How does hormonal contraceptive use and menstrual cycle phase affect the female microbiome across different body sites? 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. 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. 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. 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. 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. The microbiome samples analysed in this study were submitted to the European Nucleotide Archive under project number PRJEB37731, samples ERS4421369–ERS4422941. 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. 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. 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.

Bacterial vaginosis (BV) is a condition in which the vaginal microbiome presents an overgrowth of obligate and facultative anaerobes, which disturbs the vaginal microbiome balance. BV is a common and recurring vaginal infection among women of reproductive age and is associated with adverse health outcomes and a decreased quality of life. The current recommended first-line treatment for BV is antibiotics, despite the high recurrence rate. Live biopharmaceutical products/probiotics and vaginal microbiome transplantation (VMT) have also been tested in clinical trials for BV. In this review, we discuss the advantages and challenges of current BV treatments and interventions. Furthermore, we provide our understanding of why current clinical trials with probiotics have had mixed results, which is mainly due to not administering the correct bacteria to the correct body site. Here, we propose a great opportunity for large clinical trials with probiotic strains isolated from the vaginal tract (e.g., Lactobacillus crispatus) and administered directly into the vagina after pretreatment.

The placenta plays an important role in the modulation of pregnancy immunity; however, there is no consensus regarding the existence of a placental microbiome in healthy full-term pregnancies. This study aimed to investigate the existence and origin of a placental microbiome. A cross-sectional study comparing samples (3 layers of placental tissue, amniotic fluid, vernix caseosa, and saliva, vaginal, and rectal samples) from 2 groups of full-term births: 50 women not in labor with elective cesarean deliveries and 26 with vaginal deliveries. The comparisons were performed using polymerase chain reaction amplification and DNA sequencing techniques and bacterial culture experiments. There were no significant differences regarding background characteristics between women who delivered by elective cesarean and those who delivered vaginally. Quantitative measurements of bacterial content in all 3 placental layers (quantitative polymerase chain reaction of the 16S ribosomal RNA gene) did not show any significant difference among any of the sample types and the negative controls. Here, 16S ribosomal RNA gene sequencing of the maternal side of the placenta could not differentiate between bacteria in the placental tissue and contamination of the laboratory reagents with bacterial DNA. Probe-specific quantitative polymerase chain reaction for bacterial taxa suspected to be present in the placenta could not detect any statistically significant difference between the 2 groups. In bacterial cultures, substantially more bacteria were observed in the placenta layers from vaginal deliveries than those from cesarean deliveries. In addition, 16S ribosomal RNA gene sequencing of bacterial colonies revealed that most of the bacteria that grew on the plates were genera typically found in human skin; moreover, it revealed that placentas delivered vaginally contained a high prevalence of common vaginal bacteria. Bacterial growth inhibition experiments indicated that placental tissue may facilitate the inhibition of bacterial growth. We found no evidence to support the existence of a placental microbiome in our study of 76 term pregnancies, which used polymerase chain reaction amplification and sequencing techniques and bacterial culture experiments. Incidental findings of bacterial species could be due to contamination or to low-grade bacterial presence in some locations; such bacteria do not represent a placental microbiome per se.

The interest in and understanding of the human microbiome has grown remarkably over recent years. Advances in molecular techniques have allowed researchers to identify and study the microbiota and also use this information to develop therapeutic solutions for a spectrum of conditions. Alongside the growing interest in the microbiome, societal changes have resulted in many couples looking to start families later in life, therefore increasing the demand for assisted reproductive technologies. Combining these trends, it makes sense that clinicians are eager to understand and exploit the microbiome of their patients, i.e. the reproductive microbiome, in order to help them achieve their goal of becoming parents. This paper aims to provide an overview of the current and future research into the reproductive microbiome in relation to fertility and also share clinical practice recommendations for physicians who are new to this field or unsure about how they can utilise what is known to help their patients.

A healthy vaginal microbiome (VMB) is dominated by Lactobacillus spp. and provides the first line of defense against invading pathogens. Vaginal dysbiosis, characterized by the loss of Lactobacillus dominance and increase of microbial diversity, has been linked to an increased risk of adverse genital tract diseases, including bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, sexually transmitted infections, and pregnancy complications such as preterm birth. Currently, antibiotics and antifungals are recommended first-line treatments with high cure rates, but they also can lead to high recurrence and resistance development. As an alternative, lactobacilli have been utilized to restore the vaginal microbiota. In this review article, we discuss vaginal dysbiosis in various gynecological infections and potential interventions based on Live Biotherapeutic Products (LBPs) with a focus on those that use intravaginal treatment modalities to modulate the VMB. Based on these, we provide insights on key factors to consider in designing phenotypic and genotypic screens for selecting bacterial strains for use as vaginally administered microbiome-directed therapeutics. Lastly, to highlight current progress within this field, we provide an overview of LBPs currently being developed with published clinical trial completion for recurrent BV, VVC, and UTI. We also discuss regulatory challenges in the drug development process to harmonize future research efforts in VMB therapy.

Purpose The Swedish Maternal Microbiome (SweMaMi) project was initiated to better understand the dynamics of the microbiome in pregnancy, with longitudinal microbiome sampling, shotgun metagenomics, extensive questionnaires and health registry linkage. Participants Pregnant women were recruited before the 20th gestational week during 2017–2021 in Sweden. In total, 5439 pregnancies (5193 unique women) were included. For 3973 pregnancies (73%), samples were provided at baseline, and for 3141 (58%) at all three timepoints (second and third trimester and postpartum). In total, 38 591 maternal microbiome samples (vaginal, faecal and saliva) and 3109 infant faecal samples were collected. Questionnaires were used to collect information on general, reproductive and mental health, diet and lifestyle, complemented by linkage to the nationwide health registries, also used to follow up the health of the offspring (up to age 10). Findings to date The cohort is fairly representative for the total Swedish pregnant population (data from 2019), with 41% first-time mothers. Women with university level education, born in Sweden, with normal body mass index, not using tobacco-products and aged 30–34 years were slightly over-represented. Future plans The sample and data collection were finalised in November 2021. The next steps are the characterisation of the microbial DNA and linkage to the health and demographic information from the questionnaires and registries. The role of the microbiome on maternal and neonatal outcomes and early-childhood diseases will be explored (including preterm birth, miscarriage) and the role and interaction of other risk factors and confounders (including endometriosis, polycystic ovarian syndrome, diet, drug use). This is currently among the largest pregnancy cohorts in the world with longitudinal design and detailed and standardised microbiome sampling enabling follow-up of both mothers and children. The findings are expected to contribute greatly to the field of reproductive health focusing on pregnancy and neonatal outcomes.