Investigations of microbiota composition and neuroactive pathways in association with symptoms of stress and depression in a cohort of healthy women.

Background: Despite mounting evidence of gut-brain involvement in psychiatric conditions, functional data remain limited, and analyses of other microbial niches, such as the vaginal microbiota, are lacking in relation to mental health. This aim of this study was to investigate if the connections between the gut microbiome and mental health observed in populations with a clinical diagnosis of mental illness extend to healthy women experiencing stress and depressive symptoms. Additionally, this study examined the functional pathways of the gut microbiota according to the levels of psychological symptoms. Furthermore, the study aimed to explore potential correlations between the vaginal microbiome and mental health parameters in young women without psychiatric diagnoses. Methods: In this cross-sectional study, 160 healthy Danish women (aged 18-40 years) filled out questionnaires with validated scales measuring symptoms of stress and depression and frequency of dietary intake. Fecal and vaginal microbiota samples were collected at the beginning of the menstrual cycle and vaginal samples were also collected at cycle day 8-12 and 18-22. Shotgun metagenomic profiling of the gut and vaginal microbiome was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for functional profiling and 56 Gut Brain Modules were analyzed in the fecal samples. Results: The relative abundance in the gut of the genera Escherichia, Parabacteroides, and Shigella was higher in women with elevated depressive symptoms. Women with high perceived stress showed a tendency of increased abundance of Escherichia, Shigella, and Blautia. Amongst others, the potentially pathogenic genera, Escherichia and Shigella correlate with alterations in the neuroactive pathways such as the glutamatergic, GABAeric, dopaminergic, and Kynurenine pathways. Vaginosis symptoms were more prevalent in women reporting high levels of stress and depressive symptoms. Conclusions: The findings of this study support the concept of a microbiota-associated effect on the neuroactive pathways even in healthy young women. This suggest, that targeting the gut microbiome could be a promising approach for future psychiatric interventions.

AI supported fetal echocardiography with quality assessment.

This study aimed to develop a deep learning model to assess the quality of fetal echocardiography and to perform prospective clinical validation. The model was trained on data from the 18-22-week anomaly scan conducted in seven hospitals from 2008 to 2018. Prospective validation involved 100 patients from two hospitals. A total of 5363 images from 2551 pregnancies were used for training and validation. The model's segmentation accuracy depended on image quality measured by a quality score (QS). It achieved an overall average accuracy of 0.91 (SD 0.09) across the test set, with images having above-average QS scoring 0.97 (SD 0.03). During prospective validation of 192 images, clinicians rated 44.8% (SD 9.8) of images as equal in quality, 18.69% (SD 5.7) favoring auto-captured images and 36.51% (SD 9.0) preferring manually captured ones. Images with above average QS showed better agreement on segmentations (p < 0.001) and QS (p < 0.001) with fetal medicine experts. Auto-capture saved additional planes beyond protocol requirements, resulting in more comprehensive echocardiographies. Low QS had adverse effect on both model performance and clinician's agreement with model feedback. The findings highlight the importance of developing and evaluating AI models based on 'noisy' real-life data rather than pursuing the highest accuracy possible with retrospective academic-grade data.

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

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.

The microbiome in reproductive health: protocol for a systems biology approach using a prospective, observational study design.

STUDY QUESTION: What is the microbiome profile across different body sites in relation to the normal menstrual cycle (with and without hormonal contraception), recurrent pregnancy loss (RPL) (before and during pregnancy, pregnancy loss or birth) and endometriosis (before, during and after surgery)? How do these profiles interact with genetics, environmental exposures, immunological and endocrine biomarkers? WHAT IS KNOWN ALREADY: The microbiome is a key factor influencing human health and disease in areas as diverse as immune functioning, gastrointestinal disease and mental and metabolic disorders. There is mounting evidence to suggest that the reproductive microbiome may be influential in general and reproductive health, fertility and pregnancy outcomes. STUDY DESIGN SIZE DURATION: This is a prospective, longitudinal, observational study using a systems biology approach in three cohorts totalling 920 participants. Since microbiome profiles by shot-gun sequencing have never been investigated in healthy controls during varying phases of the menstrual cycle, patients with RPL and patients with endometriosis, no formal sample size calculation can be performed. The study period is from 2017 to 2024 and allows for longitudinal profiling of study participants to enable deeper understanding of the role of the microbiome and of host-microbe interactions in reproductive health. PARTICIPANTS/MATERIALS SETTING METHODS: Participants in each cohort are as follows: Part 1 MiMens-150 healthy women with or without hormonal contraception; Part 2 MiRPL-200 couples with RPL, 50 healthy couples with prior uncomplicated pregnancy and 150 newborns; Part 3 MiEndo-120 patients with endometriosis requiring surgery with or without hormonal treatment. Microbiome profiles from saliva, faeces, rectal mucosa, vaginal fluid and endometrium will be studied, as well as the Omics profile, endocrine disrupting chemicals and endocrine and immune factors in blood, hair, saliva and urine. Pregnancy loss products, seminal microbiome, HLA types, endometriotic tissue and genetic risk and comprehensive questionnaire data will also be studied, where appropriate. Correlations with mental and physical health will be evaluated. STUDY FUNDING/COMPETING INTERESTS: This work is supported by funding from Ferring Pharmaceuticals ([#MiHSN01] to H.S.N., M.C.K., M.E.M., L.E.V., L.E., I.S.-K., F.B., L.W.H., E.F. and M.H.), Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K. and [#E-22222-06] to S.B.), Niels and Desiree Yde's Foundation (S.B., endocrine analyses [#2015-2784]), the Musikforlæggerne Agnes and Knut Mørk's Foundation (S.B., endocrine and immune analyses [#35108-001]) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). Medical writing assistance with this manuscript was provided by Caroline Loat, PhD, and funded by Ferring Pharmaceuticals. H.S.N. reports personal fees from Ferring Pharmaceuticals, Merck Denmark A/S, Ibsa Nordic, Astra Zeneca and Cook Medical outside the submitted work. K.W. is a full-time employee of Ferring Pharmaceuticals. No other conflicts are reported. TRIAL REGISTRATION NUMBER: N/A. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar.

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.

Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome.

The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing.IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as gynecological cancers and pregnancy outcomes. This has led to a thriving research environment but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.