Changes In Vaginal Microbiome Research Articles

Human papillomavirus molecular prevalence in south China and the impact on vaginal microbiome of unvaccinated women.

In this study, we first investigated the prevalence of different HPV genotypes in south China, which could provide more information for HPV vaccinations. Then, a total of 185 subjects were selected from HPV-negative, high-risk, low-risk, multiple hr-hr HPV infection, and mixed hr-lr HPV infection populations to explore the vaginal microbiome changes. This study displayed that HPV52, HPV58, and HPV16 were the most prevalent high-risk variants in south China. In addition, high-risk HPV infection was featured by Lactobacillus iners, while low-risk HPV infection was by Lactobacillus crispatus. Further sub-group analysis showed that Lactobacillus jensenii was significantly reduced in patients with cervical lesions. Finally, CST clustering showed that CST IV was the most common type in HC group, while CST I accounted the most in H group. In a word, this study for the first time systemically profiled vaginal microbiome of different HPV infections, which may add bricks to current knowledge on HPV infection and lay the foundation for novel treatment/prevention development.

The Vaginal Microbiome Changes During Various Fertility Treatments.

This study aimed to investigate the influence of hormonal treatment on the vaginal microbiome during fertility treatments. Bacterial vaginosis (BV) could affect fecundity, particularly in the in vitro fertilization (IVF) population, where negative effects on pregnancy outcomes have been reported. It is hypothesized that the hormone treatment during fertility treatments could influence the abundance of Lactobacilli, with negative effects on the pregnancy results. A total of 53 couples attending a fertility clinic in the Netherlands between July 2019 and August 2022 were included in this prospective cohort study. Vaginal samples were collected at start of treatment, oocyte retrieval or insemination from subjects undergoing intra uterine insemination (IUI) with mild ovarian stimulation, and IVF or intra cytoplasmatic sperm injection (ICSI) with controlled ovarian hyperstimulation. AmpliSens® Florocenosis/Bacterial vaginosis-FRT qPCR and 16S rRNA gene-based amplicon sequencing were performed on all samples. In total, 140 swabs were analyzed, with a median of two swabs per person. 33 (24%) tested qPCR BV positive. Lactobacilli percentage decreased during fertility treatments, leading to changes in the vaginal microbiome. Shannon diversity index was not significantly different. Of the total of 53 persons, nine switched from qPCR BV negative to positive during treatment. The persons switching to qPCR BV positive had already a (not significant) higher Shannon diversity index at start of treatment. If the vaginal microbiome of persons deteriorates during fertility treatments, timing of following treatments, lifestyle modifications, or a freeze all strategy could be of possible benefit.

VagiBIOM Lactobacillus suppository improves vaginal health index in perimenopausal women with bacterial vaginosis: a randomized control trial

Bacterial vaginosis (BV) can cause vaginal dysbiosis that may influence general vaginal health and pregnancy complications. Balancing vaginal microbiome using Lactobacillus spp. may be a new way to prevent and treat mild BV. We conducted a randomized, double-blind, placebo-controlled pilot study aimed at evaluating the effect of the product VagiBIOM, a multi-Lactobacillus vaginal suppository, on peri- and premenopausal women with BV in restoring vaginal pH and overall vaginal health by resetting the vaginal microbiome composition. Sixty-six peri- and premenopausal women with BV symptoms were randomized with a 2:1 ratio to be treated with VagiBIOM or placebo suppositories. Vaginal pH, VAS itching score, total Nugent score, and vaginal health index (VHI) were measured. Vaginal microbiome changes before and after the treatment were analyzed by 16S rRNA sequencing and bioinformatics analysis. After 4 weeks of intervention with VagiBIOM or a placebo, the mean score for vaginal pH, VAS itching, and total Nugent score was significantly decreased from the baseline. Compared to the baseline scores, the VHI scores improved significantly following 28-day intervention (p < 0.001). Our results revealed two Lactobacillus species, L. hamsteri, and L. helveticus, as indicator species occurring differentially in the VagiBIOM-treated group. Furthermore, the regression and species network analyses revealed significant bacterial associations after VagiBIOM treatment. Lactobacillus hamsteri was positively associated with the Nugent score and negatively associated with vaginal pH. L. iners and L. salivarius were positively and inversely associated with VHI. As is typical, Bacteroides fragilis was positively associated with vaginal pH and negatively associated with the Nugent score. Interestingly, the Lactobacillus spp. diversity improved after VagiBIOM treatment. The VagiBIOM suppository treatment for peri- and premenopausal women with BV significantly relieved vaginal itching by decreasing vaginal pH and Nugent scores and improving the overall VHI after 4 weeks’ intervention. This effect was primarily the result of VagiBIOM improving vaginal Lactobacillus diversity.Trial Registration ClinicalTrials.gov registration: NCT05060029, first registration 09/28/2021: Title: A Pilot Study to Evaluate the Efficacy and Safety of Lactobacillus Species Suppositories on Vaginal Health and pH.

Host-microbiome interactions in distinct subsets of preterm labor and birth

Preterm birth, the leading cause of perinatal morbidity, often follows premature labor, a syndrome whose prevention remains a challenge. To better understand the relationship between premature labor and host-microbiome interactions, we conducted a mechanistic investigation using three preterm birth models. We report that intra-amniotic delivery of LPS triggers inflammatory responses in the amniotic cavity and cervico-vaginal microenvironment, causing vaginal microbiome changes and signs of active labor. Intra-amniotic IL-1α delivery causes a moderate inflammatory response in the amniotic cavity but increasing inflammation in the cervico-vaginal space, leading to vaginal microbiome disruption and signs of active labor. Conversely, progesterone action blockade by RU-486 triggers local immune responses accompanying signs of active labor without altering the vaginal microbiome. Preterm labor facilitates ascension of cervico-vaginal bacteria into the amniotic cavity, regardless of stimulus. This study provides compelling mechanistic insights into the dynamic host-microbiome interactions within the cervico-vaginal microenvironment that accompany premature labor and birth.

P-672 Vaginal microbiome changes in women with polycystic ovary syndrome: a large cross-sectional study

Abstract Study question How the vaginal microbiome changes in women with polycystic ovary syndrome? Summary answer The PCOS women had a higher diversity in the vaginal microbiome and showed an enhanced level of heterogeneity. What is known already PCOS is a common reproductive endocrine disorder with a highly heterogeneous clinical presentation. Recently, few studies with limited sample size have reported that the inconsistent vaginal microbiota composition changes in PCOS. Study design, size, duration The cross-sectional study conducted at a single academic university-affiliated center. A total of 1,446 subjects, including 713 PCOS cases and 733 controls, were recruited. Participants/materials, setting, methods Vaginal swabs were collected for subsequent 16S rRNA gene sequencing. The microbiome diversities, community distances, vaginal bacterial relative abundances, and microbial interactions network were compared between the PCOS group and control group. Main results and the role of chance The PCOS group had a higher alpha diversity in the vaginal microbiome than the control (P &amp;lt; 0.05), while higher intra-group variability was observed in PCOS (P &amp;lt; 0.05). At the genus level, the abundance of Lactobacillus in the PCOS group decreased, while the abundance of Gardnerella and Ureaplasma increased (FDR&amp;lt;0.2). Gardnerella vaginalis, Prevotella buccalis, and Prevotella timonensis were identified as differential species and were significantly associated with clinical parameters of PCOS, like AMH. The microbial interaction network analysis revealed that Prevotella and Lactobacillus could be key drivers for network rewiring in PCOS. Notable alterations of predicted pathways, which significantly differed between PCOS and control women, mainly enriched in amino acid metabolism. Limitations, reasons for caution The study only included Chinese Han population data. Due to the structure of the vaginal flora may vary between ethnic groups, generalizing the results of this study to other populations should be taken caution. Wider implications of the findings This study with large samples could enhance our understanding of the PCOS vaginal microbiome, and provide a basis for future research on the potential mechanism by which pathogenic bacteria are involved in PCOS vaginal microbial imbalances. Trial registration number Not applicable

94 Changes in Vaginal Microbiome of Beef Cows Enrolled in an Estrous Synchronization Protocol

Abstract Vaginal pH and microbiome have been reported to influence fertility in women. In cattle, estrous synchronization protocols are a great tool to overcome infertility; however, the effects of the hormones administered in the protocol on vaginal microbiome and pH are unknown. A total of 24 cows were enrolled in the 7-d Co-Synch + controlled internal drug release (CIDR) protocol and had individual vaginal flushes collected during the handling events of the protocol on days 0, 7 and on day 10 at timed-artificial insemination (TAI). Flushes were used for pH analysis and DNA extraction. Extracted DNA was submitted to analysis by whole genome shotgun sequencing. Pregnancy status was assessed by ultrasonography 35 days after TAI. Firmicutes was the most abundant phylum, followed by Proteobacteria, and Actinobacteria for all collection days. Bacteria from the Clostridia genus were the most abundant in all samples. Greater alpha Shannon diversity (within sample diversity) was observed for pregnant animals on day -10 compared with non-pregnant animals at the phylum, class, and order (P &amp;lt; 0.01), as well as family, and genus level (P &amp;lt; 0.001). Overall, Shannon diversity was decreased on day 7 compared with day 0 (P &amp;lt; 0.05 at class and order level; P&amp;lt; 0.01 at genus and family level) and was greater at TAI compared with day 7 (P &amp;lt; 0.01 at the phylum, class, and order level; P &amp;lt; 0.001 at family and genus level). No differences were observed for Shannon diversity between samples from D0 and TAI (P &amp;gt; 0.05). No correlations between Shannon diversity were observed in relation to vaginal pH (P &amp;gt; 0.1). Bray-Curtis analysis (between sample diversity) revealed clustering by day (P &amp;lt; 0.05 at the order, family, and genus level) and a tendency (0.1 &amp;lt; P &amp;lt; 0.05) at the phylum and class level. In conclusion, greater fertility was observed for animals with greater within sample diversity prior to protocol initiation. Furthermore, vaginal bacterial diversity changes throughout the protocol, with decreased diversity observed on day 7 of the protocol, but restored diversity on day0 at TAI.

Changes in vaginal microbiome after focused ultrasound treatment of high-risk human papillomavirus infection-related low-grade cervical lesions

BackgroundIn this study, the changes of vaginal microbiome after focused ultrasound (FU) treatment were evaluated to explore the possible mechanism of FU in the treatment of high-risk human papillomavirus (HR-HPV) infection.MethodsThis study was nested in the FU arm of a prospective cohort study. A total of 37 patients diagnosed with HR-HPV infection-related cervical low-grade squamous intraepithelial lesion (LSIL) who met the inclusion criteria were enrolled in this study from October 2020 to November 2021, and these patients were treated with FU. We used 16S ribosomal RNA (16S rRNA) gene amplicon sequencing to profile the vaginal microbiota composition of patients before and 3 months after FU treatment.ResultsAfter FU treatment, HR-HPV was cleared in 24 patients, with a clearance rate of 75.0% (24/32). Lactobacillus iners was the predominant species among all samples. No significant difference was found in alpha-diversity index before and 3 months after FU treatment (P > 0.05), but the rarefaction curves showed that the vaginal microbial diversity before FU treatment was higher than that after FU treatment. Linear discriminant analysis (LDA) effect size (LEfSe) showed that Bifidobacterium contributed the most to the difference between the two groups at the genus level, and the abundance after FU treatment was significantly higher than that before treatment (P = 0.000).ConclusionsThe decrease of vaginal microbial diversity may be related to the clearance of HR-HPV infection, and FU treatment contributed to the decrease of vaginal microbial diversity. Increased Bifidobacterium abundance in the vaginal microbiome may be associated with clearance of HR-HPV infection, and FU treatment may contribute to the increase in Bifidobacterium abundance.Trial registration number: This study was registered in the Chinese Clinical Trial Registry on 23/11/2020 (ChiCTR2000040162).

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products.

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep.Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1β, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels.Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal.Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.

A preliminary study on the associations between Ureaplasma, Mycoplasma and the vaginal microbiome

Ureaplasma genus (including U. urealyticum and U. parvum) and M. hominis are common opportunistic pathogens in vaginal microenvironments, which would lead to bacterial vaginosis, infertility and adverse pregnancy outcomes. Ascertaining associations between these elements and the vaginal microbiome can help us identify pathogenic mechanisms, and target control measures for different colonization levels. 92 childbearing-age, non-pregnant women were included, with vaginal swabs collected. DNA was extracted and 16S rRNA genes were sequenced using the IlluminaHiseq 2500 platform. A quantitative PCR method quantified bacterial loads. The colonization rates for the Ureaplasma genus and M. hominis were 87.0 ​% (80/92) and 29.3 ​% (27/92), respectively. No M. genitalium was detected. There were no statistical differences for Simpson, Shannon and Chao1 indices between Ureaplasma negative and positive groups (P ​> ​0.05). Based on the quartile classification of relative abundance for Ureaplasma, the third quartile group had the highest relative abundance of Lactobacillus. The Simpson index for the M. hominis positive group was statistically lower than the negative group (P ​= ​0.038). The Lactobacillus abundance appeared to decrease when the M. hominis relative abundance was >0.03 ​%. From a microbiome perspective, Ureaplasma vaginal colonization, at low levels appeared to be harmless, whereas M. hominis colonization was associated with vaginal microbiome changes. Further studies are required to confirm the diagnostic and treatment thresholds for Ureaplasma and M. hominis infections, and to explore in-depth associations between these organisms and the vaginal microbiome.

Changes in Vaginal Microbiome in Pregnant and Nonpregnant Women with Bacterial Vaginosis: Toward Microbiome Diagnostics?

Bacterial vaginosis (BV) is highly common, adversely affecting the health of millions of women. New therapeutic targets and diagnostics are urgently needed for BV. Microbiome research offers new prospects in this context. We report here original findings on changes in the vaginal microbiome in pregnant and nonpregnant women with BV. Reproductive age women were recruited for this study after a clinical examination. The total sample (N = 33) included four study groups: (1) healthy nonpregnant women (n = 9), (2) nonpregnant women with symptomatic BV (n = 11), (3) healthy pregnant women without BV (n = 6), and (4) pregnant women with symptomatic BV (N = 7). The vaginal microbiota in healthy women was less diverse, with dominance of a single genus, Lactobacillus. Six major phyla appeared upon taxonomic analysis of the bacterial sequences: Firmicutes, Actinobacteria, Proteobacteria, Tenericutes, Bacteroidetes, and Fusobacteria. For instance, Firmicutes had a significantly higher abundance (98.3%) in the nonpregnant healthy group and 94.3% in pregnant healthy group, compared with nonpregnant (49.7%) and pregnant (67%) women with BV (p = 0.003). Moreover, women with BV had significant increases in representation of Actinobacteria, Fusobacteria, and Bacteroidetes (p = 0.0003, 0.004, and 0.01, respectively). Although the Lactobacillus genus was predominant in healthy women, Gardnerella, Atopobium, Sneathia, and Prevotella significantly increased in nonpregnant women with BV (p = 0.001, 0.014, 0.004, and 0.012, respectively). Dysbiosis of Lactobacillus in pregnant women with BV was accompanied by increased prevalence of the Streptococcus genus. These findings contribute new insights toward microbiome diagnostics and therapeutics innovation in BV.

Characterization of the Vaginal Microbiome in Women with Infertility and Its Potential Correlation with Hormone Stimulation during In Vitro Fertilization Surgery.

Perturbation of vaginal microbiome of reproductive-age women influences all the phases of a woman's reproductive life. Although studies have shown that dynamic changes in vaginal microbiome can affect pregnancy, its role in secondary infertility (i.e., inability to become pregnant or to carry a pregnancy successfully after previous success in delivering a child) and in vitro fertilization (IVF) remains to be unraveled. To determine the vaginal microbiome in women undergoing in vitro fertilization and embryo transfer (IVF-ET) and investigate its potential correlations with hormone stimulation, we recruited 30 patients with secondary infertility and receiving IVF and 92 matched healthy women and analyzed their vaginal microbiome composition using 16S rRNA gene sequencing. Our results show that women suffering from infertility (infertile women) exhibit a significant decrease in microbiome diversity and richness compared with healthy women during the nonovulation period (follicular phase) (P < 0.01), whereas vaginal microbiome of healthy women reveals dramatic fluctuations during ovulation (P < 0.05). Interestingly, infertility patients show no change of the vaginal microbiome under conditions of gonadotropin-releasing hormone (GnRH) agonist and recombinant human chorionic gonadotropin (r-hCG) induction (P > 0.05). Moreover, our results indicate that infertile women show characteristic variations in vaginal microbiome, such as increased abundance of Atopobium, Aerococcus, and Bifidobacterium and decreased abundance of Lactobacillus and Leuconostoc IMPORTANCE The microbiome had been hypothesized to be involved in the physiology and pathophysiology of assisted reproduction before the first success in IVF, while the data supporting or refuting this hypothesis were less than conclusive. Thanks to sequencing data from the 16S rRNA subunit, we characterized the microbiome in the reproductive tract of infertile women, and we found that changes in the vaginal microbiome are related to female infertility. We also found that the characteristic microbiome bacteria are mainly members of several genera and that the vaginal microbiome of infertile women is not sensitive to hormonal changes during IVF. In conclusion, our report provides data that can be used for discovering the role of the vaginal microbiome in patients suffering from secondary infertility.

Daily Vaginal Microbiota Fluctuations Associated with Natural Hormonal Cycle, Contraceptives, Diet, and Exercise.

The microorganisms of the vaginal tract are critical for vaginal and reproductive health. However, the regulation of these microorganisms is not well understood. Therefore, we investigated whether different factors regulate the vaginal microbiota of healthy college-aged women (n = 26) with high temporal resolution by collecting daily self-administered vaginal swabs and using 16S rRNA sequencing for bacterial identification. As expected, vaginal microbiota clustered into five predefined community state types. Vaginal microbial diversity, stability, and Lactobacillus abundances were associated with the menstrual cycle and hormonal contraceptive use. Vaginal microbial diversity, as measured using the Shannon index, increased during menses (P < 0.001), while Lactobacillus abundances decreased (P = 0.01). The covariance of these microbial measures with previously established estradiol levels suggests that estrogens can regulate vaginal microbiota. Moreover, the use of hormonal contraceptives may alter the temporal dynamics of the vaginal microbiota and decrease Lactobacillus abundances, depending on hormonal content and release method. Interestingly, intrasample diversity was greater in participants on a vegetarian diet (P = 0.004) and among participants who exercised more (P = 0.04). These findings indicate that ovarian hormones, diet, and exercise can regulate vaginal microbial composition and stability and may impact vaginal and reproductive health.IMPORTANCE The vaginal microbiome is a critical component of women's sexual and reproductive health, with variations in microbial composition, particularly the loss of Lactobacillus species, being implicated in gynecologic and obstetric diseases. Given that the vaginal microbiome is so crucial, why do vaginal microbial profiles vary strikingly from person to person and even change over time within the same person? In the present study, which tracked the daily vaginal microbiomes of young healthy women through different lifestyles, we found that use of a locally released progestin contraceptive, a vegetarian diet, and intense exercise appear to lead to vaginal microbiome alterations and loss of Lactobacillus species. The impact of these vaginal microbiome changes on immediate and long-term health remain to be investigated.

Comparison of vaginal microbiota in gynecologic cancer patients pre- and post-radiation therapy and healthy women.

BackgroundWhile the importance of commensal microbes in vaginal health is well appreciated, little is known about the effects of gynecological cancer (GynCa) and radiation therapy (RT) on the vaginal microbiome (VM) of postmenopausal women.MethodsWe studied women with GynCa, pre‐ (N = 65) and post‐RT (N = 25) and a group of healthy controls (N = 67) by sequencing the V4 region of the 16S rRNA gene from vaginal swabs and compared the diversity and composition of VMs between the three groups accounting for potential confounding factors in multivariate analysis of variance.ResultsComparisons of cancer vs healthy groups revealed that Lactobacillus and Bifidobacterium have significantly higher relative abundance in the healthy group, while the cancer group was enriched in 16 phylogroups associated with bacterial vaginosis (BV) and inflammation, including Sneathia, Prevotella, Peptoniphilus, Fusobacterium, Anaerococcus, Dialister, Moryella, and Peptostreptococcus. In our sample, RT affected the α‐diversity and correlated with higher abundance of typically rare VM species, including several members of the Lacnospiraceae family, a taxon previously linked to vaginal dysbiosis. In addition to cancer and treatment modalities, age and vaginal pH were identified as significant parameters that structure the VM.ConclusionsThis is among the first reports identifying VM changes among postmenopausal women with cancer. RT alone seems to affect several phylogroups (12 bacterial genera), while gynecological cancer and its treatment modalities are associated with even greater significant shifts in the vaginal microbiota including the enrichment of opportunistic bacterial pathogens, which warrants further attention.

Comparison of Vaginal Microbiota in Gynecologic Cancer Patients Pre- and Post-Radiation Therapy and Healthy Women

Importance of microbes in vaginal health well appreciated, yet little is known about effects of gynecological cancer (GynCa) and radiation therapy (RT) vaginal microbiome (VM). Pilot study conducted to establish rigorous methods in bacterial vs human DNA extraction, quality assurance (Q/A), VM sequencing and beginning assessment of differences in VM among women with GynCa pre & post RT compared to healthy controls. Women with GynCa (cervical or endometrial) treated for RT curative intent, had vaginal swabs collected according Human Microbiome Project protocol (T0) pre-RT and 1-2 mo post-RT (T1). Controls were sampled (T0) routine gyn visit. Quality assurance (QA) conducted on first 7 cancer pts. VM sequencing assessed V3-V4 region of 16S rRNA gene. Cohorts compared in pairwise mode of 3 groups: healthy, pre- and post-RT, accounting for potential confounders (e.g., age & ethnicity) in ADONIS analysis. Alpha/beta diversity estimates computed in non-rarefied OTU libraries. Chao-Shen correction for alpha-diversity applied to account for differences in library size, and beta-diversity was computed in CSS-normalized OTU matrix. OTU richness estimated using Chao-1 index; diversity calculated as Shannon Index. Alpha diversity metric was statistically compared between GynCa and control groups using the Wilcoxon test. Beta-diversity analyses included Bray-Curtis and Jaccard. Final study included 69 VM samples obtained from 27 cancer patients (cervical n=13; endometrial n=14) and 30 healthy controls. QA showed high reproducibility of VM data. While some pre-RT communities overlapped with healthy, most post-RT VM communities did not overlap with the healthy, especially when dichotomized as “presence/absence”- distance metric (Jaccard), indicating RT as important factor contributing to differences. Trend toward lower microbial richness observed in healthy samples relative to GynCa. Characterization at genus level revealed 13 phylogroups differentially abundant between pre-RT and healthy groups: of note are- Streptococcus anginosus (p=0.01), Corynebacterium spp (p=0.01), Actinomyces spp (p=0.005), Faecalibacterium prausnitzii (p=0.04), and Bacteroides ovatus (p=0.02) significantly higher in relative abundance in healthy vs. cancer: opposite pattern or only present in the cancer seen in Sneathia spp (p<0.001), Methylobacterium adhaesivum (p<0.001), Bacillus spp (p<0.001), Ruminococcus spp (p=0.02), Clostridium spp (p=0.02), Peptostretococcus anaerobius (p=0.006), Moryella spp (p=0.01), and Shuttleworthia spp (p=0.02). Note: Sneathia, potential female reproductive tract pathogen enriched (>10% relative abundance) in the GynCa vs healthy group (<1% relative abundance). Analysis showed rigorous methods and trends in detecting RT as significant determinant of the variation observed between post-RT and control samples. Patients treated by RT likely experience vaginal injuries that may be related to changes in VM.

Generalized Hotelling's test for paired compositional data with application to human microbiome studies.

The human microbiome is a dynamic system that changes due to diseases, medication, change in diet, etc. The paired design is a common approach to evaluate the microbial changes while controlling for the inherent differences between people. For example, microbiome data may be collected from the same individuals before and after a treatment. Two challenges exist in analyzing this type of data. First, microbiome data are compositional such that the reads for all taxa in each sample are constrained to sum to a constant. Second, the number of taxa can be much larger than the sample size. Few statistical methods exist to analyze such data besides methods that test one taxon at a time. In this paper, we propose to first conduct a log-ratio transformation of the compositions, and then develop a generalized Hotelling's test (GHT) to evaluate whether the average microbiome compositions are equivalent in the paired samples. We replace the sample covariance matrix in standard Hotelling's statistic by a shrinkage-based covariance, calculated as a weighted average of the sample covariance and a positive definite target matrix. The optimal weighting can be obtained for many commonly used target matrices. We develop a permutation procedure to assess the statistical significance. Extensive simulations show that our proposed method has well-controlled type I error and better power than a few ad hoc approaches. We apply our method to examine the vaginal microbiome changes in response to treatments for menopausal hot flashes. An R package " GHT" is freely available at https://github.com/zhaoni153/GHT.

Fractional CO2 laser for genitourinary syndrome of menopause in breast cancer survivors: clinical, immunological, and microbiological aspects.

The composition of vaginal microbiome in menopause and cancer survivor women changes dramatically leading to genitourinary syndrome of menopause (GSM) in up to 70% of patients. Recent reports suggest that laser therapy may be valuable as a not hormonal therapeutic modality. The aim of the present study was to evaluate the effects of fractional CO2 laser treatment on the vaginal secretory pathway of a large panel of immune mediators, usually implicated in tissue remodeling and inflammation, and on microbiome composition in postmenopausal breast cancer survivors. The Ion Torrent PGM platform and the Luminex Bio-Plex platform were used for microbiome and immune factor analysis. The significant reduction of clinical symptoms and the non-significant changes in vaginal microbiome support the efficacy and safety of laser treatment. Moreover, the high remodeling status in vaginal epithelium is demonstrated by the significant changes in inflammatory and modulatory cytokine patterns. Laser therapy can be used for the treatment of GSM symptoms and does not show any adverse effects. However, further studies will be needed to clarify its long-term efficacy and other effects.

Association of changes in vaginal microbiome with oligoclonal T-cell expansion and early response to chemoradiation for cervical cancer.

8 Background: The composition of the vaginal microbiome has been shown to affect clearance of HPV virus and transformation to invasive cancer. Clinical studies correlating the vaginal microbiome with immune activation and response to cancer treatment are lacking. We profiled intratumoral T-cell clonality during radiation therapy and correlated it with the diversity of the vaginal flora. Methods: Thirty patients with newly diagnosed locally advanced cervical cancer were enrolled on a prospective study to characterize changes in the cervical microbiome during chemoradiation. Cervical samples were obtained before radiation therapy and during the 1st, 3rd, and 5th week of radiation therapy. The vaginal microbiome was characterized using 16 sRNA gene sequencing to produce operational taxonomic units (OTU’s) representing individual bacterial species. Disease response was categorized as early response (ER), late response (LR), or nonresponse (NR) on the basis of clinical examination at brachytherapy and 3-month PET/CT. Twenty patients had T-cell receptor β sequencing of DNA performed using the ImmunoSEQ platform. The maximum productive frequency of the top three clones (MP3) was used to assess T-cell clonality. Results: Early response was associated with clonal T-cell expansion with an increase of MP3 of 11.1% during treatment as compared to a decline of 6.1% in patients with LR/NR (p = 0.05). Early response was also associated with lower quantity of observed OTU’s of vaginal microbiota (25.0 [SD 12.68]) vs patients with LR/NR (41.15 [SD = 23.3]) (p = 0·03). Increased MP3 was associated with increased abundance of Corynebacteriales (R = 0.90; p &lt; .0001) , Actinomycetales (R = 0.83; p &lt; .0001) and Bifidobacteriales (R = 0.82; p &lt; .0001) . Decreased MP3 was associated with increased abundance of lactobacillus (R = -0.61; p &lt; .0001). Conclusions: Increased diversity of the vaginal microbiome is negatively associated with outcome, supporting previous clinical studies in non-cancer settings. Specific vaginal bacterial species are associated with increased or decreased T-cell clonality at completion of radiation.

Short- and long-term influence of the levonorgestrel-releasing intrauterine system (Mirena®) on vaginal microbiota and Candida.

Recurrent vulvovaginal infections are a frequent complaint in young women in need of contraception. However, the influence of the contraceptive method on the course of the disease is not well known. To investigate the influence of the levonorgestrel-releasing intrauterine-system (LNG-IUS) on the vaginal microflora. Short-term (3 months) and long-term (1 to 5 years) changes of vaginal microbiota were compared with pre-insertion values in 252 women presenting for LNG-IUS insertion. Detailed microscopy on vaginal fluid was used to define lactobacillary grades (LBGs), bacterial vaginosis (BV), aerobic vaginitis (AV) and the presence of Candida. Cultures for enteric aerobic bacteria and Candida were used to back up the microscopy findings. Fisher's test was used to compare vaginal microbiome changes pre- and post-insertion. Compared to the pre-insertion period, we found a temporary worsening in LBGs and increased rates of BV and AV after 3 months of LNG-IUS. After 1 and 5 years, however, these changes were reversed, with a complete restoration to pre-insertion levels. Candida increased significantly after long-term carriage of LNG-IUS compared to the period before insertion [OR 2.0 (CL951.1-3.5), P=0.017]. Short-term use of LNG-IUS temporarily decreases lactobacillary dominance, and increases LBG, AV and BV, but after 1 to 5 years these characteristics return to pre-insertion levels, reducing the risk of complications to baseline levels. Candida colonization, on the other hand, is twice as high after 1 to 5 years of LNG-IUS use, making it less indicated for long-term use in patients with or at risk for recurrent vulvovaginal candidosis.

Vaginal microbiome changes with levonorgestrel intrauterine system placement

ObjectiveTo investigate changes in female genital tract bacterial ecology associated with levonorgestrel intrauterine system (LNG IUS) use. Study DesignStudy participants were regularly cycling Caucasian women without recent history of pregnancy or exogenous hormone use. Vaginal, cervical and uterine samples were obtained at nine time intervals, from 1 week before to 12 weeks after LNG IUS placement. Replicate paired vaginal and cervical specimens were collected to determine the consistency of specimen collection and processing. ResultsA total of 406 samples from 11 women were available for analysis after DNA extraction, amplification and species identification. Of the 355 bacterial species or genera detected, Lactobacillus crispatus was most prevalent representing 48.9% of over 6 million total reads. L. crispatus reads of replicate vaginal samples were not significantly different [odds ratio (OR) 0.79, 0.36–1.73]. In addition, L. crispatus reads of vaginal and cervical samples from the same visit were not significantly different (OR 0.69, 0.31–1.51). Compared to sampling visits prior to LNG IUS placement, sampling visits after LNG IUS insertion were more likely to have L. crispatus reads greater than 50% of total reads (OR 2.13, 1.01–4.48). Of the 63 uterine samples demonstrating bacteria, Burkholderia genus proteobacteria, a common environmental contaminant, were most prevalent both before and after LNG IUS insertion accounting for 48.0% of all uterine sample reads. ConclusionThe vaginal microbiome changes very little in response to LNG IUS placement. Further study is needed to place this finding in context with clinical outcomes. ImplicationsThere do not appear to be any clinically important differences in the vaginal microbiome in response to insertion of the LNG IUS.