) Scatter plot of a two-dimensional non-metric multidimensional scaling (NMDS) of distances between samples (Bray-Curtis). Samples separate primarily by sample type, and not by phase of the cycle. Each colour represents a body site, and sampling time-point is shown in the shape of the points. (b) Genera with significant shared strains across body sites. Genera with at least 10 strains in every body site were analysed (n = 63 genera). For each genus, we tested whether separate body sites within the same individual shared strains to a larger extent than body sites from different individuals. Genera for which a few strains are prevalent over the majority of individuals can therefore not be shown to be significantly shared in this analysis.

Figure 5.

Menstrual cycle and hormonal contraceptive usage are not main drivers of sample separation, but vaginal samples present distinct clusters. (a) Scatter plots of two-dimensional non-metric multidimensional scaling of distances between samples for vaginal samples, saliva, faecal and rectal samples. None of the body sites presents a clear clustering structure, nor do samples segregate by phase of the menstrual cycle nor contraceptive. Vaginal samples present a striking triangular separation structure. (b) The same scatter plot of two-dimensional non-metric multidimensional scaling of distances between vaginal samples in Fig. 5a is presented again with a different annotation. In each panel, the relative abundance of one key taxon is depicted in colour scale, from 0 (gray) to 100% (purple): Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis and Prevotella spp. The first three of these taxa are more or less mutually exclusive, while Prevotella spp. can be found in mixtures with other taxa, but most prominently with G. vaginalis. COC, combined oral contraceptives; LNG-IUS, levonorgestrel intrauterine system; NHC, non-hormonal contraceptives.

Figure 6.

The human microbiome is most variable in the vagina and least stable during the menstrual phase. Violin plots of the within-subject Bray-Curtis sample distance in the transition between each phase of the menstrual cycle for (a) vaginal samples (b) saliva and (c) fecal samples. The phase transition is given in the x-axis. Significant differences within a phase transition are all in relation to non-hormonal contraception, and differences between phase transitions are in relation to the menstrual/follicular transition. *P < 0.05; ***P < 0.001; Pink: non-hormonal contraceptive. Yellow: combined oral contraceptive. Blue: levonorgestrel intrauterine system.

Figure 7.

Over the menstrual cycle, the vaginal microbiome of more women becomes dominated by Lactobacillus crispatus. Alluvial plot showing the fate of each sample across the menstrual cycle. ‘Dominance’ is defined as >60% of reads coming from the same genus or species. The ‘Lactobacillus’ groups comprise samples with a mixture of Lactobacillus species. Samples lacking a dominant group are classified as ‘mixed’. The three groups not dominated by Lactobacillus species are shaded grey. The lines depicting the trajectory of each sample are coloured after the contraceptive used by that individual. Pink: non-hormonal contraceptive. Yellow: combined oral contraceptive. Blue: levonorgestrel intrauterine system.

Figure 8.

Species whose abundance in the vagina varies over the menstrual cycle and correlate with oestradiol levels. (a) Violin plots showing the relative abundance of species whose abundance is significantly correlated to the phase of the menstrual cycle, adjusted for contraceptive usage and lifestyle variables. Y-axis in each panel is adjusted to show the differences in the data, so scale varies. (1) Menstrual phase, (2) follicular phase, (3) luteal phase. *P < 0.05, **P < 0.01, ***P < 0.001. (b) Both genus Lactobacillus and the species Lactobacillus crispatus are more abundant in the presence of high oestradiol levels, although some women may present low Lactobacillus spp. abundance regardless of their oestradiol level.