Comparing cervical cerclage, pessary and vaginal progesterone for prevention of preterm birth in women with a short cervix (SuPPoRT): A multicentre randomised controlled trial.

BACKGROUND: Cervical cerclage, cervical pessary, and vaginal progesterone have each been shown to reduce preterm birth (PTB) in high-risk women, but to our knowledge, there has been no randomised comparison of the 3 interventions. The SuPPoRT "Stitch, Pessary, or Progesterone Randomised Trial" was designed to compare the rate of PTB <37 weeks between each intervention in women who develop a short cervix in pregnancy. METHODS AND FINDINGS: SuPPoRT was a multicentre, open label 3-arm randomised controlled trial designed to demonstrate equivalence (equivalence margin 20%) conducted from 1 July 2015 to 1 July 2021 in 19 obstetric units in the United Kingdom. Asymptomatic women with singleton pregnancies with transvaginal ultrasound cervical lengths measuring <25 mm between 14+0 and 23+6 weeks' gestation were eligible for randomisation (1:1:1) to receive either vaginal cervical cerclage (n = 128), cervical pessary (n = 126), or vaginal progesterone (n = 132). Minimisation variables were gestation at recruitment, body mass index (BMI), and risk factor for PTB. The primary outcome was PTB <37 weeks' gestation. Secondary outcomes included PTB <34 weeks', <30 weeks', and adverse perinatal outcome. Analysis was by intention to treat. A total of 386 pregnant women between 14+0 and 23+6 weeks' gestation with a cervical length <25 mm were randomised to one of the 3 interventions. About 67% were of white ethnicity, 18% black ethnicity, and 7.5% Asian ethnicity. Mean BMI was 25.6. Around 85% of women had prior risk factors for PTB; 39.1% had experienced a spontaneous PTB or midtrimester loss (>14 weeks gestation); and 45.8% had prior cervical surgery. Data from 381 women were available for outcome analysis. Using binary regression, randomised therapies (cerclage versus pessary versus vaginal progesterone) were found to have similar effects on the primary outcome PTB <37 weeks (39/127 versus 38/122 versus 32/132, p = 0.4, cerclage versus pessary risk difference (RD) -0.7% [-12.1 to 10.7], cerclage versus progesterone RD 6.2% [-5.0 to 17.0], and progesterone versus pessary RD -6.9% [-17.9 to 4.1]). Similarly, no difference was seen for PTB <34 and 30 weeks, nor adverse perinatal outcome. There were some differences in the mild side effect profile between interventions (vaginal discharge and bleeding) and women randomised to progesterone reported more severe abdominal pain. A small proportion of women did not receive the intervention as per protocol; however, per-protocol and as-treated analyses showed similar results. The main study limitation was that the trial was underpowered for neonatal outcomes and was stopped early due to the COVID-19 pandemic. CONCLUSIONS: In this study, we found that for women who develop a short cervix, cerclage, pessary, and vaginal progesterone were equally efficacious at preventing PTB, as judged with a 20% equivalence margin. Commencing with any of the therapies would be reasonable clinical management. These results can be used as a counselling tool for clinicians when managing women with a short cervix. TRIAL REGISTRATION: EU Clinical Trials register. EudraCT Number: 2015-000456-15, clinicaltrialsregister.eu., ISRCTN Registry: ISRCTN13364447, isrctn.com.

Predicting Preterm Birth Using Cell-Free Ribonucleic Acid.

Spontaneous preterm birth (sPTB) is a complex and clinically heterogeneous condition that remains incompletely understood, leading to insufficient interventions to effectively prevent it from occurring. Cell-free ribonucleic acid signatures in the maternal circulation have the potential to identify biologically relevant subtypes of sPTB. These could one day be used to predict and prevent sPTB in asymptomatic individuals, and to aid in prognosis and management for individuals presenting with threatened preterm labor and preterm prelabor rupture of membranes.

Corrigendum: Microbiota dynamics, metabolic and immune interactions in the cervicovaginal environment and their role in spontaneous preterm birth.

[This corrects the article DOI: 10.3389/fimmu.2023.1306473.].

Omega-3 fatty acid supply in pregnancy for risk reduction of preterm and early preterm birth.

This clinical practice guideline on the supply of the omega-3 docosahexaenoic acid and eicosapentaenoic acid in pregnant women for risk reduction of preterm birth and early preterm birth was developed with support from several medical-scientific organizations, and is based on a review of the available strong evidence from randomized clinical trials and a formal consensus process. We concluded the following. Women of childbearing age should obtain a supply of at least 250 mg/d of docosahexaenoic+eicosapentaenoic acid from diet or supplements, and in pregnancy an additional intake of ≥100 to 200 mg/d of docosahexaenoic acid. Pregnant women with a low docosahexaenoic acid intake and/or low docosahexaenoic acid blood levels have an increased risk of preterm birth and early preterm birth. Thus, they should receive a supply of approximately 600 to 1000 mg/d of docosahexaenoic+eicosapentaenoic acid, or docosahexaenoic acid alone, given that this dosage showed significant reduction of preterm birth and early preterm birth in randomized controlled trials. This additional supply should preferably begin in the second trimester of pregnancy (not later than approximately 20 weeks' gestation) and continue until approximately 37 weeks' gestation or until childbirth if before 37 weeks' gestation. Identification of women with inadequate omega-3 supply is achievable by a set of standardized questions on intake. Docosahexaenoic acid measurement from blood is another option to identify women with low status, but further standardization of laboratory methods and appropriate cutoff values is needed. Information on how to achieve an appropriate intake of docosahexaenoic acid or docosahexaenoic+eicosapentaenoic acid for women of childbearing age and pregnant women should be provided to women and their partners.

Adrenal volumes in fetuses delivering prior to 32 weeks' gestation: An MRI pilot study.

INTRODUCTION: Spontaneous preterm birth prior to 32 weeks' gestation accounts for 1% of all deliveries and is associated with high rates of morbidity and mortality. A total of 70% are associated with chorioamnionitis which increases the incidence of morbidity, but for which there is no noninvasive antenatal test. Fetal adrenal glands produce cortisol and dehydroepiandosterone-sulphate which upregulate prior to spontaneous preterm birth. Ultrasound suggests that adrenal volumes may increase prior to preterm birth, but studies are limited. This study aimed to: (i) demonstrate reproducibility of magnetic resonance imaging (MRI) derived adrenal volumetry; (ii) derive normal ranges of total adrenal volumes, and adrenal: body volume for normal; (iii) compare with those who have spontaneous very preterm birth; and (iv) correlate with histopathological chorioamnionitis. MATERIAL AND METHODS: Patients at high risk of preterm birth prior to 32 weeks were prospectively recruited, and included if they did deliver prior to 32 weeks; a control group who delivered an uncomplicated pregnancy at term was also recruited. T2 weighted images of the entire uterus were obtained, and a deformable slice-to-volume method was used to reconstruct the fetal abdomen. Adrenal and body volumes were obtained via manual segmentation, and adrenal: body volume ratios generated. Normal ranges were created using control data. Differences between groups were investigated accounting for the effect of gestation by use of regression analysis. Placental histopathology was reviewed for pregnancies delivering preterm. RESULTS: A total of 56 controls and 26 cases were included in the analysis. Volumetry was consistent between observers. Adrenal volumes were not higher in the case group (p = 0.2); adrenal: body volume ratios were higher (p = 0.011), persisting in the presence of chorioamnionitis (p = 0.017). A cluster of three pairs of adrenal glands below the fifth centile were noted among the cases all of whom had a protracted period at risk of preterm birth prior to MRI. CONCLUSIONS: Adrenal: body volume ratios are significantly larger in fetuses who go on to deliver preterm than those delivering at term. Adrenal volumes were not significantly larger, we hypothesize that this could be due to an adrenal atrophy in fetuses with fulminating chorioamnionitis. A straightforward relationship of adrenal size being increased prior to preterm birth should not be assumed.

Pancreatitis in RYR1-related disorders.

Mutations in RYR1 encoding the ryanodine receptor (RyR) skeletal muscle isoform (RyR1) are a common cause of inherited neuromuscular disorders. Despite its expression in a wide range of tissues, non-skeletal muscle manifestations associated with RYR1 mutations have only been rarely reported. Here, we report three patients with a diagnosis of Central Core Disease (CCD), King-Denborough Syndrome (KDS) and Malignant Hyperthermia Susceptibility (MHS), respectively, who in addition to their (putative) RYR1-related disorder also developed symptoms and signs of acute pancreatitis. In two patients, episodes were recurrent, with severe multisystem involvement and sequelae. RyR1-mediated calcium signalling plays an important role in normal pancreatic function but has also been critically implicated in the pathophysiology of acute pancreatitis, particularly in bile acid- and ethanol-induced forms. Findings from relevant animal models indicate that pancreatic damage in these conditions may be ameliorated through administration of the specific RyR1 antagonist dantrolene and other compounds modifying pancreatic metabolism including calcium signalling. These observations suggest that patients with RYR1 gain-of-function variants may be at increased risk of developing acute pancreatitis, a condition which should therefore be considered in the health surveillance of such individuals.

Amniotic fluid sludge is associated with earlier preterm delivery and raised cervicovaginal interleukin 8 concentrations.

BACKGROUND: Preterm birth is the leading cause of global neonatal mortality. Amniotic fluid sludge, thought to indicate intra-amniotic infection, may have potential as a clinical biomarker of preterm birth risk. OBJECTIVE: This study aimed to analyze whether the presence of amniotic fluid sludge in pregnant participants with a known short cervical length can help improve the understanding of the etiology and guide management choice. STUDY DESIGN: This was a retrospective cohort study analyzing the effects of amniotic fluid sludge presence on the risk of preterm birth in high-risk asymptomatic pregnant participants with a short cervical length (<25 mm) at a large tertiary referral maternity center in London. Amniotic fluid sludge was detected on a routine transvaginal ultrasound scan. RESULTS: Overall, 147 pregnant participants with a short cervical length were identified, 54 of whom had amniotic fluid sludge. Compared with pregnant participants without amniotic fluid sludge, pregnant participants with amniotic fluid sludge were more likely to have a short cervical length (19 vs 14 mm, respectively; P<.0001) and increased cervicovaginal fetal fibronectin concentrations at diagnosis (125 vs 45 ng/mL, respectively; P=.0006). Pregnant participants with amniotic fluid sludge were at increased risk of midtrimester loss and delivery before 24 weeks of gestation (relative risk, 3.4; 95% confidence interval, 1.2-10.3). Furthermore, this study showed that pregnant participants with amniotic fluid sludge have increased cervicovaginal interleukin 8 concentrations, supporting the concept of amniotic fluid sludge as an indicator of an inflammatory response to microbial invasion (P=.03). Neonatal outcomes were similar between the 2 groups. CONCLUSION: In our cohort of high-risk asymptomatic pregnant participants with a short cervical length, the presence of amniotic fluid sludge is associated with an increased risk of delivery before 24 weeks of gestation. Moreover, pregnant participants with amniotic fluid sludge were more likely to have raised fetal fibronectin levels and inflammatory cytokines, particularly interleukin 8, in the cervicovaginal fluid, supporting the concept that amniotic fluid sludge is associated with an infective or inflammatory process. Future research should aim to further establish the clinical significance of amniotic fluid sludge presence and guide subsequent management.

Longitudinal change in cervical length following vaginal or abdominal cervical cerclage: a randomized comparison.

BACKGROUND: Cervical cerclage has been shown to reduce the risk of recurrent spontaneous preterm birth in a high-risk patient population; however, the mechanism is not well understood. Transabdominal cerclage is superior to low and high vaginal cerclage in reducing early spontaneous preterm birth and fetal loss in women with previous failed vaginal cerclage. Cervical length measurements are commonly used to monitor high-risk women and may explain the mechanism of success. OBJECTIVE: This study aimed to evaluate the rate of change in longitudinal cervical length after randomized placement of low transvaginal, high transvaginal, or transabdominal cerclage in women with a previous failed vaginal cerclage. STUDY DESIGN: This was a planned analysis of longitudinal transvaginal ultrasound cervical length measurements from patients enrolled in the Vaginal Randomised Intervention of Cerclage trial, a randomized controlled trial comparing transabdominal cerclage or high transvaginal cerclage with low transvaginal cerclage. Cervical length measurements at specific gestational ages were compared over time and between groups, using generalized estimating equations fitted using the maximum-likelihood random-effects estimator. In addition, cervical length measurements were compared in women with transabdominal cerclage placed before and during pregnancy. The diagnostic accuracy of cervical length as a predictor of spontaneous preterm birth at <32 weeks of gestation was explored. RESULTS: This study included 78 women who underwent longitudinal cervical length assessment (70% of the analyzed cohort) with a history of failed cerclage, of whom 25 (32%) were randomized to low transvaginal cerclage, 26 (33%) to high transvaginal cerclage, and 27 (35%) to transabdominal cerclage. Abdominal cerclage was superior to low (P=.008) and high (P=.001) vaginal cerclage at maintaining cervical length over the surveillance period (14 to 26 weeks of gestation) (+0.08 mm/week, 95% confidence interval, -0.40 to 0.22; P=.580). On average, the cervical length was 1.8 mm longer by the end of the 12-week surveillance period in women with transabdominal cerclage (+1.8 mm; 95% confidence interval, -7.89 to 4.30; P=.564). High vaginal cerclage was no better than low cervical cerclage in the prevention of cervical shortening; the cervix shortened by 13.2 mm over 12 weeks in those with low vaginal cerclage (95% confidence interval, -21.7 to -4.7; P=.002) and by 20 mm over 12 weeks in those with high vaginal cerclage (95% confidence interval, -33.1 to -7.4; P=.002). Preconception transabdominal cerclage resulted in a longer cervix than those performed during pregnancy; this difference was significant after 22 weeks of gestation (48.5 mm vs 39.6 mm; P=.039). Overall, cervical length was an excellent predictor of spontaneous preterm birth at <32 weeks of gestation (receiver operating characteristic curve, 0.92; 95% confidence interval, 0.82-1.00). CONCLUSION: In women with a previous failed cervical cerclage, in the next pregnancy, the cervical length in women treated with vaginal cerclage funneled and shortened over time, whereas there was preservation of cervical length in women who receive transabdominal cerclage. Cervical length remained longer in transabdominal procedures performed before pregnancy than in transabdominal procedures performed during pregnancy. Overall, cervical length was an excellent predictor of spontaneous preterm birth in our cohort. Our findings may explain the mechanism of benefit for transabdominal cerclage, with its high placement better maintaining the structural integrity of the cervix at the level of the internal os.

The neutrophil-to-lymphocyte ratio: A low-cost antenatal indicator of placental chorioamnionitis in women who deliver preterm without clinical signs and symptoms of infection.

Chorioamnionitis is present in up to 70% of spontaneous preterm births and is associated with poor maternal, fetal and neonatal outcomes. OBJECTIVE: To explore the relationship between the neutrophil-to-lymphocyte ratio and histological chorioamnionitis in women who delivered preterm with no clinical signs or symptoms of infection. STUDY DESIGN: This was a retrospective analysis of a cohort of women who delivered spontaneously between 16 and 36+6 weeks at a tertiary UK hospital. Only women with placental histology and no signs of clinical infection were included. The neutrophil-to-lymphocyte ratio was calculated from a full blood count sample taken routinely within 24 h of delivery. The neutrophil-to-lymphocyte ratio was also calculated from first trimester booking bloods (<13 + 6 weeks) in a subgroup. Placental histopathology was categorised as either inflammatory (i.e. histologic chorioamnionitis, with or without evidence of fetal inflammatory response) or non-inflammatory (vascular pathology or a normal placenta). RESULTS: 169 women had available placental pathology and were included in the analysis. 70 % (118/169) had confirmed placental inflammation. The mean neutrophil-to-lymphocyte ratio was significantly raised in this group compared to those with normal (n = 24) or vascular (n = 27) pathology (inflammatory neutrophil-to-lymphocyte ratio 9.81 vs non-inflammatory neutrophil-to-lymphocyte ratio 6.53, p = 0.002. The delivery neutrophil-to-lymphocyte ratio had an area under the receiver operating characteristic curve of 0.69 (0.60 to 0.78) for predicting placental inflammation. A raised neutrophil-to-lymphocyte ratio (>6) was associated with an odds ratio of 5.2 (95 % CI 2.55 to 10.56) for histological chorioamnionitis, with a sensitivity of 80 % and negative predictive value of 86 %. A higher cut-off of 9 had a negative predictive value of 79 % for fetal inflammatory response. CONCLUSIONS: A raised neutrophil-to-lymphocyte ratio is associated with a 5-fold increased risk of histological chorioamnionitis in women who delivered early without signs or symptoms of infection. It was also raised at the time of preterm labour compared to the first trimester. A full blood count is an almost universal investigation in women admitted in preterm labour, often repeated, making this inexpensive and non-invasive ratio a useful additional antenatal biomarker in women admitted in spontaneous preterm labour at risk of subclinical chorioamnionitis and its associated poor outcomes.

Microbiota dynamics, metabolic and immune interactions in the cervicovaginal environment and their role in spontaneous preterm birth.

Differences in the cervicovaginal microbiota are associated with spontaneous preterm birth (sPTB), a significant cause of infant morbidity and mortality. Although establishing a direct causal link between cervicovaginal microbiota and sPTB remains challenging, recent advancements in sequencing technologies have facilitated the identification of microbial markers potentially linked to sPTB. Despite variations in findings, a recurring observation suggests that sPTB is associated with a more diverse and less stable vaginal microbiota across pregnancy trimesters. It is hypothesized that sPTB risk is likely to be modified via an intricate host-microbe interactions rather than due to the presence of a single microbial taxon or broad community state. Nonetheless, lactobacilli dominance is generally associated with term outcomes and contributes to a healthy vaginal environment through the production of lactic acid/maintenance of a low pH that excludes other pathogenic microorganisms. Additionally, the innate immunity of the host and metabolic interactions between cervicovaginal microbiota, such as the production of bacteriocins and the use of proteolytic enzymes, exerts a profound influence on microbial populations, activities, and host immune responses. These interplays collectively impact pregnancy outcomes. This review aims to summarize the complexity of cervicovaginal environment and microbiota dynamics, and associations with bacterial vaginosis and sPTB. There is also consideration on how probiotics may mitigate the risk of sPTB and bacterial vaginosis.

Neutrophils Dominate the Cervical Immune Cell Population in Pregnancy and Their Transcriptome Correlates With the Microbial Vaginal Environment.

The cervicovaginal environment in pregnancy is proposed to influence risk of spontaneous preterm birth. The environment is shaped both by the resident microbiota and local inflammation driven by the host response (epithelia, immune cells and mucous). The contributions of the microbiota, metabolome and host defence peptides have been investigated, but less is known about the immune cell populations and how they may respond to the vaginal environment. Here we investigated the maternal immune cell populations at the cervicovaginal interface in early to mid-pregnancy (10-24 weeks of gestation, samples from N = 46 women), we confirmed neutrophils as the predominant cell type and characterised associations between the cervical neutrophil transcriptome and the cervicovaginal metagenome (N = 9 women). In this exploratory study, the neutrophil cell proportion was affected by gestation at sampling but not by birth outcome or ethnicity. Following RNA sequencing (RNA-seq) of a subset of neutrophil enriched cells, principal component analysis of the transcriptome profiles indicated that cells from seven women clustered closely together these women had a less diverse cervicovaginal microbiota than the remaining three women. Expression of genes involved in neutrophil mediated immunity, activation, degranulation, and other immune functions correlated negatively with Gardnerella vaginalis abundance and positively with Lactobacillus iners abundance; microbes previously associated with birth outcome. The finding that neutrophils are the dominant immune cell type in the cervix during pregnancy and that the cervical neutrophil transcriptome of pregnant women may be modified in response to the microbial cervicovaginal environment, or vice versa, establishes the rationale for investigating associations between the innate immune response, cervical shortening and spontaneous preterm birth and the underlying mechanisms.

Antenatal diagnosis of chorioamnionitis: A review of the potential role of fetal and placental imaging.

Chorioamnionitis is present in up to 70% of spontaneous preterm births. It is defined as an acute inflammation of the chorion, with or without involvement of the amnion, and is evidence of a maternal immunological response to infection. A fetal inflammatory response can coexist and is diagnosed on placental histopathology postnatally. Fetal inflammatory response syndrome (FIRS) is associated with poorer fetal and neonatal outcomes. The only antenatal diagnostic test is amniocentesis which carries risks of miscarriage or preterm birth. Imaging of the fetal immune system, in particular the thymus and the spleen, and the placenta may give valuable information antenatally regarding the diagnosis of fetal inflammatory response. While ultrasound is largely limited to structural information, MRI can complement this with functional information that may provide insight into the metabolic activities of the fetal immune system and placenta. This review discusses fetal and placental imaging in pregnancies complicated by chorioamnionitis and their potential future use in achieving non-invasive antenatal diagnosis.

Activation of Lymphocytes in Healthy Neonates Within Hours of Birth.

It is now established that immune maturation occurs along a defined trajectory in the weeks and months after birth, but the immediate changes that occur within immune cells following birth is less clear. In this study, we monitored the immune profile of neonates via analysis of paired samples (n= 28) of cord blood and heel prick blood taken at varying times post term delivery by planned elective caesarean section. This paired approach accounted for the between-subject variability often observed over the first week of life. We identified rapid changes in immune cell populations within hours of birth. Specifically, we observed increased proliferation in effector T cells (but not regulatory T cells) that exhibited an increase in cytokine producing ability and also an increase in the percentage of CD3 T cells over this short time frame. This indicates that the mobilisation of the immune system is immediate post birth, presumably as a response to sudden exposure to the external environment, antigen or stress. Hence, immune development may start to occur more rapidly than previously proposed and as such, to study this trajectory, blood sampling should begin as soon after birth as possible.

Nuclear Magnetic Resonance Metabolomics of Symbioses between Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome, characterized by low levels of lactobacilli and overgrowth of a diverse group of bacteria, associated with higher risk of a variety of infections, surgical complications, cancer, and preterm birth (PTB). Despite the lack of a consistently applicable etiology, Prevotella spp. are often associated with both BV and PTB, and Pr. bivia has known symbiotic relationships with both Peptostreptococcus anaerobius and Gardnerella vaginalis. Higher risk of PTB can also be predicted by a composite of metabolites linked to bacterial metabolism, but their specific bacterial source remains poorly understood. Here, we characterize diversity of metabolic strategies among BV-associated bacteria and lactobacilli and the symbiotic metabolic relationships between Pr. bivia and its partners and show how these influence the availability of metabolites associated with BV/PTB and/or pro- or anti-inflammatory immune responses. We confirm a commensal relationship between Pe. anaerobius and Pr. bivia, refining its mechanism, which sustains a substantial increase in acetate production. In contrast, the relationship between Pr. bivia and G. vaginalis strains, with sequence variant G2, is mutualistic, with outcome dependent on the metabolic strategy of the G. vaginalis strain. Taken together, our data show how knowledge of inter- and intraspecies metabolic diversity and the effects of symbiosis may refine our understanding of the mechanism and approach to risk prediction in BV and/or PTB. IMPORTANCE Bacterial vaginosis (BV) is the most common vaginal infection for women of childbearing age. Although 50% of women with BV do not have any symptoms, it approximately doubles the risk of catching a sexually transmitted infection and also increases the risk of preterm delivery in pregnant women. Recent studies of the vaginal microbiota have suggested that variation between species in the same genus or between strains of the same species explain better or poorer outcomes or at least some coexistence patterns for bacteria of concern. We tested whether such variation is manifested in how vaginal bacteria grow in the laboratory and whether and how they may share nutrients. We then showed that this affected the overall cocktail of chemicals they produce, including bacterially derived chemicals that we have previously shown are linked to a higher risk of preterm delivery.

Predictive RNA profiles for early and very early spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth remains the main driver of childhood morbidity and mortality. Because of an incomplete understanding of the molecular pathways that result in spontaneous preterm birth, accurate predictive markers and target therapeutics remain elusive. OBJECTIVE: This study sought to determine if a cell-free RNA profile could reveal a molecular signature in maternal blood months before the onset of spontaneous preterm birth. STUDY DESIGN: Maternal samples (n=242) were obtained from a prospective cohort of individuals with a singleton pregnancy across 4 clinical sites at 12-24 weeks (nested case-control; n=46 spontaneous preterm birth <35 weeks and n=194 term controls). Plasma was processed via a next-generation sequencing pipeline for cell-free RNA using the Mirvie RNA platform. Transcripts that were differentially expressed in next-generation sequencing cases and controls were identified. Enriched pathways were identified in the Reactome database using overrepresentation analysis. RESULTS: Twenty five transcripts associated with an increased risk of spontaneous preterm birth were identified. A logistic regression model was developed using these transcripts to predict spontaneous preterm birth with an area under the curve =0.80 (95% confidence interval, 0.72-0.87) (sensitivity=0.76, specificity=0.72). The gene discovery and model were validated through leave-one-out cross-validation. A unique set of 39 genes was identified from cases of very early spontaneous preterm birth (<25 weeks, n=14 cases with time to delivery of 2.5±1.8 weeks); a logistic regression classifier on the basis of these genes yielded an area under the curve=0.76 (95% confidence interval, 0.63-0.87) in leave-one-out cross validation. Pathway analysis for the transcripts associated with spontaneous preterm birth revealed enrichment of genes related to collagen or the extracellular matrix in those who ultimately had a spontaneous preterm birth at <35 weeks. Enrichment for genes in insulin-like growth factor transport and amino acid metabolism pathways were associated with spontaneous preterm birth at <25 weeks. CONCLUSION: Second trimester cell-free RNA profiles in maternal blood provide a noninvasive window to future occurrence of spontaneous preterm birth. The systemic finding of changes in collagen and extracellular matrix pathways may serve to identify individuals at risk for premature cervical remodeling, with growth factor and metabolic pathways implicated more often in very early spontaneous preterm birth. The use of cell-free RNA profiles has the potential to accurately identify those at risk for spontaneous preterm birth by revealing the underlying pathophysiology, creating an opportunity for more targeted therapeutics and effective interventions.

RNA profiles reveal signatures of future health and disease in pregnancy.

Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.

Regulatory T Cells in Pregnancy Adverse Outcomes: A Systematic Review and Meta-Analysis.

Background: Several studies report the role of Regulatory T-cells (Tregs) in the pathophysiology of pregnancy adverse outcomes. Objective: The aim of this systematic review and meta-analysis was to determine whether there is an association between regulatory T cell levels and pregnancy adverse outcomes (PAOs), including pre-eclampsia and preterm birth (PTB). Method: Literature searches were conducted in PubMed/MEDLINE, Embase, and Cochrane CENTRAL databases. Inclusion criteria were original articles (clinical trials, case-control studies and cohort studies) comparing Tregs, sampled from the decidua or maternal blood, in healthy pregnant women versus women with pre-eclampsia or PTB. The outcome was standardised mean difference (SMD) in Treg numbers. The tau-squared (Tau²), inconsistency index (I²), and chi-squared (χ²) test quantified heterogeneity among different studies. Analyses were performed in RevMan software V.5.4.0 for Mac using a random-effects model with outcome data reported with 95% confidence intervals (CI). This study was prospectively registered with PROSPERO (CRD42020205469). PRISMA guidelines were followed. Results: From 4,085 unique studies identified, 36 were included in qualitative synthesis, and 34 were included in quantitative synthesis (meta-analysis). In total, there were 1,783 participants in these studies: healthy controls=964, pre-eclampsia=759, PTB=60. Thirty-two studies compared Tregs in healthy pregnant women and women with pre-eclampsia, and 30 of these sampled Tregs from peripheral blood showing significantly higher Treg numbers in healthy pregnancies (SMD; 1.46; 95% CI, 1.03-1.88; I²=92%). Four studies sampled Tregs from the maternal decidua showing higher Tregs in healthy pregnancies (SMD, 0.76; 95% CI, -0.13-1.65; I²=84%). No difference was found in the number of Tregs between early versus late pre-eclampsia (SMD,-1.17; 95% CI, -2.79-0.44; I²=94%). For PTB, two studies compared Tregs sampled from the peripheral blood with a tendency for higher Tregs in healthy pregnancies but this did not reach significance (SMD, 2.18; 95% CI, -1.34-5.70; I²=96%). Subcohort analysis using Treg analysis (flow cytometry vs. qPCR vs. immunofluorescence tissue staining) showed similar associations. Conclusion: Lower Tregs in pregnancy, sampled from the maternal peripheral blood, are associated with pre-eclampsia. There is a need for further studies to confirm a relationship between low Tregs and PTB. As the precise mechanisms by which Tregs may mediate pre-eclampsia and PTB remain unclear, further fundamental research is necessary to elucidate the underlying processes and highlight the causative link. Systematic Review Registration: PROSPERO, identifier CRD42020205469.

Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues.

High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue ('bulk') RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.

The legacy of maternal SARS-CoV-2 infection on the immunology of the neonate.

Despite extensive studies into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the effect of maternal infection on the neonate is unclear. To investigate this, we characterized the immunology of neonates born to mothers with confirmed SARS-CoV-2 infection during pregnancy. Here we show that maternal SARS-CoV-2 infection affects the neonatal immune system. Despite similar proportions of B cells, CD4+ T cells and CD8+ T cells, increased percentages of natural killer cells, Vδ2+ γδ T cells and regulatory T cells were detected in neonates born to mothers with recent or ongoing infection compared with those born to recovered or uninfected mothers. Increased plasma cytokine levels were also evident in neonates and mothers within the recent or ongoing infection group. Cytokine functionality was enhanced in neonates born to SARS-CoV-2-exposed mothers, compared to those born to uninfected mothers. In most neonates, this immune imprinting was nonspecific, suggesting vertical transmission of SARS-CoV-2 is limited, a finding supported by a lack of SARS-CoV-2-specific IgM in neonates despite maternal IgG transfer.