BACKGROUND: Preterm birth remains a major public health issue affecting 10% of all pregnancies and increases risks of neonatal morbidity and mortality. Approximately 50% to 60% of preterm births are spontaneous, resulting from preterm premature rupture of membranes or preterm labor. The pathogenesis of spontaneous preterm birth is incompletely understood, and prediction of preterm birth remains elusive. Accurate prediction of preterm birth would reduce infant morbidity and mortality through targeted patient referral to hospitals equipped to care for preterm infants. Two previous studies have analyzed cervical microRNAs in association with spontaneous preterm birth and the length of gestation, but the extent to which microRNAs serve as predictive biomarkers remains unknown.

OBJECTIVE: This study aimed to examine associations between cervical microRNA expression and spontaneous preterm birth, with the specific goal of identifying a subset of microRNAs that predict spontaneous preterm birth.

STUDY DESIGN: We performed a prospective, nested, case-control study of 25 cases with spontaneous preterm birth and 49 term controls. Controls were matched to cases in a 2:1 ratio on the basis of age, parity, and self-identified race. Cervical swabs were collected at a mean gestational age of 17.1 (4.8) weeks of gestation, and microRNAs were analyzed using a quantitative polymerase chain reaction array. Normalized microRNA expression was compared between cases and controls, and a false discovery rate of 0.2 was applied to account for multiple comparisons. Histopathologic analysis of slides of cervical swab samples was performed to quantify leukocyte burden for adjustment in conditional regression models. We explored the use of Relief-based unsupervised identification of top microRNAs and support vector machines to predict spontaneous preterm birth. We performed microRNA enrichment analysis to explore potential biologic targets and pathways in which up-regulated microRNAs might be involved.

RESULTS: Of the 754 microRNAs on the polymerase chain reaction array, 346 were detected in ≥75% of participants' cervical swabs. Average cervical microRNA expression was significantly higher in cases of spontaneous preterm birth than in controls (P=.01). There were 95 significantly up-regulated individual microRNAs (>2-fold change) in cases of subsequent spontaneous preterm birth compared with term controls (P<.05; q<0.2). Notably, miR-143, miR-30e-3p, and miR-199b were all significantly up-regulated, which is consistent with the 1 previous study of cervical microRNA and spontaneous preterm birth. A Relief-based, novel variable (feature) selection machine learning approach had low-to-moderate prediction accuracy, with an area under the receiver operating curve of 0.71. Enrichment analysis revealed that identified microRNAs may modulate inflammatory cell signaling.

CONCLUSION: In this prospective nested case-control study of cervical microRNA expression and spontaneous preterm birth, we identified a global increase in microRNA expression and up-regulation of 95 distinct microRNAs in association with subsequent spontaneous preterm birth. Larger and more diverse studies are required to determine the ability of microRNAs to accurately predict spontaneous preterm birth, and mechanistic work to facilitate development of novel therapeutic interventions to prevent spontaneous preterm birth is warranted.

Background: Spontaneous preterm birth (sPTB) is a major contributor to infant mortality and its etiology remains poorly understood. Host immunity and maternal stress may play a role in the pathogenesis of sPTB but mechanisms are poorly delineated. Antimicrobial proteins in the cervicovaginal space, such as beta defensins, modulate immune responses to bacteria and have been shown to modulate the risk of sPTB from non-optimal microbiota. While stress is known to induce immunological changes, no study has examined the interplay between maternal stress and the immune response in association with sPTB.

Objectives: Our objectives were to determine whether psychosocial stress was associated with a mediator of the immune system in the cervicovaginal space, beta defensin-2, and to examine the combined impact of high stress and low cervicovaginal beta defensin-2 levels on the odds of sPTB.

Study Design: From the cohort study (n=2000), we performed a secondary, nested case-control study, frequency matched by race/ethnicity, of 519 pregnant women (110 sPTB and 409 term). Stress and cervicovaginal beta defensin-2 levels were measured at 16-20 weeks of gestation. Stress was dichotomized at a score of 30 on Cohen's Perceived Stress Scale (PSS-14). We measured cervicovaginal beta defensin-2 levels with ELISA and dichotomized at the median. We modeled associations of high stress and low cervicovaginal beta defensin-2 levels using multivariable logistic regression. We also compared the proportion of women with high stress and low cervicovaginal beta defensin-2 levels among women with spontaneous preterm and term births using Chi-Square tests. We modeled adjusted associations of stress and cervicovaginal beta defensin-2 levels with odds of sPTB using logistic regression.

Results: The majority of the study population was non-Hispanic black (72.8%), insured by Medicaid (51.1%), and had a PSS-14 score < 30 (80.2%). High stress was associated with reduced adjusted odds of low beta defensin-2 levels (aOR 0.63, 95% CI: 0.38 -0.99). In a model adjusted for race and smoking, both high stress (aOR 1.72, 95% CI: 1.03-2.90) and low beta defensin-2 (aOR 1.58, 95% CI: 1.004-2.49) were associated with increased odds of sPTB. We then built a model of the four possible combinations of low and high stress and low and high beta defensin-2 levels with the odds of sPTB. Women with either high stress (aOR 1.37, 95% CI: 0.68 - 2.78) or low beta defensin-2 (aOR 1.40, 95% CI: 0.83-2.34), had slightly elevated but not significantly increased odds of sPTB compared to women with neither exposure. However, women with both high stress and low beta defensin-2 had significantly elevated odds of sPTB compared to women with neither exposure (aOR 3.16, 95 % CI: 1.46 - 6.84).

Conclusion: High perceived stress and low cervicovaginal beta defensin-2 levels are associated with higher odds of sPTB, and when present concurrently, they result in the highest odds of sPTB in a largely non-Hispanic black cohort. Our findings warrant further work to examine social determinants of health and the host cervicovaginal immune responses that may modulate the pathogenesis of sPTB.

<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>To identify pregnancy-associated changes in cervical noncoding RNA (ncRNA), including miRNA and long noncoding RNA (lncRNA), and their potential effects on biologic processes. We enrolled 21 pregnant women with term deliveries (≥37&nbsp;weeks' gestation) in a prospective cohort and collected cervical swabs before 28&nbsp;weeks' gestation. We enrolled 21 nonpregnant controls. We analyzed miRNA, lncRNA and mRNA expression, applying a Bonferroni correction. Five miRNA and three lncRNA were significantly differentially (&gt;twofold change) expressed. Putative miRNA targets are enriched in genes mediating organogenesis, glucocorticoid signaling, cell adhesion and ncRNA machinery. Differential cervical ncRNA expression occurs in the setting of pregnancy. Gene ontology classification reveals biological pathways through which miRNA may play a biologic role in normal pregnancy physiology.</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>