Second trimester short cervix is associated with decreased abundance of cervicovaginal lipid metabolites

Abstract

BACKGROUND:

Short cervix is a risk factor for preterm birth. Molecular drivers of short cervix remain elusive. Metabolites may function as mediators of pathologic processes.

OBJECTIVES:

We sought to determine if a distinct cervicovaginal metabolomic profile is associated with short cervix (<25 mm) to unveil potential mechanisms by which premature cervical remodeling leads to short cervix.

STUDY DESIGN:

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 <25 mm as measured by transvaginal ultrasound. Unpaired t-tests were performed with a Bonferroni correction for multiple comparisons.

RESULTS:

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<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.

CONCLUSIONS:

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.

INTRODUCTION

Lactobacillus-deficient cervicovaginal microbial communities are associated with short cervix and spontaneous preterm birth (sPTB).^1–11 Short cervix serves as a sonographic proxy for premature cervical remodeling, believed to be a common biologic precursor of sPTB. While cervical length screening is utilized clinically to identify individuals at high risk of delivering preterm, mechanisms underlying these observed associations and pathogenic processes remain to be elucidated.

In other organ systems, characterization of the biochemical footprints of microbial ecosystems has illuminated host-microbial interactions. Microbiota exert a large part of their effect through the production of metabolites, which can impact inflammatory tone, cell signaling, and epithelial barriers.^12–15 In the obstetric realm, cervicovaginal epithelial barrier disruption and induction of select immune responses have been implicated in cervical remodeling.^16–18 Our laboratory has demonstrated that microbial output from microbes common in Lactobacillus-deficient communities, induces epithelial barrier disruption and pro-inflammatory cytokines in cervicovaginal epithelial cell lines.^{18 19} Observations from our clinical pregnancy cohorts corroborate these in vitro findings.² These collective data provide compelling evidence that microbial output and interactions at the host-microbial interface contribute to the molecular underpinnings of cervical remodeling leading to short cervix.

Our understanding of the cervicovaginal metabolome and its role in reproductive health is evolving. Our group and others have demonstrated that select cervicovaginal metabolites and metabolomic profiles are associated with features of vaginal ecosystems, specific host immune response, and sPTB.^20–28 It remains unknown, however, whether these small molecules are bioactive and function as drivers of cervical remodeling that result in short cervix on the pathway to sPTB. To begin unraveling these various molecular inputs, we examined the cervicovaginal metabolome in pregnancy with the goal of determining whether select cervicovaginal metabolites were associated with second trimester short cervix.

MATERIALS AND METHODS

Study Setting

This is a secondary analysis from a prospective pregnancy cohort study, Motherhood and Microbiome (M&M). A flow diagram of study participants is presented in Figure 1. For M&M, 2,000 pregnant individuals enrolled from December 2013 through February 2017. This study was approved by the Institutional Review Board at the University of Pennsylvania (IRB #818914) on October 23, 2013. The M&M methods have been previously published.² In brief, individuals receiving prenatal care at the Hospital of the University of Pennsylvania enrolled after informed consent prior to 20 weeks’ gestation. Exclusion criteria included major fetal anomalies, HIV seropositive status, history of organ transplant, chronic steroid use, enrollment into the study during a previous pregnancy, or multiple gestations. Participants were followed to delivery. Cases of preterm birth (PTB) were adjudicated by a maternal-fetal medicine physician (MAE) to distinguish spontaneous (sPTB) from medically indicated (mPTB). PTB was considered sPTB if a woman presented with either cervical dilation and/or premature rupture of membranes and delivered prior to 37 weeks of gestation. Cervicovaginal microbiota profiling was done at multiple time points. This secondary analysis includes profiling from samples collected between 20–24 weeks’ gestation (V2, mean gestational age 21.7 weeks [SD 1.4]) for 612 participants. Microbiota were analyzed by 16S rRNA gene sequencing via amplification of the V3-V4 regions of the 16S rRNA gene. Microbial communities were classified into CST as previously reported.²⁹ Classifications were assigned to each sample using hierarchical clustering with Jensen-Shannon divergence and Ward linkage. CST I is predominated with L. crispatus, CST II with L. gasseri, CST III with L. iners and CST V with L. jensenii. CST IV is defined by a paucity of Lactobacillus species and a diverse set of strict and facultative anaerobes. Lactobacillus-dominant communities included CST I, CST II, CST III, and CST V while Lactobacillus-deficient communities included CST IV. Metabolomic profiling of V2 cervicovaginal swabs was performed as described below.

Figure 1.

Flow chart of study participants.

Participants were matched by birth outcome (sPTB n=80, mPTB n=40, and term birth ≥38 weeks’ gestation n=153) as well as cervicovaginal microbiota community state type (CST IV [n=112] versus all other CSTs [n=161]). This present analysis was then restricted to participants who also had a cervical length measurement obtained in the second trimester (mean gestational age 20.2 weeks [SD 0.8]) by trained sonographers as part of routine clinical care (n=222). Cervical length was measured by transvaginal ultrasound and dichotomized at 25 mm. Short cervix was defined as a length <25 mm. Participants with a normal cervical length (≥25 mm) were used as the reference group in this analysis.

Biospecimen Collection

Specimens were self-collected by the participant or collected by a research coordinator during a clinical exam. These included an ESwabs (COPAN) stored in 1 ml of Amies Transport Medium and a Dacron swab stored without buffer. All samples were immediately frozen at −80°C until processing. Microbiome analysis was performed as previously reported and described above.²

Non-targeted Global Metabolite Profiling

Sample preparation and analysis was carried out as described previously³⁰ at Metabolon, Inc. In brief, sample preparation involved protein precipitation and removal with methanol, shaking and centrifugation. The resulting extracts were profiled on an accurate mass global metabolomics platform consisting of multiple arms differing by chromatography methods and mass spectrometry ionization modes to achieve broad coverage of compounds differing by physiochemical properties such as mass, charge, chromatographic separation, and ionization behavior. The details of this platform have been described previously.^{31 32} Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra, and were curated by visual inspection for quality control using software developed at Metabolon.^{30 33} Metabolomics data have been deposited to the EMBL-EBI MetaboLights³⁴ database (DOI: 10.1093/nar/gkz1019, PMID:31691833) with the identifier MTBLS4650. The complete dataset can be accessed here https://www.ebi.ac.uk/metabolights/MTBLS4650.

Statistical Analyses

Bivariate comparisons of maternal characteristics between cases of short cervix and normal cervical length controls were performed using Chi-square (χ2) or Student t-tests, as appropriate. Metabolite analyses were conducted on log₂-transformed data, normalized to volume available/utilized for extraction. For metabolites with levels below the limit of detection (LOD), the LOD divided by the square root of 2 was assigned. Student’s t-tests were used to compare metabolite abundance between short cervix cases and normal cervical length controls.³⁵ We performed two sensitivity analyses to ensure our findings were robust. First, we imputed the lowest detectable value for metabolites below the LOD. Second, we restricted analyses to metabolites detected in at least half of participants.

To address the potential that normalization of metabolite abundance to volume available/utilized for extraction may introduce significant sample variance, we also repeated analyses using a ratiometric mean control. Specifically, three metabolites present in all individuals with the lowest standard deviation were identified and used as reference metabolites. A normalization factor was calculated for each participant by taking the geometric mean of the three reference metabolites. Each metabolite value for each participant was then divided by their normalization factor and data subsequently log₂ transformed. Metabolites were then compared between cases of short cervix and normal cervical length controls generating a list of significant metabolites as presented in Supplemental Table 1.

Several secondary analyses were conducted. First, we restricted to individuals with a Lactobacillus-deficient microbial community. Next, we restricted to individuals with short cervix and compared those with sPTB <34 weeks’ to those with ≥34 weeks’ gestation. Finally, given that short cervix prompted clinical use of vaginal progesterone, we compared metabolic profiles among participants using vaginal progesterone for short cervix to participants not exposed, excluding the one participant using vaginal progesterone in the absence of short cervix. Then, among participants using vaginal progesterone for short cervix, we compared metabolite abundance between those with sPTB <34 weeks compared those with gestations ≥34 weeks.

Fold change was calculated as the difference in log₂-transformed abundance between groups for each metabolite. Only metabolites that met the Bonferroni threshold p-value of ≤0.05/(n metabolites compared) were considered significant. Statistical analyses were done using SAS version 9.4, Cary, NC and R version 4.1.1 (2021–08-10).³⁶

RESULTS

Demographic Characteristics of Participants

A total of 222 individuals were included in this study, of whom 27 (12.2%) had a short cervix and 195 (87.8%) had a normal cervical length (Table 1). Characteristics were similar between groups with respect to age, body mass index (BMI), and race/ethnicity, with most participants self-identifying as non-Hispanic Black. Consistent with our prior work, a Lactobacillus-deficient cervicovaginal microbiota was more prevalent in cases of short cervix.¹ Frequency of vaginal intercourse and douching in the 24 hours preceding swab collection was similar between groups. The mean difference in time between cervical length screening to swab collection was 1.9 weeks (SD 1.5) for individuals with short cervix and 1.5 weeks (SD 1.6) for those with normal cervical length (p=0.64).

Table 1.

Descriptive characteristics among participants with normal and short (<25 mm) cervical length

	Normal cervical length (n=195)	Short cervical length (n=27)	p-value
	mean (SD)
Age (years)	28.6 (5.88)	27.0 (5.18)	0.149
Pre-pregnancy BMI (kg/m 2 )	29.5 (8.18)	30.1 (8.01)	0.7
Gestational age at cervical length screening (weeks)	20.2 (0.764)	19.9 (0.818)	0.0446
	n (column %)
Obstetric history			0.00545
Multiparous with prior sPTB	26 (13.3)	9 (33.3)
Multiparous without prior sPTB	89 (45.6)	5 (18.5)
Nulliparous	80 (41.0)	13 (48.1)
Prior cervical excision surgery	7 (3.6)	0 (0)	0.68
Race			0.656
Asian	5 (2.6)	1 (3.7)
Black	140 (71.8)	22 (81.5)
Other	1 (0.5)	0 (0)
White	49 (25.1)	4 (14.8)
Hispanic	10 (5.1)	1 (3.7)	1
Lactobacillus-deficient vaginal microbiota	75 (38.5)	17 (63.0)	0.0268
Vaginal progesterone use	1 (0.5)	12 (44.4)	<0.001
Vaginal douching in prior 24 hours	4 (2.1)	1 (3.7)	1
Intercourse in prior 24 hours	43 (22.1)	4 (14.8)	0.541

Short Cervix and Metabolite Analyses

A total of 637 metabolites were detected in at least one participant, 608 metabolites detected in at least 10% of participants and 530 metabolites detected in at least 50% of participants. In the primary analysis, 26 metabolites differed between cases of short cervix and controls with normal cervical length (Table 2). Among differentially detected metabolites, 22 were decreased in abundance among cases. Notably, all but one of these metabolites (tartronate) belonged to the lipid metabolism pathway (all P<7.85E-5). Consistent with biochemical nomenclature, lipid metabolites are presented when appropriate using the family name (e.g. sphingomyelin) followed parenthetically by the common backbone then fatty acid chain length and saturation (e.g. d18:1/18:0). Sub-pathway categories within the lipid pathway included sphingosines, sphingomyelins, dihydrosphingomyelins, sphingolipid synthesis, hexosylceramines, ceramides, dihydroceramines, fatty acid and dicarboxylate, plasmalogen, phosphatidylcholine, and sterols. Notably, 15 of the 22 decreased lipid metabolites are sphingolipids or sphingolipid precursors. The four metabolites that were increased in abundance among cases of short cervix include two xenobiotics, diethanolamine and erythritol, as well as progesterone and mannitol/sorbitol. Progesterone elevation in cases was consistent with clinical practice of prescribing vaginal progesterone for individuals with short cervix. Results were similar in the two sensitivity analyses. In the analysis that imputed the lowest detectable values, only sphingomyelin (d18:1/14:0, d16:1/16:0) no longer differed significantly, while the other 25 metabolites remained significant. In the analysis restricted to metabolites present in at least 50% of the participants, only sphingomyelin (d18:1/14:0, d16:1/16:0) and progesterone no longer differed significantly, while the other 23 metabolites remained significantly different.

Table 2.

Metabolite abundance by fold change among participants with short cervix (n=27) compared to normal cervical length (n=195)

Super pathway	Sub pathway	Biochemical	Fold change	P value*
Lipid	Dihydroceramides	N-palmitoyl-sphinganine (d18:0/16:0)	−1.89	1.06E-08
Lipid	Ceramides	N-palmitoyl-sphingosine (d18:1/16:0)	−1.86	1.08E-07
Lipid	Hexosylceramides (HCER)	glycosyl-N-palmitoyl-sphingosine (d18:1/16:0)	−1.80	7.27E-06
Lipid	Sphingosines	heptadecasphingosine (d17:1)	−1.78	1.31E-07
Lipid	Sphingosines	sphingosine	−1.77	1.46E-06
Lipid	Sphingomyelins	lignoceroyl sphingomyelin (d18:1/24:0)	−1.64	3.75E-08
Lipid	Sphingomyelins	stearoyl sphingomyelin (d18:1/18:0)	−1.63	2.94E-07
Lipid	Sterol	cholesterol sulfate	−1.63	1.21E-08
Lipid	Plasmalogen	1-(1-enyl-palmitoyl)-2-linoleoyl-GPE (P-16:0/18:2)	−1.59	4.54E-06
Lipid	Sphingolipid Synthesis	phytosphingosine	−1.58	9.84E-08
Lipid	Sphingolipid Synthesis	sphinganine	−1.56	1.22E-06
Lipid	Sphingomyelins	sphingomyelin (d18:1/20:0, d16:1/22:0)	−1.55	5.35E-06
Lipid	Sphingomyelins	behenoyl sphingomyelin (d18:1/22:0)	−1.54	1.98E-05
Lipid	Dihydrosphingomyelins	palmitoyl dihydrosphingomyelin (d18:0/16:0)	−1.51	4.19E-08
Lipid	Sphingomyelins	palmitoyl sphingomyelin (d18:1/16:0)	−1.38	1.76E-07
Lipid	Phosphatidylcholine (PC)	1-stearoyl-2-oleoyl-GPC (18:0/18:1)	−1.37	1.41E-05
Lipid	Plasmalogen	1-(1-enyl-palmitoyl)-2-oleoyl-GPE (P-16:0/18:1)	−1.31	8.47E-06
Xenobiotics	Food Component/Plant	tartronate (hydroxymalonate)	−1.21	7.16E-05
Lipid	Sphingomyelins	sphingomyelin (d18:1/14:0, d16:1/16:0)	−1.18	7.02E-05
Lipid	Sphingosines	eicosanoylsphingosine (d20:1)	−1.17	1.78E-06
Lipid	Fatty Acid, Dicarboxylate	undecanedioate (C11-DC)	−0.45	1.00E-06
Lipid	Fatty Acid, Dicarboxylate	sebacate (C10-DC)	−0.37	1.11E-05
Carbohydrate	Fructose, Mannose and Galactose Metabolism	mannitol/sorbitol	0.70	8.25E-13
Lipid	Progestin Steroids	progesterone	0.83	5.81E-08
Xenobiotics	Food Component/Plant	erythritol	0.94	5.70E-05
Xenobiotics	Chemical	diethanolamine	1.08	3.72E-09

^*All presented P values were significant at the Bonferroni threshold for multiple comparisons of P<7.85E-5 (0.05/637)

To address the possibility that vaginal or intramuscular progesterone used for sPTB risk reduction contributed to the observed associations, subanalyses were performed excluding all individuals who used progesterone for this indication. Similar results were identified in terms of top differentially detected metabolites when comparing short cervix cases to normal cervical length controls. Few metabolites remained significant after applying the Bonferonni threshold, presumably due to the small number of short cervix cases (n=8) (Supplemental Table 2).

Analyses Among Individuals with a Lactobacillus-deficient Microbiota

We next restricted the analysis to those individuals colonized by a Lactobacillus-deficient cervicovaginal microbiota, known to be associated with both short cervix and sPTB (CST IV). This secondary analysis comprised 92 individuals, of whom 17 (18.5%) had a short cervix and 75 (81.5%) had a normal cervical length. Demographic characteristics were largely balanced (Supplemental Table 3). Table 3 shows the top ten metabolites that differed between the two groups; only diethanolamine and mannitol/sorbitol were significantly higher in participants with short cervix (P< 7.97E-5).

Table 3.

Metabolite abundance by P value among participants with Lactobacillus-deficient cervicovaginal microbiota comparing short cervix (n=17) to normal cervical length (n=75)

Super pathway	Sub pathway	Biochemical	Fold change	P value
Carbohydrate	Fructose, Mannose and Galactose Metabolism	mannitol/sorbitol	0.79	1.89E-08*
Xenobiotics	Chemical	diethanolamine	1.31	5.07E-07*
Lipid	Fatty Acid Synthesis	malonate	−0.40	1.79E-04
Lipid	Progestin Steroids	progesterone	0.71	1.84E-04
Lipid	Fatty Acid, Dicarboxylate	undecanedioate (C11-DC)	-0.43	2.93E-04
Lipid	Sterol	cholesterol sulfate	−1.35	3.56E-04
Xenobiotics	Food Component/Plant	erythritol	1.07	3.80E-04
Carbohydrate	Pentose Metabolism	arabinose	0.95	5.37E-04
Lipid	Fatty Acid, Dicarboxylate	sebacate (C10-DC)	−0.38	1.14E-03
Lipid	Sphingolipid Synthesis	phytosphingosine	−1.39	1.33E-03

^*P value was significant at the Bonferroni threshold for multiple comparisons of P<7.97E-5 (0.05/627)

Analyses Among Individuals with Short Cervix

To determine whether metabolites could distinguish birth outcome among individuals with short cervix, we restricted to the 27 cases of short cervix. There were 13 cases of sPTB <34 weeks and 14 participants with gestations ≥ 34 weeks. Demographic characteristics were largely balanced (Supplemental Table 4). No metabolites differed between groups (Supplemental Table 5).

Analyses Among Individuals Using Vaginal Progesterone

Among the 27 cases of short cervix, there were 12 individuals using vaginal progesterone. We examined metabolomic profiles among these 12 individuals to determine whether any metabolites were associated with progesterone use or efficacy with respect to sPTB risk reduction. Among these 12 individuals, six had sPTB <34 weeks and six had gestations ≥34 weeks. Demographic characteristics were largely balanced (Supplemental Table 6). Table 4 shows the top ten metabolites associated with use of vaginal progesterone; seven metabolites differed significantly compared to participants without vaginal progesterone exposure, including several drugs (p < 7.85E-5). Among participants who used vaginal progesterone for short cervix, no metabolites were associated with sPTB <34 weeks (Supplemental Table 7).

Table 4.

Metabolite abundance by P value among participants using vaginal progesterone for short cervix (n=12) compared to participants not using vaginal progesterone (n=210)

Super pathway	Sub pathway	Biochemical	Fold change	P value
Lipid	Progestin Steroids	progesterone	1.91	7.38E-20*
Xenobiotics	Drug - Psychoactive	citalopram N-oxide	0.04	2.09E-05*
Xenobiotics	Drug - Psychoactive	citalopram propionate	0.04	2.09E-05*
Xenobiotics	Drug - Psychoactive	citalopram/escitalopram	0.04	2.09E-05*
Xenobiotics	Drug - Psychoactive	desmethylcitalopram	0.04	2.09E-05*
Xenobiotics	Drug - Metabolic	metformin	0.04	2.09E-05*
Xenobiotics	Chemical	diethanolamine	1.09	5.50E-05*
Xenobiotics	Food Component/Plant	tartronate (hydroxymalonate)	−1.66	1.76E-04
Carbohydrate	Fructose, Mannose and Galactose Metabolism	mannitol/sorbitol	0.54	2.34E-04
Xenobiotics	Food Component/Plant	erythritol	1.11	1.06E-03

^*P value was significant at the Bonferroni threshold for multiple comparisons of P<7.85E-5 (0.05/637)

COMMENT

Principle Findings

Second trimester short cervix is associated with a decreased abundance of cervicovaginal lipid metabolites, specifically sphingolipids. This class of lipids that serves various biologic functions including stabilization of cell membranes and protection against environmental exposures.³⁷ Our observed associations between cervicovaginal metabolites and short cervix do not appear to be attributable to vaginal or intramuscular progesterone supplementation used for sPTB risk reduction. Increased cervicovaginal diethanolamine, an immunostimulatory xenobiotic metabolite, is associated with short cervix. Sugar alcohols, erythritol and mannitol/sorbitol, are also increased in cervicovaginal fluid among cases of short cervix. Whether cervicovaginal metabolites function as drivers of cervical remodeling leading to short cervix, or simply serve as biomarkers, remains to be elucidated.

Results in the Context of What is Known

Sphingolipids contain a sphingoid base and backbone amide-linked to a fatty acid residue with variable head groups. These molecules function in the organization and dynamics of cell membranes and are essential components of lipid rafts, which comprise a cluster of cell surface lipids and proteins critical for cell signaling, survival, motility, and growth.³⁷ Sphingolipids modify cytoskeletal reorganization and cell adhesion, and their presence in cell membranes may alter host immune response to various pathogens.³⁸ In other organ systems, this class of lipids play a role in barrier function; epidermal sphingolipids, including ceramide, regulate epidermal permeability and antimicrobial response in the skin.³⁹ Lipid depletion of the stratum corneum layer of the epidermis results in penetration of non-motile bacteria across this tissue barrier and induction of a pro-inflammatory host immune response.⁴⁰

Our understanding of the role of lipids in the lower reproductive tract is limited, particularly in pregnancy. Findings from this current study are consistent with previously published data from our group, which demonstrated similar biochemical fingerprinting of cervicovaginal fluid from 16 to 20 weeks’ gestation among non-Hispanic Black individuals in an untargeted metabolomics discovery study. ⁴¹ Specifically, a decrease in sphingolipids and in eicosanoid precursors within the lipid pathway were detected among individuals who delivered preterm. These data are consistent with our prior metabolomic studies of cervicovaginal fluid in pregnant individuals that identified alterations in lipid metabolism among those with sPTB.^{20 21} Despite variance in gestational age at time of sampling and metabolomic profiling, classification of cervicovaginal microbial communities, and racial diversity across our studies, we have observed consistency with respect to lipid metabolite signatures and sPTB. Our findings also corroborate recent data from Pruski et al. in which lipid metabolites emerged as a core component of the metabolomic profile of vaginal inflammation in pregnancy.²⁷

While prior studies have examined metabolites in the context of sPTB, cervicovaginal microbiota, and vaginal inflammation, this study is the first to specifically examine the outcome of short cervix. Though historic data have identified changes in various classes of lipid metabolites in the cervicovaginal space in association with clinical outcomes of interest, our observation that global changes specifically in sphingolipid biosynthesis and metabolism exist in association with short cervix is novel and contributes substantially to the existing literature.

Outside of pregnancy, characterization of lipid profiles from vaginal discharge among women with vulvovaginal candidiasis and cytolytic vaginosis has identified increased concentrations of lipids related to oxidative stress and apoptosis, whereas lipids involved in epithelial tissue integrity were more prevalent in the control group.⁴² Lipid metabolism is also altered in cases of bacterial vaginosis with increases in eicosanoids, which are known to regulate inflammatory pathways.⁴³ Though we did not detect an increase in eicosanoids in this current study or in prior work, the decrease in abundance of eicosanoid precursors in association with sPTB observed in our recent discovery study, as noted above, suggests potential increased utilization of these molecules to produce inflammatory mediators. Nonetheless, an increase in the abundance of cervicovaginal eicosanoids appears to be an important metabolomic distinction between bacterial vaginosis compared to sPTB and warrants further investigation. Administration of vaginal surfactant lipids has been shown to decrease inflammatory cytokines and increase anti-inflammatory cytokines.⁴⁴ Seemingly contrary to our findings, sphingolipids and plasmalogens positively correlate with genital inflammation in cases of invasive cervical cancer; however, this may be explained by higher rates of cell proliferation and cell membrane synthesis resulting from oncogenic pathways.⁴⁵

Diethanolamine is an aminoalcohol and a weak base used in the preparation of soaps, surfactants, laundry and dishwashing detergents, hair products, and cosmetics.⁴⁶ Longterm dermal administration of diethanolamine preparations have been shown to increase epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, hepatic and renal tumors in rodents, as well as diminish hepatic stores of the essential nutrient choline.^46–49 While data on the effect of diethanolamine in the cervicovaginal space are lacking, accumulation of this xenobiotic may result in recruitment of immune cells and induction of inflammatory mediators.

Our finding that the xenobiotic diethanolamine was elevated in cases of short cervix raises questions about trafficking of exogenous biochemicals to the cervicovaginal space and the extent to which they are biologically active. Similar to drugs, many xenobiotics are topically absorbed or ingested, thereby entering the systemic circulation. Tissue distribution depends upon vascularization, lipid content, pH, and permeability of cell membranes. It is conceivable that a lipid-deficient cervicovaginal microenvironment may compromise the epithelial barrier, permitting accumulation of select xenobiotics in this space. An alternative potentially more plausible explanation is that certain xenobiotics are present in feminine hygiene products or laundered underwear due to detergents, and that presence of these xenobiotics in the cervicovaginal space results from this physical proximity.

Mannitol and sorbitol are sugar alcohol isomers that are industrially produced as well as occur naturally in some in plants and fruits. While mannitol is predominantly utilized for medical applications, sorbitol is typically added to foods and beverages as a sugar substitute. Erythritol is a naturally occurring sugar alcohol also used as a sugar substitute. Given this context, these metabolites in the cervicovaginal space are likely exogenous in origin. Higher levels of sugar alcohols among individuals with short cervix likely reflects increased permeability of the cervicovaginal epithelial barrier, as these biochemicals are often used as a proxy for paracellular intestinal permeability.⁵⁰ Some sugar alcohols, including mannitol, exert osmotic effects, which could affect tissue hydration and mechanical properties of the cervix. It is well known that cervical remodeling and ripening precede both preterm and term delivery, and that an increase in cervical hydration is a key component of this process.^51–53

Clinical Implications

Findings from our study offer potential to enhance sPTB risk stratification in the obstetric population. In the future, cervicovaginal metabolites may be utilized in combination with cervical length measurements and other biological data, such as microbiota or immune profiles, to inform more sophisticated predictive models. If cervicovaginal metabolites serve a mechanistic role in cervical remodeling, it follows that these metabolite differences might precede sonographic short cervix and therefore be of greater clinical utility in early identification of those at risk since.

Research Implications

Our data underscore the need for mechanistic work to further define the molecular underpinnings of cervical remodeling and future clinical studies to interrogate the effects of environmental exposures on cervical biology. Indeed, delineating temporality is key to advancing our understanding and will help to determine whether sphingolipids or select xenobiotics play a causal role in cervical remodeling and short cervix. An extension of this question involves determining why the cervicovaginal space is relatively deplete of sphingolipids in high-risk individuals. Future research is also necessary to discern how sphingolipids and lipid rafts may be involved in regulating host immune response, signal transduction, epithelial barrier integrity, in addition to whether cervicovaginal lipid depletion increases susceptibility to inflammation and infection or vulnerability to environmental factors. Examining the effect of lipid repletion on cervical remodeling may unveil novel approaches for therapeutic intervention. Our findings are of further scientific relevance given the known interplay between lipids and steroidogenesis. Bioactive sphingolipids have been implicated in the production of steroid hormones, including progesterone.⁵⁴ Vaginal lipid administration may offer potential to mitigate aberrant premature cervical remodeling in select individuals.

Strengths and Limitations

Strengths of this study include the large well-phenotyped pregnancy cohort with a predominance of non-Hispanic Black individuals who are known to be at highest risk of sPTB. Our cohort incorporates microbiota data, facilitating more granular analyses among individuals colonized by a Lactobacillus-deficient cervicovaginal microbiota, a known risk factor for short cervix and sPTB. This study is unable to elucidate causality given the cross-sectional study design. While it is biologically plausible that metabolite changes precede cervical shortening given their posited role in remodeling, whether a lipid-deficient cervicovaginal microenvironment in fact precedes development of short cervix remains to be determined. Our work is limited also by the discovery approach, focusing on individual metabolites rather than reducing data dimensionality or describing broader patterns that may illuminate function. Despite this methodology, the numerous metabolites within the lipid pathway emerged as a striking difference between groups and is hypothesis-generating with respect to underlying mechanism. Finally, lipid metabolism may be affected by vulvovaginal candidiasis and bacterial vaginosis as noted above, and our study did not evaluate specifically for these conditions.

Conclusions

Our data identify a sphingolipid-deficient cervicovaginal microenvironment and select xenobiotics were both associated with second trimester short cervix in a cohort at high risk of sPTB. This research carries significant translational and clinical implications. Our findings may be leveraged to modify components of the cervicovaginal microenvironment or environmental exposures to potentially promote reproductive health. Understanding the biochemical footprint of the cervicovaginal space will have widespread consequences for reproductive health beyond pregnancy.

AJOG AT A GLANCE:

A. Why was this study conducted?

• Lactobacillus-deficient cervicovaginal microbiota are associated with short cervix, a risk factor for preterm birth, though molecular drivers of this sonographic finding remain elusive

• Characterization of biochemical footprints of microbiota through metabolomics has illuminated host-microbial interactions in other systems

• This study sought to determine whether select cervicovaginal metabolites are associated with short cervix to unveil potential mechanisms by which premature cervical remodeling leads to short cervix

B. What are the key findings?

• Short cervix is associated with decreased cervicovaginal lipid metabolites, particularly sphingolipids which are implicated in cell membrane stabilization and protection against environmental exposures

• Short cervix is associated with increased cervicovaginal xenobiotics, including immunostimulatory metabolite diethanolamine

C. What does this study add to what is already known?

• These findings identify potential mechanisms by which modifiable environmental factors may invoke cell damage in the setting of biologic vulnerability, thus promoting premature cervical remodeling in preterm birth