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Microbial-driven preterm labour involves crosstalk between the innate and adaptive immune response

Denise Chan; Phillip R. Bennett; Yun S. Lee; Samit Kundu; T. G. Teoh; Malko Adan; Saqa Ahmed; Richard G. Brown; Anna L. David; Holly V. Lewis; Belen Gimeno-Molina; Jane E. Norman; Sarah J. Stock; Vasso Terzidou; Pascale Kropf; Marina Botto; David A. MacIntyre; Lynne Sykes

2022

Abstract

There has been a surge in studies implicating a role of vaginal microbiota in spontaneous preterm birth (sPTB), but most are associative without mechanistic insight. Here we show a comprehensive approach to understand the causative factors of preterm birth, based on the integration of longitudinal vaginal microbiota and cervicovaginal fluid (CVF) immunophenotype data collected from 133 women at high-risk of sPTB. We show that vaginal depletion of Lactobacillus species and high bacterial diversity leads to increased mannose binding lectin (MBL), IgM, IgG, C3b, C5, IL-8, IL-6 and IL-1β and to increased risk of sPTB. Cervical shortening, which often precedes preterm birth, is associated with Lactobacillus iners and elevated levels of IgM, C3b, C5, C5a and IL-6. These data demonstrate a role for the complement system in microbial-driven sPTB and provide a scientific rationale for the development of live biotherapeutics and complement therapeutics to prevent sPTB.

Key points

● We demonstrate that immune activation and a dysregulated immune response involving the lectin mediated pathway, IgM/IgG complex activated classical pathway, and a pro-inflammatory cytokine milieu occurs in microbial-driven spontaneous preterm birth (sPTB)

● Concentrations of mannose binding lectin (MBL), IgG1 and IgG3 increased significantly between 12+0–16+6 and 20+0–24+6 weeks in women who delivered preterm, whereas no statistically significant changes in any mediators of microbial recognition were seen between timepoints in women who delivered at term (Fig. 4a–f)

● We found a significant increase in cervicovaginal concentrations of IL-8, IL-6 and IL-1β in women who were deplete of Lactobacillus spp., or in association with a diverse vaginal microbial composition

● Studies of cervicovaginal MBL in pregnancy are lacking, cervicovaginal fluid (CVF) MBL is increased with vulvovaginal candidiasis[32], and binding has been shown with G. vaginalis in non-pregnant women[33], supporting its role in the recognition of pathogenic organisms

● Local inflammation and/or a high-risk vaginal microbial composition does not always lead to preterm birth (Fig. 10c), we propose that a dysregulated innate and adaptive immune response, bridged by the complement cascade, is what triggers microbial-driven preterm parturition

● There was a significantly higher preterm birth rate, 57% vs. 20%, in the braided group, compared to the monofilament group

● We have demonstrated that the commensal L. crispatus does not activate inflammation, and may be protective against inflammation, which provides a rationale for its use as a vaginal live biotherapeutic product to reduce preterm birth rates

Summary

Introduction The global preterm birth rate is estimated at over 10%1. Preterm birth is the leading cause of neonatal and childhood mortality[2].

There have been limited advances in prediction, prevention, and treatment of spontaneous preterm birth over the last few decades

This is predominantly because it may be due to one of, or a combination of, multiple aetiological factors, yet research studies exploring pathophysiology or therapeutics typically fail to attempt to phenotype cases.

Decades of research has implicated inflammation and infection in the aetiology of a significant proportion of spontaneous preterm births[6], especially those occurring prior to 34 weeks[7].

Recent studies of the pregnancy vaginal microbiota have implicated Lactobacillus dominance and inhibition of pathogen colonisation of the vaginal niche as an important mediator of preterm birth risk.

Lactobacillus depleted, and high diversity vaginal microbiota are associated with an increased risk of PPROM and of sPTB13,14,15,16,17.

The wider implications of these results support the potential for therapeutic modulation of local microbial composition and the immune milieu for the prevention of sPTB

Results This study was focused upon women undergoing clinical surveillance at preterm birth prevention clinics during their pregnancy because of risk factors associated with a higher than usual risk of sPTB.

No statistically significant differences were seen in the concentration of IL-10 (Fig. 3l)

These data demonstrate that a degree of microbial-driven inflammation occurs regardless of outcome, yet preterm delivery is associated with a significantly greater degree of inflammation, alluding to a dysregulated immune response.

Concentrations of mannose binding lectin (MBL), IgG1 and IgG3 increased significantly between 12+0–16+6 and 20+0–24+6 weeks in women who delivered preterm, whereas no statistically significant changes in any mediators of microbial recognition were seen between timepoints in women who delivered at term (Fig. 4a–f).

By mid-pregnancy, in women who were Lactobacillus spp. deplete, preterm delivery was associated with significantly higher concentrations of MBL and IgM compared to women who delivered at term (Fig. 4m, n).

Discussion The authors' study identifies cross-talk between the host innate and adaptive immune response in microbial-driven cervical shortening and sPTB.

The authors report on an increase in microbial mediators of recognition and complement proteins, demonstrating cross-talk between the innate and adaptive immune response in women who deliver spontaneously preterm.

The authors found a significant increase in cervicovaginal concentrations of IL-8, IL-6 and IL-1β in women who were deplete of Lactobacillus spp., or in association with a diverse vaginal microbial composition.

The highest cytokine concentrations were seen in those women with these microbial signatures who subsequently delivered preterm, suggesting excessive innate immune response to specific vaginal microbiota as a potential mechanism leading to untimely activation of parturition in these women.

IgG1 increased during pregnancy in women who delivered preterm, there was no significant difference in concentrations in relation to vaginal microbial composition.

The authors have proposed a potential mechanism involving cross-talk between the innate and adaptive immune response in microbial-driven preterm birth, which could lead to the development of novel therapies for the prevention of preterm birth

Study design Study participants were prospectively recruited from preterm birth prevention clinics from five UK hospitals (Chelsea Westminster Hospital, Edinburgh Royal Infirmary, St Marys Hospital London, Queen Charlottes Hospital, and University College London Hospital) between February 2016 and June 2018.

Metadata was collected at each visit and stored on a secure database

This included maternal age, BMI, ethnicity, past obstetric history, medical history, any interventions for preterm birth including progesterone and cervical cerclage.

Study participants may have required a clinical intervention such as progesterone and/or cervical cerclage to prevent preterm birth.

This included women with cervical shortening on transvaginal ultrasound, defined as a cervical length ≤ 25 mm, and women with a history of cervical insufficiency and/or previous cervical cerclage.

Differences in the immune profiles in Lactobacillus dominant and deplete samples, and between vaginal microbial groups were analysed using the Mann–Whitney U-test or the Kruskal–Wallis test and Dunn’s post hoc multiple comparisons test.

The level of statistical significance was taken as a p-value ≤ 0.05

Introduction

The global preterm birth rate is estimated at over 10%1. Preterm birth is the leading cause of neonatal and childhood mortality[2]. A third of preterm births are medically indicated for maternal or fetal reasons such as preeclampsia or intrauterine growth restriction. The remainder are considered to be spontaneous, with preterm prelabour rupture of fetal membranes (PPROM) preceding 25–30% of cases[3]. Three physiological processes are required for labour: cervical shortening and dilation, uterine contractility and rupture of fetal membranes. In the case of preterm labour, these occur as a result of a pathological process, and each may occur in isolation, conferring a greater risk of preterm birth. Cervical shortening, a prerequisite of cervical dilation, when detected at 24 weeks leads to an almost tenfold increased risk of preterm birth, with ultrasound detection commonly used as a screening tool[4]. PPROM occurs in 3% of all pregnancies, of whom 50–60% will deliver within a week[5]. There have been limited advances in prediction, prevention, and treatment of spontaneous preterm birth (sPTB) over the last few decades. This is predominantly because it may be due to one of, or a combination of, multiple aetiological factors, yet research studies exploring pathophysiology or therapeutics typically fail to attempt to phenotype cases. Increased understanding of aetiology is required to improve patient stratification for targeted therapeutic intervention, and for the development of novel therapeutic strategies.

Methods

Study design

Results

Study cohort This study was focused upon women undergoing clinical surveillance at preterm birth prevention clinics during their pregnancy because of risk factors associated with a higher than usual risk of sPTB. Principal risk factors included previous sPTB, previous PPROM, previous mid-trimester pregnancy loss (MTL), previous large loop excision of the transformation zone of the cervix (LLETZ), or a combination of these. A total of 133 women, recruited from preterm birth prevention clinics in five UK Hospitals, provided a total of 385 cross sectional sampling points, 126 at timepoint A (12+0–16+6 weeks), 133 at timepoint B (20+0–24+6 weeks) and 126 at timepoint C (30+0–34+6 weeks) (Fig. 1). Longitudinal samples were targeted at timepoints A and B, since women who delivered before 30 weeks would fail to meet timepoint C. Longitudinal samples for timepoint A and B were collected in 122 women. sPTB occurred in 37 women (27.82%) < 37 weeks, 21 (15.79%) < 34 weeks, and 4 (3.01%) < 28 weeks. PPROM occurred in 22 (16.54%) women, and complicated 59.46% of preterm births. Cervical shortening occurred in 53 women, 39.85% of the study cohort, and 59.46% of women who had a sPTB. Of the 96 women who delivered at term, 40 women had intervention with cervical cerclage and/or progesterone (term intervention, TI) and 56 had no intervention (term uncomplicated, TU). Cervical cerclage was placed if there was a clinical history suggestive of an insufficient cervix or if the cervical length was found to be ≤25 mm in length on ultrasound scan. While women who delivered at term without intervention were used as a control group, it is acknowledged that our study cohort was predefined as being at high risk of preterm delivery. There was no significant difference in maternal age (p = 0.93), BMI (p = 0.20) or ethnicity (p = 0.09) between women who delivered spontaneously preterm or at term with or without intervention (Supplementary Table 1). No women were diagnosed with a sexually transmitted disease.

Discussion

Our study identifies cross-talk between the host innate and adaptive immune response in microbial-driven cervical shortening and sPTB. To date, most studies exploring the local cervicovaginal immune milieu in the context of sPTB have focused on the innate immune response, and predominantly on the role of Toll-like receptors, cytokines, and chemokines. Elevated cervicovaginal concentrations of cytokines such as IL-8, IL-6 and IL-1β, TNF-α have been consistently reported in women who subsequently deliver preterm[20]. In our study of women at high risk of preterm birth, we demonstrate that this elevation occurs between the early and late second trimester of pregnancy. Furthermore, we report on an increase in microbial mediators of recognition and complement proteins, demonstrating cross-talk between the innate and adaptive immune response in women who deliver spontaneously preterm. Recent studies of the vaginal microbiome in pregnancy have linked increased microbial diversity and the presence of pathobionts with a higher risk of preterm birth[12,14,15,18,21,22,23], while the dominance of the vaginal niche by commensals such as L. crispatus reduces the risk[11,12,18,23,24]. In this study we used amplification of the V1/V2 hypervariable region for metataxonomic analysis and clustered the resulting sequence data using the VALENCIA classifier. Amplification of the V1/V2 regions has been widely used in the study of the vaginal microbiome, including the original Ravel community state type classification study[25]. This approach has the advantage over application of other regions of improved discrimination between species of Lactobacilli, which are the most prevalent genus in the vaginal microbiota. It has the disadvantage of that if a universal forward primer is used, mismatches can lead to under representation of important vaginal bacterial genera including Gardnerella. However, as we have done in this study, this problem can be overcome through the use of a mixed formulation of the 27F forward primer, which has been shown to maintain the rRNA gene ratio of Lactobacillus spp. to Gardnerella spp[26].

Conclusion

In summary, we propose a mechanism whereby the maternal host immune response to vaginal microbiota drives inflammation, cervical shortening, and preterm labour (Fig. 10b). Although local inflammation and/or a high-risk vaginal microbial composition does not always lead to preterm birth (Fig. 10c), we propose that a dysregulated innate and adaptive immune response, bridged by the complement cascade, is what triggers microbial-driven preterm parturition. We have demonstrated that the commensal L. crispatus does not activate inflammation, and may be protective against inflammation, which provides a rationale for its use as a vaginal live biotherapeutic product to reduce preterm birth rates. We have identified the complement system as a potential key player in driving the adverse host–microbial interactions, which provides the rationale for the potential application of complement therapeutics[52], especially if delivered locally. In conclusion, we have proposed a potential mechanism involving cross-talk between the innate and adaptive immune response in microbial-driven preterm birth, which could lead to the development of novel therapies for the prevention of preterm birth.

Funding

This work was funded by the March of Dimes European Preterm Birth Research Centre at Imperial College London and supported by the National Institute of Health Research (NIHR) Imperial Biomedical Research Centre (BRC), NIHR Clinical Lectureship Scheme, and the Genesis Research Trust

A.L.D. is supported by the UCLH NIHR Biomedical Research Centre

S.J.S. is funded by a Wellcome Trust Career Development Fellowship (209560/Z/17/Z). Author information

Participants and statistics

Principal risk factors included previous sPTB, previous PPROM, previous mid-trimester pregnancy loss (MTL), previous large loop excision of the transformation zone of the cervix (LLETZ), or a combination of these. A total of 133 women, recruited from preterm birth prevention clinics in five UK Hospitals, provided a total of 385 cross sectional sampling points, 126 at timepoint A (12+0–16+6 weeks), 133 at timepoint B (20+0–24+6 weeks) and 126 at timepoint C (30+0–34+6 weeks) (Fig. 1). Longitudinal samples were targeted at timepoints A and B, since women who delivered before 30 weeks would fail to meet timepoint C

Longitudinal samples were targeted at timepoints A and B, since women who delivered before 30 weeks would fail to meet timepoint C. Longitudinal samples for timepoint A and B were collected in 122 women. sPTB occurred in 37 women (27.82%) < 37 weeks, 21 (15.79%) < 34 weeks, and 4 (3.01%) < 28 weeks. PPROM occurred in 22 (16.54%) women, and complicated 59.46% of preterm births

PPROM occurred in 22 (16.54%) women, and complicated 59.46% of preterm births. Cervical shortening occurred in 53 women, 39.85% of the study cohort, and 59.46% of women who had a sPTB. Of the 96 women who delivered at term, 40 women had intervention with cervical cerclage and/or progesterone (term intervention, TI) and 56 had no intervention (term uncomplicated, TU)

Cervical shortening occurred in 53 women, 39.85% of the study cohort, and 59.46% of women who had a sPTB. Of the 96 women who delivered at term, 40 women had intervention with cervical cerclage and/or progesterone (term intervention, TI) and 56 had no intervention (term uncomplicated, TU). Cervical cerclage was placed if there was a clinical history suggestive of an insufficient cervix or if the cervical length was found to be ≤25 mm in length on ultrasound scan

Alpha-diversity of CST I- L. crispatus was significantly lower than in every other of the seven CSTs, and richness was lower compared to CST II, IV-B and IV-C (Supplementary Fig. 2c, d). Twenty-one women delivered before 34 weeks and 16 women delivered between 34 and 37 weeks. There was a greater proportion of women with either CST III or CST IV at 12+0-16+6 (timepoint A) and 20+0–24+6 weeks (timepoint B) in those who had early-preterm birth (Fig. 2f) compared to late preterm birth (Fig. 2g), with a small increase in CST III and CST IV at timepoint B in the former group

In women with CST IV IL-8, IL-6 and IL-1β were also significantly higher in those who delivered preterm compared to those who delivered at term (Fig. 3i–k). Sample size limited the ability to compare outcome between sub-CST IV-B and IV-C0-4, however, 4 of the 6 women who delivered preterm with CST IV had high relative abundance of G. vaginalis. compared to only 3 of the 9 women who delivered at term (Supplementary Data 2). No statistically significant differences were seen in the concentration of IL-10 (Fig. 3l)

Statistical analysis was performed using a one-sided Wilcoxon matched pairs signed rank test. MBL, IgM, and IgG1-IgG4 concentrations were also compared between samples taken from women who were classed as CST I with CST II–V g–l, n = 385 samples from n = 133 women. The Kruskal–Wallis and Dunn’s multiple comparison’s test was used to determine statistical significance

The Kruskal–Wallis and Dunn’s multiple comparison’s test was used to determine statistical significance. Cervicovaginal concentrations of MBL, IgM, and Ig1-IgG4 were compared between women who delivered preterm or at term in those who were classed as being Lactobacillus deplete (n = 22), or abundant in either VMG 3 (L. iners) (n = 22) or VMG 4 (diverse) (n = 15). CST IV was subdivided into CST IV-B (n = 8), CSTIV-C1 (n = 1), CSTIV-C2 (n = 1) and CST IV-C3 (n = 5) (m–r)

Cervicovaginal concentrations of MBL, IgM, and Ig1-IgG4 were compared between women who delivered preterm or at term in those who were classed as being Lactobacillus deplete (n = 22), or abundant in either VMG 3 (L. iners) (n = 22) or VMG 4 (diverse) (n = 15). CST IV was subdivided into CST IV-B (n = 8), CSTIV-C1 (n = 1), CSTIV-C2 (n = 1) and CST IV-C3 (n = 5) (m–r). Statistical analysis was performed using a one-sided Mann–Whitney test

Statistical analysis was performed using a one-sided Wilcoxon matched pairs signed rank test. C3b, C5, and C5a concentrations were also compared between samples taken from women who were classed as CST I with CST II–V (d–f) n = 385 samples from n = 133 women. The Kruskal–Wallis and Dunn’s multiple comparison’s test was used to determine statistical significance

The Kruskal–Wallis and Dunn’s multiple comparison’s test was used to determine statistical significance. Cervicovaginal concentrations of C3b, C5, and C5a were compared between women who delivered preterm or at term in those who were classed as being Lactobacillus deplete (n = 22), or abundant in either CST III (L. iners) (n = 22) or CST IV (diverse) (n = 15) (g–i). CST IV was subdivided into CST IV-B (n = 8), CSTIV-C1 (n = 1), CSTIV-C2 (n = 1) and CST IV-C3 (n = 5)

Cervicovaginal concentrations of C3b, C5, and C5a were compared between women who delivered preterm or at term in those who were classed as being Lactobacillus deplete (n = 22), or abundant in either CST III (L. iners) (n = 22) or CST IV (diverse) (n = 15) (g–i). CST IV was subdivided into CST IV-B (n = 8), CSTIV-C1 (n = 1), CSTIV-C2 (n = 1) and CST IV-C3 (n = 5). Statistical analysis was performed using a one-sided Mann–Whitney test

This supports a mechanism whereby host–microbial-driven inflammation occurs via activation of the complement cascade in women who deliver preterm. Comparison of the cervicovaginal immune milieu between women with (n = 13) and without (n = 96) cervical shortening between 12+0–16+6 weeks of pregnancy showed increased levels IgM, C5, C5a and IL-6, and lower concentrations of IL-10 in those women with a short cervix (Fig. 7a–e). As microbial-driven inflammation is considered to be an aetiological factor for cervical shortening in a proportion of women, we next examined the interaction between microbial composition, immune mediators and cervical length

The two most common interventions used in women at high risk of preterm birth who are found to have a short cervix in the second trimester of pregnancy are cervical cerclage and vaginal progesterone therapy. Forty women were sampled before and after the insertion of a cervical cerclage. Of these, 21 women received progesterone after insertion of the cervical cerclage and 19 women did not, according to patient or clinician preference. 14 women had braided suture material, and 25 women had monofilament, and in one case it was not possible to obtain information on suture material

Forty women were sampled before and after the insertion of a cervical cerclage. Of these, 21 women received progesterone after insertion of the cervical cerclage and 19 women did not, according to patient or clinician preference. 14 women had braided suture material, and 25 women had monofilament, and in one case it was not possible to obtain information on suture material. Choice of cerclage material depended on either dual participation in the C-STICH trial (a multicentre randomised controlled trial to compare pregnancy outcomes between these two cerclage materials, https://www.isrctn.com/ISRCTN15373349), or was dependent on local standard practice

Cervicovaginal immune mediators were analysed from women pre- and post-cervical cerclage. The type of material was known for 39 women, 14 women received braided cerclage, and 25 women received monofilament. The fold change concentration of mediators of microbial recognition (a), complement proteins (b), and cytokine concentrations (c) between samples pre-cerclage and post-cerclage are compared between women who had braided cerclage (n = 14) and women who had monofilament cerclage (n = 25)

The type of material was known for 39 women, 14 women received braided cerclage, and 25 women received monofilament. The fold change concentration of mediators of microbial recognition (a), complement proteins (b), and cytokine concentrations (c) between samples pre-cerclage and post-cerclage are compared between women who had braided cerclage (n = 14) and women who had monofilament cerclage (n = 25). The percentage of women who delivered preterm with a braided cerclage was compared with the percentage who delivered preterm following a monofilament cerclage (d)

Source data are provided as a Source Data file. The influence of cerclage and cerclage material on immune mediators and vaginal microbial composition was examined more closely in 33 women who delivered preterm who had longitudinal samples at timepoint A and B. Despite most women maintaining stability of microbial composition, there was a significant increase in local production of MBL, C3b, C5a, IL-8, IL-6, and IL-1β following a braided material cerclage (Fig. 9a–i)

In contrast, monofilament insertion was not associated with immune activation (Fig. 9b–i). Of the 73 women who delivered at term with longitudinal samples between the A and B timepoint, five women had a cerclage using braided material, 15 had monofilament and 54 women had no intervention (Supplementary Fig. 6a–i). The microbial composition was relatively stable in each group between timepoints (Supplementary Fig. 6)

An upward trajectory was seen between timepoint A and B in the concentrations of MBL, IgM, C5, C5a, IL-1β (Supplementary Fig. 6b, c, e, f, i) in most women who delivered at term with a braided cerclage material, but a statistically significant increase was seen only in C5a concentrations (Supplementary Fig. 6f). Vaginal microbiota composition (a) and cervicovaginal immune mediators (b–i) were analysed in 34 women who delivered preterm between 12–16 weeks and again between 20–24 weeks. Ten women had a cerclage with braided material and 7 women had a cerclage with monofilament material between timepoints, whereas 16 women had no intervention (a)

Vaginal microbiota composition (a) and cervicovaginal immune mediators (b–i) were analysed in 34 women who delivered preterm between 12–16 weeks and again between 20–24 weeks. Ten women had a cerclage with braided material and 7 women had a cerclage with monofilament material between timepoints, whereas 16 women had no intervention (a). The concentration of MBL (b), IgM (c), C3b (d), C5 (e), C5a (f), IL-8 (g), IL-6 (h), and IL-1β (i) were compared between 12–16 and 20–24 weeks in women who had a braided cerclage, monofilament cerclage and no cerclage

The proportion of women who had an increase in immune mediators were also compared between suture material. Statistical analysis was performed using a one-sided Wilcoxon matched pairs signed rank test and a one-sided Fisher’s exact test, N = 33 women. Source data are provided as a Source Data file

Cerclage material was either monofilament or braided. The choice of cerclage material was based on either randomisation to C-STICH (REC: Cambridgeshire and Hertfordshire, ISRCTN 15373349) or according to local practice. 10 of the 59 (17%) women who received a cervical cerclage were dual participants of the C-STICH study. The BBL™ CultureSwab™ was thawed on ice

CST I was further divided to I-A and I-B sub-CST,CST III was further divided to III-A and III-B sub-CST, and CST IV-C was further divided to IV-C0, IV-C1, IV-C2, IV-C3, and IV-C4 sub-CST. Heatmaps were used to visualise the abundance data - hierarchical clustering with Ward linkage of the top 20 most abundant species. Statistical analyses were performed using Graphpad Prism 9.0.0

While women who delivered at term without intervention were used as a control group, it is acknowledged that our study cohort was predefined as being at high risk of preterm delivery. There was no significant difference in maternal age (p = 0.93), BMI (p = 0.20) or ethnicity (p = 0.09) between women who delivered spontaneously preterm or at term with or without intervention (Supplementary Table 1). No women were diagnosed with a sexually transmitted disease

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