Vaginal microbial profiling in a preterm birth high-risk cohort using shallow shotgun metagenomics

Preterm birth (PTB) is a significant health problem globally, with an estimate of 15 million cases annually. Approximately 10% of neonates born early will die prematurely, while a subset will develop severe life-long morbidities. Unfortunately, preterm birth's syndromic nature has evaded prevention strategies, and it continues to impose a high burden on healthcare systems and families. The role of vaginal bacteria in triggering biomolecular causes of PTB has been recognised for years. However, translating this knowledge to practical diagnostic and therapeutic strategies has remained elusive. New techniques in high-throughput sequencing have improved our understanding of the nature and role of the vaginal microbiome during pregnancy. Several multi-ethnic and multi-geographical studies into the vaginal microbiome have identified five distinct bacterial profiles termed community state types (CSTs), one of which is positively associated with dysbiosis and increased risk of PTB. In a small pilot study of first-trimester vaginal microbial DNA obtained from pregnant women at high-risk of PTB, we compared the CST profiles generated using standard 16S amplicon sequencing with shallow shotgun metagenomics (SSM). Both methods identified the presence of the five CSTs as has been reported previously, although the metagenomic data showed greater taxonomic resolution and more accurate CST assignation. These findings suggest that SSM is a cost-effective and potentially superior alternative to 16S sequencing for vaginal microbiome analysis

Comparison of the cardiometabolic profiles of adolescents conceived through ART with those of a non-ART cohort

STUDY QUESTION Is the cardiometabolic health of adolescents conceived through ART worse than that of their counterparts conceived without ART? SUMMARY ANSWER The majority of cardiometabolic and vascular health parameters of adolescents conceived through ART are similar or more favourable, than those of their counterparts of similar age and conceived without ART. WHAT IS KNOWN ALREADY It has been proposed that the cardiometabolic health of offspring conceived with ART may be unfavourable compared to that of their counterparts conceived without ART. The literature pertaining to cardiometabolic health of offspring conceived after ART is contradictory, but generally suggests unfavourable cardiometabolic health parameters, such as an increase in blood pressure (BP), vascular dysfunction and adiposity, as well as unfavourable glucose and lipid profiles. With over 8 million children and adults born through ART worldwide, it is important to investigate whether these early signs of adverse cardiometabolic differences persist into adolescence and beyond. STUDY DESIGN, SIZE, DURATION The Growing Up Healthy Study (GUHS) is a prospective cohort study that recruited 303 adolescents and young adults conceived after ART (aged 13–21 years) and born between 1991 and 2001 in Western Australia. Their health parameters, including cardiometabolic factors, were assessed and compared with counterparts from the Raine Study Generation 2 (Gen2). The 2868 Gen2 participants were born 1989–1992 and are representative of the Western Australian adolescent population. At ∼17 years of age (2013–2017), 163 GUHS participants replicated assessments previously completed by Gen2 at a similar age. PARTICIPANTS/MATERIALS, SETTING, METHODS Cardiometabolic parameters were compared between a total of 163 GUHS and 1457 Gen2 adolescents. Separate male (GUHS n = 81, Gen2 n = 735) and female (GUHS n = 82, Gen2 n = 722) analyses were conducted. Assessments consisted of a detailed questionnaire including health, lifestyle and demographic parameters, anthropometric assessments (height, weight, BMI, waist circumference and skinfold thickness), fasting serum biochemistry, arterial stiffness and BP (assessed using applanation tonometry). Abdominal ultrasonography was used to assess the presence and severity of hepatic steatosis, and thickness of abdominal fat compartments. Non-alcoholic fatty liver disease (NAFLD) was diagnosed if there was sonographic fatty liver in the absence of significant alcohol consumption. Chi2, Fisher’s exact and Mann–Whitney U tests, performed in SPSS V25, examined cohort differences and generalized estimating equations adjusted for the following covariates: singleton vs non-singleton pregnancy, birthweight (z-score), gestational age, BMI, smoking, alcohol consumption in the past 6 months and parent cardiovascular status. Arterial stiffness measures and waist circumference were additionally adjusted for height, and female analyses were additionally adjusted for use of oral contraceptives in the preceding 6 months. MAIN RESULTS AND THE ROLE OF CHANCE In adjusted analyses, GUHS females had a lower BMI (22.1 vs 23.3 kg/m2, P = 0.014), and thinner skinfolds (triceps, subscapular, mid-abdominal; 16.9 vs 18.7 mm, P = 0.021, 13.4 vs 15.0 mm, P = 0.027, 19.7 vs 23.2 mm, P < 0.001, respectively), whereas males were not significantly different. Waist circumference was lower in GUHS adolescents (males: 78.1 vs 81.3 cm, P = 0.008, females: 76.7 vs 83.3 cm, P = 0.007). There were no significant differences between the two groups in glucose, insulin, homeostatic model assessment for insulin resistance, low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC), alanine aminotransferase and high-sensitivity C-reactive protein in both sexes. In females, serum triglycerides were lower in GUHS adolescents (1.0 vs 1.2 mmol/l, P = 0.029). GUHS males had higher serum HDL-C (1.1 vs 1.0 mmol/l, P = 0.004) and a lower TC/HDL-C ratio (3.2 vs 3.6, P = 0.036). There were no significant differences in the prevalence of NAFLD or steatosis severity scores between the cohorts in males and females. GUHS females had less subcutaneous adipose tissue (9.4 vs 17.9 mm, P < 0.001), whereas GUHS males had greater visceral adipose thickness (44.7 vs 36.3 mm, P < 0.001). There was no significant difference in pre-peritoneal adipose thickness. Pulse wave velocity was lower in GUHS males (5.8 vs 6.3 m/s, P < 0.001) and heart rate corrected augmentation index was lower in GUHS females (−8.4 vs −2.7%, P = 0.048). There were no significant differences in BP or heart rate in males or females between the two groups. LIMITATIONS, REASONS FOR CAUTION Despite the substantial study size and the unique study design of the ART cohort, we were unable to differentiate between different types of ART, due to the low number of ICSI cycles (e.g. IVF vs ICSI), draw definite conclusions, or relate the outcomes to the cause of infertility. Considering the differences in time points when both cohorts were studied, external factors could have changed, which could not be accounted for. Given the observational nature of this study, causation cannot be proven. WIDER IMPLICATIONS OF THE FINDINGS Contrary to our hypothesis and previous findings focussing mainly on childhood, this study reports mostly similar or favourable cardiometabolic markers in adolescents conceived with ART compared to those conceived without ART. The greater visceral adipose thickness, particularly present in males, requires further investigation. While these findings are generally reassuring, future well-designed and appropriately powered studies are required to definitively address the issue of cardiometabolic health in ART adults. STUDY FUNDING/COMPETING INTEREST(S) This project was supported by NHMRC project grant number 1042269 and R.J.H. received education grant funding support from Ferring Pharmaceuticals. R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia

Targeting Toll-like receptor-4 to tackle preterm birth and fetal inflammatory injury

Every year, 15 million pregnancies end prematurely, resulting in more than 1 million infant deaths and long-term health consequences for many children. The physiological processes of labour and birth involve essential roles for immune cells and pro-inflammatory cytokines in gestational tissues. There is compelling evidence that the mechanisms underlying spontaneous preterm birth are initiated when a premature and excessive inflammatory response is triggered by infection or other causes. Exposure to pro-inflammatory mediators is emerging as a major factor in the 'fetal inflammatory response syndrome' that often accompanies preterm birth, where unscheduled effects in fetal tissues interfere with normal development and predispose to neonatal morbidity. Toll-like receptors (TLRs) are critical upstream gatekeepers of inflammatory activation. TLR4 is prominently involved through its ability to sense and integrate signals from a range of microbial and endogenous triggers to provoke and perpetuate inflammation. Preclinical studies have identified TLR4 as an attractive pharmacological target to promote uterine quiescence and protect the fetus from inflammatory injury. Novel small-molecule inhibitors of TLR4 signalling, specifically the non-opioid receptor antagonists (+)-naloxone and (+)-naltrexone, are proving highly effective in animal models for preventing preterm birth induced by bacterial mimetic LPS, heat-killed Escherichia coli, or the TLR4-dependent pro-inflammatory lipid, platelet-activating factor (PAF). Here, we summarise the rationale for targeting TLR4 as a master regulator of inflammation in fetal and gestational tissues, and the potential utility of TLR4 antagonists as candidates for preventative and therapeutic application in preterm delivery and fetal inflammatory injury.Sarah A Robertson, Mark R Hutchinson, Kenner C Rice, Peck-Yin Chin, Lachlan M Moldenhauer, Michael J Stark, David M Olson, Jeffrey A Keela

Labour-associated changes in the regulation of production of immunomodulators in human amnion by glucocorticoids, bacterial lipopolysaccharide and pro-inflammatory cytokines

Parturition is associated with changes in the production of inflammatory mediators by gestational tissues. An explant system was established to study the change in response of human amnion to various regulating factors during labour. Disks of tissue (6 mm) were excised from amnion membranes obtained either at term by Caesarian section before labour (n = 5-6) or after spontaneous vaginal delivery (n = 3-7). After 24 h equilibration in media, the tissues were treated with interleukin 1 beta (10 ng ml(-1)), tumour necrosis factor alpha (100 ng ml(-1)), lipopolysaccharide (5 mu g ml(-1)) and dexamethasone (1 mu mol l(-1)) or an appropriate vehicle control for 24 h (n = 3 wells per treatment). Media were harvested and interleukin 10, interleukin 6 and prostaglandin E-2 concentrations were determined by immunoassay. in tissues taken both before and after the onset of labour, basal interleukin 10 production by amnion explants was near to the limit of detection. Basal production rates of PGE(2) by amnion explants were significantly higher (P < 0.0012; Mann-Whitney U test) in tissues taken during labour than in tissues taken before the onset of labour, while interleukin 6 production was not significantly altered by labour. Production rates of interleukin 6 and prostaglandin E-2 were significantly increased by interleukin 1 beta, tumour necrosis factor alpha and lipopolysaccharide in explants from tissues taken during and before labour, while the responsiveness of interleukin 10 production to these treatments was inconsistent. Dexamethasone had no effect on interleukin 6 production by amnion explants, but significantly inhibited prostaglandin E-2 production, although this inhibition was approximately 30% lower in tissues obtained after the onset of labour. These results support the presence of inflammatory positive feedback cycles, coincident with a deficiency of an anti-inflammatory factor within gestational tissue, which may be involved in the progression or maintenance of labour

Interleukin (IL)-6 and IL-8 production by human amnion: Regulation by cytokines, growth factors, glucocorticoids, phorbol esters, and bacterial lipopolysaccharide

Amniotic fluid at term contains high concentrations of interleukin (IL)- 6 and IL-8. The source of these cytokines has not been identified, although the fetal membranes (amnion and chorion) are likely contributors. Amnion cytokine production was investigated by using amnion cells isolated by enzymatic digestion (from placentas delivered at term before labor) and cultured in vitro. IL-6 and IL-8 were measured in conditioned media by ELISA. Amnion cells produced detectable amounts of both IL-6 and IL-8 throughout the 7-day culture period. The ratio of IL-8 to IL-6 was approximately 5:1, similar to the ratio found in amniotic fluid. Production of both IL-6 and IL- 8 was stimulated in a concentration-dependent fashion by interleukin-1β (0.1-10 ng/ml), tumor necrosis factor-α (1-100 ng/ml), and bacterial lipopolysaccharide (0.1-10 μg/ml), and also by 100 nM phorbol 12-myristate 13-acetate. Epidermal growth factor (1-25 ng/ml) had only minimal effects on amnion cytokine production. Dexamethasone (10 nM) inhibited IL-6/-8 production by approximately 50% throughout the culture period. Production of IL-6/-8 by cultured amniotic fibroblasts, which under basal conditions was much lower than that by epithelial cells, was regulated by all the agents tested in a fashion similar to that of the epithelial cells. These findings suggest that the amnion contributes to the pool of IL-6 and IL-8 in amniotic fluid. We speculate that amnion-derived cytokines might have functions during normal human parturition that are distinct from their conventional roles as inflammatory mediators

Paradoxical proinflammatory actions of interleukin-10 in human amnion: potential roles in term and preterm labour

IL-10 is regarded predominantly as an inhibitor of cell-mediated inflammatory reactions. As much, it has been suggested that IL-10 could have therapeutic potential, including the treatment of preterm labor. Using explant cultures of gestational membranes we have found that IL-10 does indeed exert anti-inflammatory properties in choriodecidua, but that in the adjacent amnion it has remarkable pro-inflammatory actions. Amnion prostaglandin (PG) E(2) production was significantly increased following 24-h treatment with IL-10 (5-100 ng/ml). Production of IL-8 also showed a significant stimulation at 100 ng/mL IL-10. In contrast, choriodecidual production of IL-8 and tumor necrosis factor (TNF)-alpha was dramatically inhibited by IL-10, confirming the ability of this tissue to exhibit a classical IL-10 response IL-10 retained its stimulatory actions on amnion in the presence of IL-1beta and TNF-alpha stimulation. These findings suggest that the fetal membranes can exhibit opposing responses to IL-10, depending on whether the inflammatory insult occurs at the maternal or fetal face. While inflammatory reactions are negatively regulated by IL-10 in choriodecidua, if the pathogen reaches the amnion and threatens the fetus, pro-inflammatory reactions may predominate to ensure successful labor to spare and protect the fetus

Labor-associated changes in interleukin-10 production and its regulation by immunomodulators in human choriodecidua

Parturition is associated with increased production of proinflammatory mediators by gestational tissues. Interleukin-10 (IL-10) is an antiinflammatory cytokine produced by human chorion, decidual, and trophoblast tissues. To study the effects of immunomodulators on IL-10, IL-6, and PGE(2) production by human choriodecidua before and after labor, an organ explant system was established. Tissue disks (6 mm) were excised from choriodecidual membranes obtained at term by cesarean section before labor (n = 6-7) or after spontaneous vaginal delivery (n = 7-8). After 24-h equilibration in medium, the tissues were treated with IL-1 beta (10 ng/mL), tumor necrosis factor-tu (100 ng/mL), Lipopolysaccharide (5 mu g/mL), dexamethasone (1 mu mol/L), or an appropriate vehicle control (n = 8 wells/treatment) for 24 h. Media were harvested, and IL-10, IL-6, and PGE(2) concentrations were determined by immunoassay. Basal choriodecidual production rates of IL-10 mere significantly decreased with labor (P < 0.001), whereas PGE(2) and IL-6 production rates increased. The production of all three substances was increased by IL-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide, but inhibited by dexamethasone. In contrast to PGE(2) and IL-6, there was significantly increased responsiveness of IL-10 production to inflammatory stimuli after labor, but decreased responsiveness to the inhibitory effects of dexamethasone. These data indicate that IL-10 could play a role in modulating or promoting resolution of the inflammatory processes associated with labor at term and with intrauterine infection-associated preterm labor. Media were harvested, and IL-10, IL-6, and PGE(2) concentrations were determined by immunoassay. Basal choriodecidual production rates of IL-10 mere significantly decreased with labor (P < 0.001), whereas PGE(2) and IL-6 production rates increased. The production of all three substances was increased by IL-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide, but inhibited by dexamethasone. In contrast to PGE(2) and IL-6, there was significantly increased responsiveness of IL-10 production to inflammatory stimuli after labor, but decreased responsiveness to the inhibitory effects of dexamethasone. These data indicate that IL-10 could play a role in modulating or promoting resolution of the inflammatory processes associated with labor at term and with intrauterine infection-associated preterm labor

Key enzymes of prostaglandin biosynthesis and metabolism. Coordinate regulation of expression by cytokines in gestational tissues: A review

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues