<p><strong>BACKGROUND: </strong>Short cervix is a risk factor for preterm birth. Molecular drivers of short cervix remain elusive. Metabolites may function as mediators of pathologic processes.</p>

<p><strong>OBJECTIVES: </strong>We sought to determine if a distinct cervicovaginal metabolomic profile is associated with short cervix (&lt;25 mm) to unveil potential mechanisms by which premature cervical remodeling leads to short cervix.</p>

<p><strong>STUDY DESIGN: </strong>This was a secondary analysis of a completed prospective pregnancy cohort. Cervicovaginal fluid was obtained between 20-24 weeks' gestation. Participants selected for metabolomic profiling were frequency matched by birth outcome and cervicovaginal microbiota profile. This analysis included the 222 participants with cervical length measured. Short cervix was defined as &lt;25 mm as measured by transvaginal ultrasound. Unpaired t-tests were performed with a Bonferroni correction for multiple comparisons.</p>

<p><strong>RESULTS: </strong>There were 27 participants with short cervix and 195 with normal cervical length. Of the 637 metabolites detected, 26 differed between those with short cervix and normal cervical lengths; 22 were decreased, of which 21 belonged to the lipid metabolism pathway (all P&lt;7.85E-5). Diethanolamine, erythritol, progesterone and mannitol/sorbitol were increased in cases of short cervix. Among participants with a Lactobacillus-deficient microbiota, only diethanolamine and mannitol/sorbitol differed between short cervix (n=17) and normal cervical length (n=75), both increased.</p>

<p><strong>CONCLUSIONS: </strong>Short cervix is associated with decreased cervicovaginal lipid metabolites, particularly sphingolipids. This class of lipids stabilizes cell membranes and protects against environmental exposures. Increased diethanolamine, an immunostimulatory xenobiotic, is associated with short cervix. These observations begin to identify potential mechanisms by which modifiable environmental factors may invoke cell damage in the setting of biologic vulnerability, thus promoting premature cervical remodeling in spontaneous preterm birth.</p>

<p>Biomechanical and molecular processes of premature cervical remodeling preceding spontaneous preterm birth (sPTB) likely result from interactions between the cervicovaginal microbiota and host immune responses. A non-optimal cervicovaginal microbiota confers increased risk of sPTB. The cervicovaginal space is metabolically active in pregancy; microbiota can produce, modify, and degrade metabolites within this ecosystem. We establish that cervicovaginal metabolomic output clusters by microbial community in pregnancy among Black individuals, revealing increased metabolism within the amino acid and dipeptide pathways as hallmarks of a non-optimal microbiota. Few differences were detected in metabolomic profiles when stratified by birth outcome. The study raises the possibility that metabolites could distinguish women with greater risk of sPTB among those with similar cervicovaginal microbiota, and that metabolites within the amino acid and carbohydrate pathways may play a role in this distinction.</p>

<p><strong>BACKGROUND: </strong>Water total trihalomethanes (TTHMs) are disinfectant byproducts found in municipal water supplies. TTHM exposure has been linked to cancer and may be associated with adverse reproductive outcomes. A non-optimal cervicovaginal microbiota and low cervicovaginal beta-defensin-2 levels are associated with increased risk of spontaneous preterm birth. Whether TTHM exposure increases the risk of spontaneous preterm birth or alters the cervicovaginal microbial or immune state is unknown.</p>

<p><strong>OBJECTIVE: </strong>Investigate associations of water TTHM levels with spontaneous preterm birth, a non-optimal cervicovaginal microbiota, and beta-defensin-2 levels in a completed, diverse, urban pregnancy cohort. We hypothesized that higher TTHM levels would be associated with spontaneous preterm birth, a non-optimal cervicovaginal microbiota, and lower beta-defensin-2 levels.</p>

<p><strong>DESIGN: </strong>/Methods: This was a secondary analysis of participants (n=474) in the Motherhood &amp; Microbiome (M&amp;M) study (n=2000), who lived in Philadelphia and had cervicovaginal samples analyzed for cervicovaginal microbiota composition and beta-defensin-2 levels. The microbiota was classified into community state types (CSTs). CST IV (non-optimal microbiota) is characterized by a paucity of Lactobacillus species and wide array of anaerobes. Municipal water TTHM levels were obtained from 16 sites monthly across the city of Philadelphia to establish mean residential water supply levels for each participant for the first four months of pregnancy (prior to vaginal swab collection at 16-20 weeks' gestation). Associations of water TTHM levels with spontaneous preterm birth and a non-optimal cervicovaginal microbiota birth were analyzed using multivariable logistic regression. Multivariable linear regression was used to model associations of water TTHM levels with log-transformed cervicovaginal beta-defensin-2 levels. Since water TTHM levels vary by season and Beta-defensin-2 levels have been shown to differ by race, stratified models by warm (April-September) and cold (October-March) seasons as well as by self-identified race were utilized.</p>

<p><strong>RESULTS: </strong>Participants' water supply TTHM levels (mean μg/L [SD]) were higher in the warm (53.5 [9.4]) than cold (33.4 [7.5]) season (p&lt;0.0001). TTHM levels were non-significantly higher among Black participants than non-Black participants (44.8[13.5] vs. 41.8[11.8], p=0.07). No associations were detected between TTHM with spontaneous preterm birth (per SD increment of TTHM, aOR 0.94, 95%CI: 0.66, 1.34) or with CST IV (aOR 0.94, 95%CI: 0.86, 1.16). Counter to our hypothesis, we observed positive associations of water TTHM with log-transformed cervicovaginal beta-defensin-2 levels in unadjusted models (β 0.20 [95%CI: 0.02, 0.39)] per SD increment of TTHM), but the association was null after adjustment for season. However, in models adjusted for covariates including season and stratified by race, TTHM was significantly associated with lower beta-defensin-2 levels among non-Black participants (β -0.75 [95%CI: -1.43, -0.08]) but not among Black participants (β 0.17 [95%CI: -0.15, 0.49]), interaction p=0.013).</p>

<p><strong>CONCLUSION: </strong>We did not detect associations of water TTHM levels with spontaneous preterm birth or the structure of the cervicovaginal microbiota. However, the finding of a significant interaction between TTHM and race on beta-defensin-2 levels suggest that environmental exposures may contribute to differences in reproductive tract innate immune function by race. Future studies to delineate environmental contributions to the cervicovaginal microbial-immune state, a potentially important biologic underpinning for preterm birth, are warranted.</p>

<p><strong>OBJECTIVE: </strong> While select cervicovaginal microbiota and psychosocial factors have been associated with spontaneous preterm birth, their effect on the risk of recurrence remains unclear. It is also unknown whether psychosocial factors amplify underlying biologic risk. This study sought to determine the effect of nonoptimal cervicovaginal microbiota and perceived stress on the risk of recurrent spontaneous preterm birth.</p>

<p><strong>STUDY DESIGN: </strong> This was a secondary analysis of a prospective pregnancy cohort, . The Cohen's Perceived Stress Scale (PSS-14) was administered and cervical swabs were obtained between 16 and 20 weeks of gestation. PSS-14 scores ≥30 reflected high perceived stress. We analyzed cervicovaginal microbiota using 16S rRNA sequencing and classified microbial communities into community state types (CSTs). CST IV is a nonoptimal cervicovaginal microbial community characterized by anaerobes and a lack of . The final cohort included a predominantly non-Hispanic Black population of women with prior spontaneous preterm birth who had recurrent spontaneous preterm birth or term birth and had stress measurements ( = 181). A subanalysis was performed in the subset of these women with cervicovaginal microbiota data ( = 74). Multivariable logistic regression modeled adjusted associations between CST IV and recurrent spontaneous preterm birth, high stress and recurrent spontaneous preterm birth, as well as high stress and CST IV.</p>

<p><strong>RESULTS: </strong> Among the 181 women with prior spontaneous preterm birth, 45 (24.9%) had high perceived stress. We did not detect a significant association between high stress and recurrent spontaneous preterm birth (adjusted odds ratio [aOR] 1.67, 95% confidence interval [CI]: 0.73-3.85). Among the 74 women with prior spontaneous preterm birth and cervicovaginal microbiota analyzed, 29 (39.2%) had CST IV; this proportion differed significantly among women with recurrent spontaneous preterm birth (51.4%) compared with women with term birth (28.2%) ( = 0.04). In models adjusted for race and marital status, the association between CST IV and recurrent spontaneous preterm birth persisted (aOR 3.58, 95% CI: 1.25-10.24). There was no significant interaction between stress and CST IV on the odds of spontaneous preterm birth ( = 0.328). When both stress and CST IV were introduced into the model, their associations with recurrent spontaneous preterm birth were slightly stronger than when they were in the model alone. The aOR for stress with recurrent spontaneous preterm birth was 2.02 (95% CI: 0.61-6.71) and for CST IV the aOR was 3.83 (95% CI: 1.30-11.33). Compared to women with neither of the two exposures, women with both high stress and CST IV had the highest odds of recurrent spontaneous preterm birth (aOR = 6.01, 95% CI: 1.002-36.03).</p>

<p><strong>CONCLUSION: </strong> Among a predominantly non-Hispanic Black cohort of women with a prior spontaneous preterm birth, a nonoptimal cervicovaginal microbiota is associated with increased odds of recurrent spontaneous preterm birth. Adjustment for perceived stress may amplify associations between CST IV and recurrent spontaneous preterm birth. Identification of modifiable social or behavioral factors may unveil novel nonpharmacologic interventions to decrease recurrent spontaneous preterm birth among women with underlying biologic risk.</p>

<p><strong>KEY POINTS: </strong>· CST IV, a nonoptimal microbiota, is associated with increased odds of recurrent spontaneous preterm birth.. · Adjustment for perceived stress amplified associations between CST IV and recurrent spontaneous preterm birth.. · Identification of modifiable psychosocial factors may unveil novel nonpharmacologic interventions to decrease recurrent preterm birth..</p>