The Vaginal Microbiome During Pregnancy Christina A. Muzny, MD, MSPH Associate Professor of Medicine Division of Infectious Diseases University of Alabama at Birmingham SSS STAR STI CTG 2018 Programmatic Meeting on STIs in Pregnancy & Reproductive Health April 19, 2018

Disclosures K23AI106957 (NIH/NIAID) – BV pathogenesis study R01AI097080 (NIH/NIAID) – TV treatment trial

The “Normal” Vaginal Microbiota Lactobacillus spp. dominate a healthy vaginal community Primarily L. crispatus, L. jensenii, L. gasseri, and L. iners Produce lactic acid maintaining a low (hostile) vaginal pH < 4.5 May play a role in preventing urogenital diseases Bacterial vaginosis (BV) Vaginal yeast infections Acquisition and transmission of STIs (including HIV) Urinary tract infections BMC Infectious Diseases 2011;11:200

Vaginal Microbiome of Asymptomatic, Reproductive Age Women, n=396 CST IV: Lactobacillus-poor CST IV-A: higher abundance of Peptoniphilus, Anaerococcus, Corynebacterium, Finegoldia, Gardnerella, Prevotella, etc. CST IV-B: higher abundance of Atopobium CST V: L. jensenii - dominated Composed of 5 Community State Types: CST I: L. crispatus - dominated CST II: L. gasseri - dominated CST III: L. iners - dominated The vaginal bacterial communities of 396 asymptomatic North American women who represented four ethnic groups (white, black, Hispanic, and Asian) were sampled and the species composition characterized by pyrosequencing of barcoded 16S rRNA genes. The communities clustered into five groups: four were dominated by Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii, whereas the fifth had lower proportions of lactic acid bacteria and higher proportions of strictly anaerobic organisms, indicating that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities. The major difference between CST IV-A and IV-B is that CST IV-B has a higher abundance of Atopobium, while CST IV-A has a more even microbial composition including the following phylotypes: Peptoniphilus, Anaerococcus, Corynebacterium, Finegoldia and Prevotella. Proc Natl Acad Sci USA 2011;108 Suppl 1:4680-7

The Proportions of Community State Types Vary among Ethnic Groups in Healthy, Reproductive Age Women (n=396) The proportions of each community group varied among the four ethnic groups and these differences were statistically significant [χ2(10)=36.8, P < 0.0001]. No statistically significant associations were observed between age and community types within or across ethnic groups. The inherent differences within and between women in different ethnic groups strongly argues for a more refined definition of the kinds of bacterial communities normally found in healthy women and the need to appreciate differences between individuals so they can be taken into account in risk assessment and disease diagnosis. Proc Natl Acad Sci USA 2011;108 Suppl 1:4680-7

The 4 Major Lactobacilli in the Vaginal Microbiota L. crispatus: associated with stable VMF1 L. jensenii: elicits intermediate stability; conversion to abnormal VMF is more likely to be associated with its disappearance1 L. gasseri: proliferates with intermediate VMF2 L. iners: proliferates with BV2; recolonization following an episode of BV might be a risk factor for recurrence3 1BMC Microbiology 2009; 9:116; 2BMC Microbiology 2007;7:115; 3Curr Infect Dis Rep 2007; 9:485

“Abnormal” Vaginal Microbiota Loss of lactic acid-producing lactobacilli Higher proportion of facultative (Gardnerella vaginalis) and strict anaerobes pH > 4.5 (less protective) Correlated with higher Nugent scores (used in BV diagnosis) BV associated with preterm birth in pregnant women independent of other risk factors1 Nugent score 0-3: normal vaginal flora Nugent score 4-6: intermediate flora Nugent score 7-10: BV 1N Engl J Med 1995; 333:1737-42; Photos courtesy of Charles Rivers, PhD, MSPH

Longitudinal Analysis of the Vaginal Microbiota in Pregnancy Prospective cohort study of 110 Caucasian pregnant women in Belgium Lateral vaginal wall swabs obtained once during 1st, 2nd, and 3rd trimesters 77% had normal or Lactobacillus-dominated vaginal microflora (VMF) during the 1st trimester, of which 18 had L. crispatus-dominated VMF, 16 had L. crispatus and other Lactobacillus spp., and 43 had non-L. crispatus lactobacilli (L. gasseri and/or L. iners) 13 women with normal VMF at baseline converted to abnormal VMF (non-Lactobacillus spp.) in the 2nd or 3rd trimester Compared to L. crispatus-dominated VMF and L. crispatus and other Lactobacillus spp.- dominated VMF, women with L. gasseri and/or L. iners were 10 times more likely to convert from normal to abnormal VMF during pregnancy Conclusion: L. gasseri and/or L. iners VMF offer significantly less stability than L. crispatus VMF Limitations: no sexual behavioral data collected, pregnancy outcomes not reported The presence of different Lactobacillus species with the normal vaginal microflora is a major determinant to the stability of this microflora in pregnancy: L. crispatus promotes the stability of the normal vaginal microflora while L. gasseri and/or L. iners predispose to the occurrence of abnormal vaginal microflora. Due to the choice of the BstUI restriction enzyme, which generates equal-sized terminal fragment lengths for L. gasseri and L. iners on tRFLP, the investigators were unable to differentiate between L. gasseri and L. iners, also because we failed to culture these species consistently. BMC Microbiology 2009; 9:116

Composition and Stability of the Vaginal Microbiota of Normal Pregnant Women is Different from Healthy, Non-Pregnant Women 22 healthy, pregnant women (delivering at term) and 32 healthy, non-pregnant women followed longitudinally with serial samples of vaginal fluid from posterior vaginal fornix Pregnant women sampled q4weeks until 24 weeks of gestation, than q2weeks until delivery Non-pregnant women sampled 2x weekly for 16 weeks Pregnant women had a higher abundance of L. crispatus, L. gasseri, and L. jensenii and a lower abundance of 22 other phylotypes (many associated with CST IV-A and IV-B) The stability of the vaginal microbiota of normal pregnant women was higher than that of non-pregnant women but did undergo some change over time --V1-V2 sequenced: 27F primer does not detect G. vaginalis well. --The relative abundance of L. iners was not significantly different between the two groups. This might reflect a lack of optimal protection by this common Lactobacillus spp. and requires further investigation. --This is the first longitudinal study of the vaginal microbiota in normal pregnancy where samples have been frequently collected and microbial composition has been characterized using high-throughput pyrosequencing of the 16S rRNA gene. Microbiome 2014; 2:4

During pregnancy, bacterial communities shifted from one CST dominated by Lactobacillus spp. to another CST dominated by Lactobacillus spp. but rarely to CST IV-A or CST IV-B This may confer a protective role against ascending infection of the genital tract during pregnancy Microbiome 2014; 2:4

Dynamics of vaginal CSTs in 32 women at Johns Hopkins University over 16 weeks The frequency of CST IV-B was significantly higher in non-pregnant women; 7 women persisted in this state for 16 weeks Sci Transl Med 2012;4(132):132ra52

Temporal and Spatial Variation of the Vaginal Microbiota During Pregnancy 49 pregnant women followed prospectively at Stanford University, including 15 with preterm delivery Weekly vaginal specimens obtained during gestation, then monthly after delivery Presence of CST IV inversely correlated with gestational age at delivery (p=0.0039) Risk for preterm birth more pronounced for those with CST IV and high levels of Gardnerella Most women experienced a post-delivery vaginal flora disturbance characterized by a decrease in Lactobacillus spp. and an increase in diverse anaerobes such as Peptoniphilus, Gardnerella, Prevotella, and Anaerococcus spp. Unrelated to gestational age at delivery and persisted for up to 1 year Pregnancy outcomes might be predicted by features of the microbiota early in gestation. NO SEXUAL BEHAVIOR DATA Proc Natl Acad Sci USA 2015;112(35):11060-5

Diversity of the Vaginal Microbiome Correlates with Preterm Birth Prospective cohort study of 88 pregnant women (some of whom had a history of preterm birth) at UCSF Posterior vaginal fornix swabs collected L. crispatus highly abundant in the vaginal microbiome of Caucasian women L. iners highly abundant in African American and Hispanic women Species diversity was greatest among African American women (p=0.0045) Preterm birth occurred in 19% of women (15% Hispanic, 18% Caucasian, 25% African American, 25% Asian) (National average 12%) Diversity of the vaginal microbiome correlated with preterm birth and race/ethnicity is an important variable Inclusion criteria included pregnant women who had experienced 1 spontaneous PTBs, pregnant women who had one previous term pregnancy, nulliparious women, and women in the index pregnancy without significant comorbidities (see exclusion criteria in the subsequent paragraph); entry into the study during any trimester, 18 years of age or older, able to provide informed consent, willing to undergo serial speculum examinations and vaginal specimen collection with a swab, and willing to abstain 3 days from intercourse prior to specimen collection. No sexual behavior data recorded. There was no correlation between the vaginal microbiome with little or no Lactobacillus and PTB. Reprod Sci 2014;21:32–40

Vaginal Microbiome during Pregnancy and the Postpartum Period in a European Population (Imperial College of London) British cohort of women (n=542) with uncomplicated term deliveries Vaginal samples obtained from posterior vaginal fornix longitudinally throughout pregnancy (8–12, 20–22, 28–30 and 34–36 weeks gestation) and 6 weeks postpartum Pregnancy vaginal microbiome dominated by L. jensenii in Asian and Caucasian women L. gasseri absent in samples from Black women Detailed medical and gynaecological history was taken by the research team, in particular to include information including time since last sexual intercourse and douching practices. Exclusion criteria included women who had sexual activity within 72 h of sampling, reported vaginal bleeding in the preceding week, used antibiotics in the preceding 2 weeks, multiple pregnancies, were HIV positive or under the age of 18 years. Sci Rep 2015;5:8988

Vaginal Microbiome during Pregnancy and the Postpartum Period in a European Population (Imperial College of London) Vaginal microbiome composition changed dramatically postpartum to become less Lactobacillus spp.-dominant, irrespective of the community structure during pregnancy and independent of ethnicity Rapid reduction of estrogen levels in the postpartum period leads to a decrease in glycogen This causes a decrease in glycogen break-down products utilized by lactate-producing bacteria, which likely plays a role in reducing the community stability and resilience of the vaginal microbiome Sci Rep 2015;5:8988

Prevalence of Vaginal Micro-Organisms among Pregnant Women According to Trimester and Association with Preterm Birth (Korea) 593 pregnant women underwent vaginal culture in 1st, 2nd, and 3rd trimesters Abnormal VMF rate significantly decreased with advancing trimester of pregnancy (21.7% 1st, 21.0% 2nd, 14.5% 3rd, p=0.048) Abnormal VMF detected in the 2nd trimester (but not in 1st) was associated with a significant increase in preterm delivery before 28 weeks gestation (6.9% vs. 0%; p=0.006) Klebsiella pneumonia associated with preterm delivery before 28 weeks, Streptococcus agalactiae related to late miscarriage As pregnancy progresses, the increased estrogen level has a positive effect on lactobacillary activity and proliferation by increasing glycogen availability. Obstet Gynecol Sci 2018;61(1):38-47

Summary: Vaginal Microbiome in Pregnancy The vaginal microbiome becomes less diverse and more stable during a normal pregnancy and is largely dominated by Lactobacillus spp. The distribution of dominate vaginal CSTs during pregnancy varies by population/race/ethnicity Dysbiosis (including BV) is associated with complications of pregnancy, particularly preterm birth The influence of sexual activities on the vaginal microbiota during pregnancy is not well defined

What Precipitates the Shift from Normal  Intermediate  Abnormal Vaginal Flora? (i.e. What is the Pathogenesis of BV?) Initial loss of vaginal lactobacilli (caused by behavioral activities or other inciting events)? (Sexual) acquisition of a keystone pathogen that leads to the complex changes in the vaginal flora associated with BV? (Sexual) acquisition of a polymicrobial consortium of micro-organisms that leads to the complex changes in the vaginal flora associated with BV? Something else? J Infect Dis 2016;214 Suppl 1:S1-5

K23 Vaginal Microbiome Study Objective: use molecular methods to determine the sequence of microbiological events culminating in incident BV among sexually active WSW Prospective study at the Jefferson Country Department of Health (JCDH) STD clinic WSW with normal vaginal flora (no Amsel criteria and Nugent score 0-3) followed for 90 days with daily vaginal swab specimens for development of incident BV (Nugent score 7- 10 on at least 2-3 consecutive days) Methods: for women with incident BV and women maintaining normal vaginal flora for the majority (≥95% of days) of the study, 16S rRNA gene sequencing targeting V4 was performed on vaginal swab specimens WSW=women who have sex with women Muzny et al, under review

Muzny et al, under review

Day 0 = First day of incident BV Y-axis=difference in average relative abundance between incident BV cases and women maintaining normal vaginal flora for the majority of the study Day 0 = First day of incident BV Muzny et al, under review

Day 0 = First day of incident BV Muzny et al, under review

Day 0 = First day of incident BV Muzny et al, under review

Summary – K23 Data The mean relative abundance of L. crispatus became significantly lower in cases starting 14 days before incident BV The mean relative abundance of P. bivia, G. vaginalis, A. vaginae, and Megasphaera-type I became significantly higher in women with incident BV 4 days before, 3 days before, and on the day of incident BV (A. vaginae and Megasphaera-type I), respectively Synergy between P. bivia and G. vaginalis may be an important event prior to incident BV Muzny et al, under review

Questions/Comments? cmuzny@uabmc.edu