Few studies have evaluated the role of methylation-pathway nutrients involved in fetal growth . These one-carbon metabolism (OCM) nutrients are essential for DNA methylation in the periconception period. We aimed to characterize dietary patterns of 1638 women from the EDEN mother-child cohort in the year before pregnancy according to the contribution of OCM nutrients and to study the association of such patterns with anthropometric measurements at birth. Dietary intake before pregnancy was assessed by using a semi-quantitative food frequency questionnaire. We used the reduced-rank regression (RRR) method to identify dietary patterns using OCM nutrients as intermediate variables. We ran linear regressions models to study the association between dietary patterns scores and birth weight, length, head circumference, gestational age, and sex-specific z-scores, adjusting for maternal characteristics and vitamin supplementation before and during pregnancy. Three patterns, “varied and balanced”, “vegetarian tendency”, and “bread and starchy food” were identified, explaining 58% of the variability in OCM nutrient intake. Higher scores on the “varied and balanced” pattern tended to be associated with higher birth length and weight. In mainly well-nourished young French women, we did not find evidence that variability in OCM nutrient intake has major effects on fetal growth. Among the multiple factors involved in the physiological process guaranteeing a healthy pregnancy, adequate micronutrient intake during the pregnancy is necessary to support placenta homeostasis and fetal development ,2. Micror = 0.13) but also with a lower risk of large-for-gestational age birth weight (LGA)  [Some studies have reported associations between individual OCM micronutrients and fetal development. Folate intake during pregnancy (dietary folate and folic acid supplementation) has been associated with higher birth weight. The average effect has been estimated to be a 2% increase in birth weight for every two-fold increase in folate intake  and furt1, 0·89) ,17.The above studies often evaluated the impact of a single nutrient on fetal growth, but recent randomized and observational studies highlighted the positive effect of multiple-micronutrient supplementation on placental vascular function and for preventing fetal growth restriction in low- and middle-income countries ,18,19.The OCM cycle comprises different interlinked metabolic pathways and is of interest to investigate the effect of the combination of micronutrients provided by dietary intake on fetal development . The varSo far, studies investigating the role of OCM nutrients in relation to fetal development have focused on dietary intake during the pregnancy period. However, the pre-conception period also deserves attention because it is an important time window for epigenetic programming of development and health due to major epigenetics processes taking place during this period . Therefore, after identifying dietary patterns characterizing OCM micronutrient intake  in the year before pregnancy, our objective was first to study maternal characteristics associated with these patterns in the pre-conception period, and second to investigate their association with birth anthropometry. This study is reported according to the Strengthening the Reporting of Observational Studies in Nutritional Epidemiology (STROBE-nut) guidelines   of Kremlin Bicêtre Hospital (02-70) and the Commission Nationale de l’Informatique et des Libertés (CNIL), the French data privacy institution. The usual diet of women during the year before pregnancy was assessed retrospectively at inclusion  by using a validated food-frequency questionnaire (FFQ) . This FFNo food composition database was available to estimate direct intake of the nutrients implicated in OCM based on the EDEN FFQ items. Therefore, we used different sources for the food composition database and established correspondence with the Individual and National Food Consumption Survey (INCA 2)  to use fWhen we matched the EDEN FFQ food items and the INCA2 food items, we sometimes had to combine several INCA2 items to characterize a more generic EDEN FFQ one . In these cases, we used information for the frequency of food consumption by adult French women 18 to 50 years old for each of these food items in the INCA2 study  to compuThese weights were then applied in estimating each nutrient or vitamin intake per EDEN FFQ item see .z-scores for birth weight, length, and head circumference according to the French Audipog reference [We collected parity, gestational age, child sex, and anthropometry at birth from medical records. We calculated eference , which a2 (m2) and classified into 4 categories according to the World Health Organization thresholds: underweight (<18.5 kg/m2), normal weight (18.5 to <25 kg/m2), overweight (25.0 to <30 kg/m2), and obese (≥ 30 kg/m2). Sociodemographic and pregnancy characteristics were recorded by interviews with the mothers by a midwife investigator at 6 months of pregnancy and after delivery. Maternal characteristics studied were country of birth , maternal age , employment status , maternal education level  according to the International Standard Classification of Education ), livingInformation on dietary supplements was collected from maternal self-completed questionnaires: at inclusion for supplementation in the 3 months before pregnancy and after delivery for supplementation during pregnancy. We had information on consumption of mixed multivitamins/minerals or the use of single micronutrients, among which we selected vitamins B2, B6, and B9 (folic acid).We classified the variable “vitamin supplementation” into the following four categories: no supplementation, supplementation only before pregnancy, supplementation only during pregnancy, and supplementation before and during pregnancy. n = 37), with more than 3 items missing (N = 155), with implausible food group values (N = 2), or with extreme total energy intake ≤ 1000 or ≥ 5000 kcal/day (N = 101) were excluded from the RRR analysis. Those for whom infant birth weight was not available  were excluded (N = 69) from subsequent analyses. Mothers who did not complete the FFQ (We performed multiple imputation of missing data for confounders and outcomes (birth length and head circumference) . We inclThe RRR method was used to determine the linear combinations of predictors (food groups) that explain as much as possible of the variation in the response, or nutrients variables . Before using the RRR method, the 137 food items in the EDEN cohort were grouped into 36 major food groups or specific food items for those that were particularly rich in specific OCM nutrients, such as whole grain bread, avocado, and broccoli . More thWe used the 36 food items as predictor variables and the log-transformed nutrient variables  as response variables. Confounding by total energy intake is a major point to address because women who consume more energy may also have higher consumption of specific nutrients . Therefop < 0.05 considered statistically significant.Characteristics of mothers and their children are described with mean ± SD and n (%) before imputation. Unadjusted associations between demographic, socioeconomic and health maternal characteristics, and the scores for each selected dietary pattern were tested by ANOVA, with p > 0.05).We analyzed the association between selected dietary pattern scores and birth weight, length, and head circumference z-scores  and gestational age by multivariable linear regression. Because the effect of micronutrient intake might differ whether or not they were provided by supplementation, interaction terms between the pattern scores and maternal supplement intake before pregnancy were systematically tested . We also tested interactions terms between each pattern scores and offspring sex . No interaction terms were significant neither for birth weight nor for birth length  or LGA (as compared with non-LGA), and prematurity. We adjusted gestational age and prematurity for infant sex. Betas (β) or odds ratios (ORs) and 95% confidence intervals (CIs) were calculated per 1 SD of the dietary pattern score. Potential confounding factors were selected from the literature and their bivariate associations with our factors of interest. Therefore, all models were adjusted for center, maternal education level, maternal age, employment status, monthly household income, parity, smoking during pregnancy, BMI, and vitamin supplementation before and during pregnancy if appropriate. Maternal BMI was taken into account with a power three factor. As a complementary analysis, we used dietary pattern score quintiles to illustrate the shape of the association with birth size. Finally, as a sensitivity analysis, we re-ran all the analyses restricted to complete cases .N = 1638) are summarized in The characteristics of the women selected for analysis (Three of the seven patterns resulting from the reduced rank regression explained 58% of the considered variation in intake of nutrient (response) variables . The firp = 0.05).Younger women and those with a lower education level or who were unemployed had lower scores for the “varied and balanced” pattern than their counterparts, whereas women with a high monthly household income had higher scores for this pattern . We founp = 0.02) and birth length (p < 0.01), and positively but not significantly associated with head circumference at birth (p = 0.06) (p = 0.14), birth length (p = 0.08), and head circumference at birth (p = 0.43) (p for trend = 0.06).On unadjusted linear regression, adherence to the “varied and balanced” pattern was positively associated with both birth weight ( = 0.06) . After a = 0.43) . Using q = 0.43) , we founp = 0.05) and high scores on the “varied and balanced” pattern were associated with increased risk of LGA (p < 0.01). These associations were still significant in the adjusted model (p = 0.03). On unadjusted logistic regression, the “bread and starchy food” pattern was associated with reduced probability of SGA (ed model . We founp = 0.05), but the risk of prematurity was not significantly higher (p > 0.05).In adjusted analyses, vitamin supplementation during the three months before pregnancy was not significantly associated with birth size. Gestational length was shorter on average for women with supplementation before pregnancy  and birth length . Consistent results were found in the complete-case analysis, although with weaker associations between the “varied and balanced” pattern and both birth weight  of the variation in intake of these micronutrients before pregnancy. Importantly, our study identified three patterns of maternal diet as the main source of OCM variability. A high score on the “varied and balanced diet” pattern correspond to a high intake of the main OCM micronutrients and a likely adequation with recommended intake for major OCM nutrients. The “vegetarian-tendency” pattern is related to high intake of vitamin B6, B9, and betaine to the detriment of others nutrients such as vitamin B12, choline, and methionine. The “bread and starchy food” pattern, is mainly characterized by betaine intake and a low level of B9 vitamins.The nutrient intake according to dietary patterns that we found is consistent with previous studies showing that a high intake of plant-based foods may lead to high folate levels . The INCThe association of dietary patterns with socioeconomic and other behaviors was consistent with results from other studies of dietary patterns of young pregnant or non-pregnant women ,44,45. CImportantly, our study further focused on dietary patterns that explained the variability in intake of the OCM nutrients combined and fetal development during the pre-conception period, which is a crucial time window for fetal programming. Some studies showed the association of single OCM micronutrients provided by the maternal diet or supplementation with both brain development and optimal birth weight ,8,11,46.Our results suggest little evidence for an association between the “varied and balanced diet” score before pregnancy and birth weight, length, and head circumference. A high adherence to this pattern was also associated with an increased risk of LGA, which might be related more to longer length than increased adiposity. However, we found no relation between scores on the “vegetarian tendency” pattern and anthropometric measurements at birth. A pre-pregnancy varied and balanced diet providing a variety of nutrients may be best to support fetal development. Conversely, the vegetarian tendency pattern, although characterized by high intake of vitamins B6 and B9 also featured low vitamin B12 consumption, which may be less optimal for fetal growth. These results are consistent with studies highlighting the effect of multiple micronutrient supplementation for fetal growth ,19. Our finding of a high adherence to the “vegetarian tendency” pattern associated with reduced risk of prematurity is consistent with a review showing the importance of adequate folate status to avoid risk of prematurity . HoweverThe third pattern, “bread and starchy food,” contributed mainly to the variability in betaine intake and was associated with reduced risk of SGA not modified by adjustments for confounders. This pattern was less associated with maternal characteristics. Conversely, two studies showed that plasma betaine concentration during the second trimester was associated with smaller infant birth size, birth weight, and less abdominal fat mass ,16. Few Although our dietary patterns were driven by the variability in content of OCM nutrients in the maternal diet, other aspects of the diet  may also explain the observed associations, especially for the “varied and balanced” and “vegetarian tendency” patterns. We did not find any association between vitamin supplementation and anthropometry at birth. Only a few women (8%) had vitamin supplementation before pregnancy and maintained this consumption during pregnancy. We did not have information on the duration of supplementation and whether it concerned specific trimesters during pregnancy. This lack of information precluded a more detailed analysis of the different vitamins or minerals used for supplementation. However, we found a positive association between vitamin supplementation before pregnancy and high scores on the “varied and balanced” pattern. A study of the NutriNet-Santé cohort showed that dietary supplement users had a high dietary intake of most vitamins and minerals . Pre-conceptional vitamin supplementation was significantly associated with reduced gestational length, three days on average, with no association with risk of prematurity. This finding may be explained by reverse causation: women more at risk of gestational complications are more likely to be followed before pregnancy and then initiate early vitamin supplementation. This study provides new insights into the characteristics of the diet of women of childbearing age before pregnancy in light of its contribution to the combined intake of micronutrients essential for fetal development. As compared with other dietary pattern approaches, the RRR technique was particularly useful because of its multivariate approach combined with existing knowledge of diet–disease associations and may help provide a novel understanding of the pathways by which diet influences health. Associations between food groups and nutrients can be used to interpret the effects of food groups as components of dietary patterns. We also used a multiple imputation technique to limit bias due to missing data for our variables of interest and confounders.Among the limitations is the questionable accuracy of self-reported data on diet before pregnancy and supplementation before pregnancy and during pregnancy. Women may have under- or over-reported their consumption of specific food groups . However, the accuracy might be similar for information collected by a health professional. In our study, women with disadvantaged social status and possibly more nutritional deficiencies were under-represented, which may explain in part why we did not find stronger associations with fetal growth. Moreover, residual confounding cannot be ruled out, and thus, these results need replication.The role of nutrition in OCM and DNA methylation has been more extensively studied in animal models than epidemiological studies. However, recent human research has indicated that altered consumption of methyl donors may affect the fetal epigenome . One stuThis study identified three patterns describing the variability of the combined intake of OCM nutrients in the diet of French women of childbearing age. High adherence to a pattern associated with the combined intake of all major OCM nutrients may have a small contribution to fetal linear growth. In this sample of French women without large nutritional deficiencies, we did not find any evidence that the variation in intake of micronutrients implicated in methylation-related pathways, have major effect on fetal growth.


Preterm birth is associated with a high risk of morbidity and mortality including brain damage and cerebral palsy. The development of brain injury in the preterm infant may be influenced by many factors including perinatal asphyxia, infection/inflammation, chronic hypoxia and exposure to treatments such as mechanical ventilation and corticosteroids. There are currently very limited treatment options available. In clinical trials, magnesium sulfate has been associated with a small, significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood but no effect on the combined outcome of death or disability, and longer-term follow up to date has not shown improved neurological outcomes in school-age children. Recombinant erythropoietin has shown neuroprotective potential in preclinical studies but two large randomized trials, in extremely preterm infants, of treatment started within 24 or 48 h of birth showed no effect on the risk of severe neurodevelopmental impairment or death at 2 years of age. Preclinical studies have highlighted a number of promising neuroprotective treatments, such as therapeutic hypothermia, melatonin, human amnion epithelial cells, umbilical cord blood and vitamin D supplementation, which may be useful at reducing brain damage in preterm infants. Moreover, refinements of clinical care of preterm infants have the potential to influence later neurological outcomes, including the administration of antenatal and postnatal corticosteroids and more accurate identification and targeted treatment of seizures. Premature birth, defined as birth before 37 weeks completed gestation, represents 11.1% of all live births worldwide. The rate of premature birth increased in almost all countries from 1990 to 2010 [Hypoxia-ischemia (HI) before, during or shortly after birth can contribute to brain injury in at least some preterm infants ,3,4. OldSome of the controversy surrounding the prevalence of asphyxia in preterm infants may stem from the fact that identifying that events have occurred is more challenging than in infants over 36 weeks gestational age, as reviewed in . For exath percentile [In addition to acute HI as discussed above, many preterm infants are exposed to chronic hypoxia before birth, as shown by intrauterine growth restriction (IUGR)/small for gestational age (SGA), defined as birthweight below the 10rcentile ,12. The rcentile , likely rcentile .Preterm infants may also experience intermittent HI after birth, due to conditions such as apnea of prematurity causing repeated periods of mild hypoxia, which are associated with neurodevelopmental and motor impairment . FurtherChorioamnionitis  is associated with 11–40% of all preterm births . The incDuring exposure to intrauterine infection/inflammation, the fetal immune system responds by releasing pro-inflammatory cytokines that induce a fetal inflammatory response syndrome (FIRS) . Fetal sFinally, and not least, the preterm infant is understandably not fully adapted to the ex-utero environment, because of reduced access to suitable nutrition, high oxygen exposure, large blood pressure fluctuations related to fluid shifts, immune system challenges, and exposure to a highly pro-inflammatory environment.Moreover, there are some unique characteristics that contribute to the vulnerability of the preterm brain to injury. Firstly, cell division and maturation are in progress. There is considerable evidence suggesting that pre-oligodendrocytes are particularly susceptible to injury and death . OligodeThe relative vulnerability of the pre-oligodendrocyte to HI compared to the immature oligodendrocyte may, in part, be mediated by greater susceptibility to oxidative stress, as shown in vitro by greater death of pre-oligodendrocytes in response to oxidative stress induced by depletion of intracellular glutathione . PregnanNext, the preterm brain has a comparatively underdeveloped vasculature, which matures relatively late in gestation; these increases in vascular density and cross-sectional area continue well into adulthood ,36. The Infants born preterm are at high risk for neurodevelopmental disorders, including cerebral palsy . The risPreterm infants examined at term equivalent age show widespread changes in white matter diffusivity, indicating compromised white matter integrity, which is often observed in neurodevelopmental disorders such as cerebral palsy . CriticaA key challenge in developing treatment strategies to reduce brain injury in preterm infants is the large number of different injurious phenomena that these infants can be exposed to before, during or after birth. As highlighted above, these include preterm birth itself, developmental vulnerability to injury at a particular gestational age, chronic hypoxia in utero, acute asphyxia around the time of birth, infection/inflammation and poor respiratory function after birth. Further, many therapies required for neonatal intensive care such as ventilation, may decrease mortality but increase risk of brain injury . To dateMaternal glucocorticoid therapy is recommended for cases of threatened or current preterm labor to promote fetal lung development. The safety and benefits of antenatal glucocorticoids were observed in the EPIPAGE cohort study , which sOf greater potential concern, studies in preterm fetal sheep suggest potential deleterious effects after acute HI. For example, maternal dexamethasone given 15 min after fetal asphyxia was associated more severe neuronal loss in the hippocampus and basal ganglia and greater loss of both total and immature/mature oligodendrocytes in the periventricular white matter . In the When a single i.m. injection of maternal dexamethasone was given 4 h before asphyxia, induced by 25 min of complete umbilical cord occlusion in preterm fetal sheep, severe, cystic brain injury was seen, with increased numbers of seizures, worse recovery of brain activity and increased arterial glucose levels compared to diffuse injury after asphyxia alone . ImportaPreterm infants with BPD have a prolonged oxygen requirement after birth, with greatest risk of BPD with decreasing gestational age. Approximately 50% of extremely preterm infants (<28 weeks) and up to 80% of infants born <25 weeks develop BPD ,77. GiveMore recent studies suggest that key factors affecting the relative benefits and risks of postnatal glucocorticoid therapy, include dosing and timing of treatment as well as the underlying relative risk of developing cerebral palsy. High-dose dexamethasone exposure decreased brain weight, delayed neurological development , reducedRecent systematic reviews of the relative benefits and risks of both postnatal early (<8 days) or late (>7 days) glucocorticoid therapy concluded that the benefits may not outweigh the risks of treatment ,66. SystMagnesium is an endogenous anti-excitotoxic agent that acts by binding to the magnesium site on the NMDA receptor, inhibiting these glutamatergic channels . This ha4 before or after HI. However, it is critical to appreciate that magnesium is a potent vasodilator and so can increase heat loss, leading to iatrogenic hypothermia in small animals. Systematic review of the literature suggests no benefit in rodents, piglets and fetal sheep in studies with good temperature control [Experimentally, some studies in neonatal rodents have suggested neuroprotection with MgSO control .4 administered to women in preterm labor found that it was associated with a small but significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood  has a central role in erythropoiesis and has been widely used to prevent postnatal anemia in premature infants . In addirEpo treatment has shown potential to support the restoration of neural structure and function over the long-term recovery from HI. In P7 rat pups, treatment with repeated injections of rEpo (1000 U/Kg) from 48 h after HI, increased neurogenesis and oligodendrogenesis, improved oligodendrocyte maturation, and restored myelination two weeks after HI . SimilarImportantly, rEpo also appears to be beneficial in models of preterm infection/inflammation. A single injection of rEpo (5000 IU/Kg) after maternal LPS injection in rats at 18–19 days gestation was associated with reduced IL6, IL1 and TNF-α concentrations, apoptosis and demyelination at p7 . In pretClinically, phase 1 and 2 trials have provided strong evidence of the safety of rEpo treatment in low birth weight infants, prematurely born infants and full-term neonates with HIE ,123. In One potential solution to the need for frequent dosing with rEpo could be the use of darbepoetin (Darbe), the long-acting erythropoiesis-stimulating agent, which has been shown to have a half-life that is 3-fold longer than rEpo in term neuroprotection studies ,125. In Mild therapeutic hypothermia induced by cooling the head or body of an infant is now routine clinical care for term infants suffering HIE ,128. TheThere is compelling clinical and experimental evidence that therapeutic hypothermia can reduce neuronal loss and improve neurological outcome in term and near-term infants with moderate to severe HIE ,128. MetPreclinical evidence suggests that hypothermia is also effective at reducing brain damage in the preterm brain. For example, mild cerebral hypothermia started 90 min after asphyxia induced by complete umbilical cord occlusion and continued for 3 days was associated with a marked reduction in loss of neurons and immature oligodendrocytes, restored carotid artery blood flow and EEG frequency to sham control levels in 0.7 gestation fetal sheep . SimilarAlthough it seems plausible that hypothermia will be effective at reducing brain damage in the preterm brain after an acute hypoxic–ischemic event such as perinatal asphyxia, concerns remain regarding its safety. Mild hypothermia (33−35 °C) has been associated with slowing of the atrial pacemaker and intracardiac conduction, with sinus bradycardia, decreased left ventricular contractility and cardiac output, as reviewed in . A smallThe Preemie Hypothermia for Neonatal Encephalopathy trial (NCT01793129) is currently active, investigating the use of mild therapeutic hypothermia for preterm infants between 33 and 35 weeks gestational age who present at less than 6 h postnatal age with moderate to severe neonatal encephalopathy . Infants are randomized to receive 72 h of whole-body hypothermia or normothermia.N-acetyl-5-methoxytryptamine) is a naturally occurring indolamine secreted by the pineal gland to regulate circadian rhythm that also has antioxidant properties [Melatonin  in conjunction with therapeutic hypothermia, significantly improved recovery of aEEG activity, improved cerebral energy metabolism seen using magnetic resonance spectroscopy and reduced TUNEL-positive cell death after asphyxia . HoweverPretreatment with maternal melatonin has been shown to be beneficial in a mouse model of maternal inflammation, with reduced preterm birth and preterm brain injury seen when pregnant mothers of embryonic day 16.5 mice received melatonin before the induction of inflammation using lipopolysaccharide . A limitMelatonin given in combination with erythropoietin prevented post-hemorrhagic hydrocephalus of prematurity in rat pups . In thisMaternal pre-treatment with melatonin has been shown to reduce preterm birth and perinatal brain injury in a mouse model of maternal inflammation induced by lipopolysaccharide . MelatonSmall clinical studies suggest that melatonin is not associated with adverse outcomes and may improve survival of neonates with septic shock and reduce lung injury associated with ventilation in preterm infants ,147. A sMaternal vitamin D deficiency appears to be a significant risk factor for preterm birth ,150, priVitamin D plays several important roles during development. Vitamin D has been shown to influence normal fetal brain development, including cell differentiation, neurotrophic factor expression, cytokine regulation, neurotransmitter synthesis, intracellular calcium signaling, antioxidant activity and expression of genes and proteins involved in neuronal differentiation, structure and metabolism . MaternaVitamin D supplementation has shown a range of neuroprotective effects in preclinical models of brain injury, including immunomodulatory and anti-inflammatory effects. Indeed, vitamin D has been shown to be a key immunomodulator and regulator of pro-inflammatory Th 17 lymphocytes in adult stroke patients ,169. In Although there is currently no evidence to suggest that vitamin D supplementation is neuroprotective in preterm infants, monitoring vitamin D status should be considered as a focus for future research . FurtherThere is increasing preclinical evidence that “stem” cell  therapy may be a viable neuroprotective strategy for the preterm brain, particularly after exposure to HI or infection/inflammation ,176,177.There are many different stem cells available. hAECs offer a number of practical advantages. They can be readily harvested from the amnion membranes, which surround the fetus in utero and are discarded at birth, therefore they do not require invasive extraction, making them readily available for rapid treatment in the early postnatal phase . hAECs aIn adult mice, hAECs have been shown to reduce infarct development, reduce inflammation and functional deficits after stroke, while in the adult marmoset, hAECs also prevented infarct development . FurtherhAECs also appear to be neuroprotective in the preterm brain after asphyxia. Delayed intranasal infusion of hAECs, given at 24 h and 3 and 10 days after 25 min of complete umbilical cord occlusion, was associated with improved brain weight, improved oligodendrocyte maturation and myelination and reduced microglia and astrocyte number after 21 days recovery in the preterm fetal sheep . FurtherUCB contains a diverse mix of stem cells and progenitor cells, with the potential to generate a wide variety of cell types . AdvantaEarly preclinical trials in neonatal rat pups showed that UCB administration at 24 h post-insult, decreased reactive gliosis and normalized connexin 43 expression, increased tissue repair and cognitive improvements and enhanced endogenous neural stem cell proliferation after HI induced by the Rice-Vannucci model of unilateral carotid ligation and inhalational hypoxia ,193,194.Interestingly, the gestational age at which UCB cells are harvested may have an impact on the neuroprotective mechanisms. When preterm fetal sheep received UCB cells from either term or preterm sheep, both reduced white matter injury, cell death and microgliosis. However, only preterm cord blood prevented upregulation of plasma tumor necrosis factor alpha, while term cord blood increased the anti-inflammatory cytokine Il-10 and reduced oxidative stress .Preterm birth is associated with a high burden of neurological impairment and lifelong disability. Developing therapies for brain injury in the preterm infant is particularly complex given the wide range of potentially injurious phenomena that preterm fetuses and infants may be exposed to before, during and after birth, including chronic antenatal and postnatal hypoxia, perinatal asphyxia, prenatal and postnatal infection/inflammation, poor respiratory function after birth, ventilation-induced brain injury and a variety of treatments with potential side effects, including anticonvulsants and antenatal and postnatal glucocorticoids. It is important to appreciate that each individual infant may be exposed to different combinations of these factors. The heterogeneous nature of preterm brain injury presents many challenges in the quest to develop treatment strategies for these infants. It is likely that different treatment strategies may be more effective in treating brain damage stemming from particular types of insults. For example, it is plausible that therapeutic hypothermia may alleviate brain damage after acute perinatal asphyxia but not chronic hypoxia. The multifactorial nature of preterm brain injury also presents a major challenge to the development of suitable preclinical models. Many preclinical studies, particularly those in large animal translational models focus on brain injury induced by acute asphyxia or infection/inflammation but studying the pathology associated with preterm birth itself or combinations of these factors is practically difficult in these paradigms. This has implications for the translation of potential new therapies into clinical trials where infants are likely to have been exposed to a variety of these potentially damaging factors.Current treatments for preterm infants that may have beneficial effects on neurological outcome include antenatal glucocorticoids, which in addition to improving respiratory function, have been shown to reduce the incidence of IVH in preterm infants. The use of postnatal corticosteroids remains controversial due to concerns around increased risk of cerebral palsy but this appears to be influenced by dose and timing of administration. The use of anticonvulsants to treat seizures in the preterm brain remains controversial, due to poor efficacy and concerns around adverse effects in the developing brain raised by preclinical studies. Better understanding of the cellular mechanisms underlying seizure activity in the developing brain may aid in the development of safer and more effective anticonvulsant treatments for preterm neonates. There is conflicting evidence around the efficacy of MgSo4 for reducing preterm brain injury, with its use having been shown to be associated with a small but significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood but no overall improvement in death or disability and studies with long-term follow up to school age to date have shown no long-term benefit.A number of promising neuroprotective therapies are currently being investigated for use in the preterm brain. Preclinical studies suggest that therapeutic hypothermia is effective at reducing brain damage after asphyxia in the preterm brain but concerns remain around its safety. The results of the randomized controlled trial of therapeutic hypothermia in preterm infants are eagerly awaited to determine whether it is safe and effective for use in preterm infants who may have been exposed to a period of asphyxia around the time of birth. Further, preclinical studies suggest that recombinant erythropoietin may be a useful treatment option to reduce brain damage in the preterm infant but this has not yet translated successfully in clinical trials, perhaps due to the frequency of treatment. Preclinical studies suggest that melatonin, although the evidence for benefit is confound by the common use of ethanol as a diluent, human amnion epithelial cells and umbilical cord blood cells may also be viable treatment options for preventing brain damage in preterm infants. However, to ensure the greatest chance of these potential neuroprotective treatments translating into clinical practice, well-designed research in a range of translational models are essential to guide the development of evidence-based dosing regimens for use in clinical trials to ensure that they have the highest chance of being successful.Despite the challenges associated with studying preterm brain injury and the development of treatment strategies, there are now a number of potential neuroprotective treatments showing promise in preclinical studies and/or in clinical trials. With further well-designed, translational research, these treatment strategies may help to reduce the high burden of brain damage and disability associated with preterm birth.


Epidermoid and dermoid cysts in the floor of the mouth are uncommon. A large cyst can lead to functional morbidities including airway, swallowing and speech problems. The treatment of choice is surgical excision. To obtain a good result, the decision for optimal surgical technique is valuable.A young female patient presented with a slowly growing mass at the submandibular area and swelling in the floor of mouth. The MRI showed large sublingual cyst extended posteriorly to the parapharyngeal space. She underwent surgical excision of the cyst size 6.5 × 3.2 × 2.5 cm via an intraoral approach. The pathological examination revealed a benign stratified squamous epithelium-lined cyst with no evidence of adnexal structure consistent with an epidermoid cyst. The patient remained disease-free after two years of follow-up.Generally, large sublingual cysts over 6 cm located or transgress below the mylohyoid muscle are removed by an extraoral approach. Our team reported the first case of a large lateral sublingual epidermoid cyst with parapharyngeal extension removed by an intraoral approach. Preoperative imaging is important for surgical approach selection.Successful management of sublingual epidermoid cyst extended to the parapharynx is feasible by an intraoral approach with excellent functional and cosmetic outcomes. The pathogenesis is the entrapment of the ectodermal tissue along embryonic fusion lines. The clinical presentation is a progressive swelling in the floor of the mouth and neck. If large enough, the cyst can potentially compromise the airway, speech, and swallowing. The main treatment is surgical excision by either intraoral or extraoral approach depend on the size and location of the cyst.We present the case of a large sublingual epidermoid cyst with parapharyngeal space extension excised by an intraoral approach. The patient was managed at our academic institution. The present case is reported in accordance with the SCARE criteria .2A 26-year-old-female presented with a painless, slowly growing mass over 6 months at the left submandibular area. She reported no increased swelling of the mass or pain during eating. Her speech and swallowing were normal. She had taken two courses of antibiotics from other hospitals, but her symptoms persisted. There was no previous history of surgery or trauma to the oral cavity or neck. Clinical examination revealed a well-defined, firm, non-tender mass size 4 × 4 cm in the left submandibular area. Bimanual palpation of the left submandibular gland showed no stone or pus from the Wharton’s duct. Oral cavity examination revealed swelling in the left floor of mouth with normal tongue movement. We scheduled her for MRI imaging and fine-needle aspiration (FNA) for further investigation. The MRI showed a well-defined hypointense T1 and hyperintense T2-weighted image cystic lesion at the left sublingual space above the mylohyoid muscle that extended to the left submandibular and left parapharyngeal space displacing the left submandibular gland downward . The pre3Dermoid cysts in the floor of the mouth are uncommon entities. New and Erich et al. found 7% of dermoid cysts in the head and neck area of which only 1.6 % occurred on the floor of the mouth . The floAnatomically, the floor of mouth dermoid cyst can be divided into three regions as sublingual, submental and lateral according to the anatomic relationship between cyst and floor of mouth muscle. The majority of the cysts (78 %) are in the midline, 16 % involve more than one space and only 6% are located in the submandibular space . The sigMRI imaging allows us to define the location of the cyst and its relationship to geniohyoid and mylohyoid muscle. This information is important for selecting the optimal surgical approach. Dermoid cysts are isointense or hypointense to muscle on T1-weighted image and hyperintense or heterogeneous on T2-weighted image.Fine needle aspiration cytology is useful in differentiating the cyst as it did in our case. However, aspiration of the high viscosity content cyst may be difficult and inadequate for interpretation. A definite pathologic examination is essential for confirming the diagnosis.Surgical excision is the treatment of choice. An aspiration to decompress the cyst before endotracheal intubation may be useful for a very large cyst obstructing the airway. The surgical approach can be an intraoral or extraoral approach depending on the size and location of the cyst. The intraoral approach is recommended for a small to a medium sublingual cyst which is less than 6 cm in size and above the mylohyoid muscle, whereas an extraoral approach is appropriate for large cysts over 6 cm located or transgress below the mylohyoid muscle . Althoug4The epidermoid and dermoid cyst should be considered one of the differential diagnoses in the patient presented with a cystic mass in the floor of mouth or neck mass. Preoperative imaging is important for localizing the lesion and determining the surgical approach. Surgical excision by an intraoral approach is feasible and has an excellent cosmetic outcome.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.This research has been approved by Research Ethics Committee 4, Faculty of Medicine, Chiang Mai University.Study Code : ENT-2563-07145 / Research ID : ENT-2563-07145Patient/guardian consent was obtained for publication of this case report.Hanpon Klibngern: Conceptualization, Investigation, Resources, Manuscript writing and revisionChanisa Pornchaisakuldee: Manuscript writing and revision, Manuscript submission1. Name of the registry: -2. Unique identifying number or registration ID: -3. Hyperlink to your specific registration (must be publicly accessible and will be checked): -Hanpon KlibngernNot commissioned, externally peer-reviewedThe authors declare that we have no conflict of interest.


Background: Preterm birth remains a major cause of neonatal morbidity and mortality. Several potential pathways and pathophysiologic processes can lead to preterm birth, complicating efforts to screen for the risk of preterm birth and making implementation of prevention strategies difficult.Methods: Based on a review of the literature, this article addresses screening strategies for preterm birth risk stratification and interventions for preterm birth prevention.Results: In women with a history of a prior spontaneous preterm birth, cervical cerclage placement in the setting of short cervix reduces the rate of recurrent spontaneous preterm birth. Weekly injections of 17-hydroxyprogesterone caproate (17-P) have been used as standard treatment for the prevention of recurrent preterm birth since 2011. However, results of a replication study of 17-P published in 2020 have raised questions regarding the effectiveness of this drug, and it is under review by the US Food and Drug Administration. Among women with no history of preterm birth, cervical length appears to be the best predictor of risk for preterm birth in asymptomatic women. In women with a cervical length <25 mm, vaginal progesterone has been demonstrated to reduce the risk of preterm birth.Conclusion: Strategies including cervical length screening, vaginal progesterone administration, cervical cerclage placement, and, potentially, 17-P administration may help reduce rates of preterm birth when used in the appropriate patient populations. Development of protocols for patient evaluation and risk stratification will help identify patients at highest risk for preterm birth and allow use of the best available therapeutic interventions. While the rate of preterm birth declined nationally from 2007 to 2014, the rate rose to 9.93% in 2017.7 In Louisiana, the rate of preterm birth at <37 weeks of gestation was 12.7% in 2017. Other states in the southeastern United States have similar challenges with high rates of preterm birth.7Preterm birth remains the most common cause of neonatal morbidity and mortality in the United States.10 Therefore, other strategies are needed to identify women at highest risk in the absence of a history of prior preterm birth.Although the rate of preterm birth has risen, some interventions have been demonstrated to have a potential role in reducing the likelihood of recurrent preterm birth in women with prior preterm births. Even with these interventions, however, the impact on the overall rate of preterm birth is minimal because 90% of deliveries prior to 34 weeks of gestation and 85% of deliveries prior to 37 weeks of gestation occur in women with no previous history of preterm birth.The focus of this review is the current (2020) data on interventions used to reduce the risk of recurrent preterm birth in women with a prior spontaneous preterm birth, as well as risk assessment methods and interventions to reduce the risk of preterm birth in the obstetric population with no history of prior preterm birth.4 While classification of preterm birth has historically been limited to births that occur between 20 weeks 0 days and 36 weeks 6 days of gestation, many of the etiologies of deliveries at 16 weeks 0 days to 20 weeks are similar to those of later preterm births, and these women appear to have a high risk of recurrent preterm birth.12 While the true definition of preterm birth applies only to births occurring at 20 weeks of gestation or later, based on the similar etiologies and the high risk for recurrence, preterm birth screening and prevention approaches have been recommended for women with a prior preterm birth as early as 16 weeks’ gestation.13Preterm birth has typically been classified as either spontaneous, which includes preterm labor and premature rupture of membranes, or indicated because of a maternal or fetal complication. Approximately two-thirds of preterm births are spontaneous.8 Spontaneous preterm birth recurs in 35% to 50% of women, and the probability of spontaneous preterm birth increases with the number of prior spontaneous preterm births.14 Several other risk factors have been associated with spontaneous preterm birth, including non-Hispanic Black race, low socioeconomic status, midtrimester cervical length <25 mm, history of dilation and evacuation procedure, maternal smoking, late prenatal care, uterine overdistension, decidual hemorrhage, and short interpregnancy interval.19The strongest predictor of spontaneous preterm birth is a history of spontaneous preterm birth.21 A further hypothesis suggests that differences in risk-predisposing genetic variant frequencies may partly explain differences in rates of preterm birth among women of different races and ethnicities.25Despite the magnitude of the problem, the underlying etiologies of spontaneous preterm birth remain poorly understood. Strong evidence suggests a genetic contribution to spontaneous preterm birth. Women who were born prematurely are at increased risk to have a spontaneous preterm birth.26 The prevalence of short cervix in women without a prior spontaneous preterm birth ranges from 1.7% to 7.9%.28 Risk stratification with cervical length measurement has, therefore, been suggested to help identify potential candidates for intervention.As already noted, the majority of preterm births occur in women who have no history of prior preterm birth—that is, they occur in women who are nulliparous or have had prior term births. Development of short cervix has been associated with subsequent preterm birth, and midtrimester cervical length assessment by transvaginal ultrasound is the best clinical predictor of subsequent spontaneous preterm birth.29 Women with singleton gestations with a cervical length ≤25 mm at 24 weeks of gestation have a 6-fold increased risk of preterm birth, and the risk is along a continuum with the risk of preterm birth increasing as cervical length decreases.29 In one study of unselected pregnant women at 22 to 24 weeks of gestation, only 1.7% had a cervical length <15 mm, but these women accounted for 86% of preterm births at <28 weeks of gestation and 58% of preterm births at <32 weeks of gestation.28A cervical length measurement below the 10th centile for gestational age has been used as an arbitrary cutoff to diagnose short cervix. At 18 to 24 weeks of gestation, the 10th centile corresponds to a cervical length <26 mm.32 A 2017 observational study of nulliparous women reported that a cervical length cutoff of 25 mm identified only 23.3% of women with spontaneous preterm birth at <37 weeks of gestation.27 The specificity of short cervical length is also related to the cutoff used. In an unselected cohort and using a cervical length ≤20 mm, the specificity was 99.9%  for preterm birth at <34 weeks, but specificity decreased to 90.1% (95% CI 89.0-91.2) for a cervical length ≤30 mm.32 Regardless of the cutoff used, the positive predictive value of short cervix remains relatively low.The positive and negative predictive values of transvaginal cervical length screening for preterm birth are highly dependent on the population being screened, especially at later gestational ages. The positive predictive value of a cervical length <25 mm in identifying subsequent preterm birth at <34 to <35 weeks is good (as high as 81%) in high-risk cohorts of women with a prior spontaneous preterm birth, but it is only fair in the general obstetric population, ranging from 26.3% to 39.1%.33Despite these limitations, the finding of short cervical length, irrespective of prior pregnancy history, has been consistently associated with an elevated risk of spontaneous preterm birth. No universal agreement on a threshold for diagnosis of short cervix has been reached; most clinicians use 15, 20, or 25 mm as the threshold at which to offer treatment for women with no history of prior preterm birth. As of 2020, neither the American College of Obstetricians and Gynecologists (ACOG) nor the Society for Maternal-Fetal Medicine (SMFM) recommends a universal cervical length screening for all patients but instead state that cervical length screening can be considered as part of a strategy for preterm birth prevention.40 Studies of pessary and cerclage have produced conflicting results, while 17-P treatment has failed to demonstrate benefit when prescribed for the indication of cervical shortening. Results have, however, been consistently more salutary for vaginal progesterone.Given the association of cervical shortening with preterm birth, several interventions aimed at reducing the risk of preterm birth have been investigated, including 17-hydroxyprogesterone caproate (17-P), cerclage, pessary, and vaginal progesterone.36 Two smaller studies produced conflicting results on the efficacy of 17-P in the setting of short cervix, with one study demonstrating benefit similar to vaginal progesterone and the other demonstrating no reduction in preterm birth.42 In aggregate, in the absence of a history of prior preterm birth, 17-P has not been consistently demonstrated to reduce the occurrence of spontaneous preterm birth, even in the setting of short cervical length.In a large multicenter trial, women with a cervical length <30 mm at 16 to 22 weeks were randomized to receive weekly injections of 17-P or placebo. The rate of preterm birth was similar between groups , and no improvement was seen in neonatal outcomes.43Cervical cerclage, an operative procedure in which a suture is placed in purse-string fashion around the cervix, is most traditionally used for the prevention of recurrent cervical insufficiency (painless dilation). Cerclage has historically been reserved for patients with a diagnosis of cervical insufficiency. However, cervical insufficiency is now recognized to be along a spectrum, with some women demonstrating cervical shortening prior to painless dilation or clinically apparent signs such as labor or rupture of membranes.48 In a study published in 2004, 253 women with very short midtrimester cervical lengths (<15 mm) were randomized to cerclage (n=127) vs expectant management (n=126); the incidence of preterm birth at <33 weeks’ gestation was not improved with cerclage placement .48 A meta-analysis of 5 randomized trials that included 419 asymptomatic women with cervical length <25 mm and no prior preterm birth (n=224 in the cerclage group) found that cerclage placement did not prevent preterm birth.49 These studies excluded women with visible membranes, and these findings therefore may not extend to this subgroup of women. However, a 2018 retrospective study examined the utility of cerclage placement in women being treated with vaginal progesterone because of short cervix who developed further cervical shortening to <10 mm. The cohort who received vaginal progesterone plus cerclage delivered 7 weeks later than those treated with progesterone alone , and the rate of spontaneous preterm birth was reduced even after controlling for confounders .50 Neonatal outcomes, including neonatal intensive care unit admission, respiratory distress syndrome, and necrotizing enterocolitis, were all significantly improved as well. Given the conflicting results in these studies, the role of ultrasound-indicated cerclage placement in women without a history of preterm birth who develop further cervical shortening despite treatment with vaginal progesterone needs additional investigation. Neither the ACOG nor the SMFM provides recommendations for this subgroup of patients.While compelling data demonstrate the benefit of cerclage in the setting of short cervix among women with a prior preterm birth, in the absence of a history of prior preterm birth, cerclage has not generally been demonstrated to result in a reduction of preterm birth.52As cervical length screening has become more common, interest has grown in nonpharmacologic and nonsurgical interventions to reduce preterm birth. The Arabin pessary, a silicone ring-shaped pessary that encircles the cervix, has been studied. Putative mechanisms of action include alteration in the uterine-cervical angle, displacement of the weight of the gravid uterus, prevention of opening of the internal cervical os, and protection of the cervical mucus plug.54 Goya and colleagues randomized 385 women with singleton gestations and a cervical length ≤25 mm to Arabin pessary or usual care between 18 and 22 weeks of gestation. Women treated with a pessary had an 82% reduction in preterm birth at <34 weeks compared to the usual care group , as well as a similar reduction in adverse neonatal outcomes .35 In a study funded by the Fetal Medicine Foundation, 932 women with a cervical length ≤25 mm were randomized to pessary or usual care.53 Cervical length was measured at 20 weeks 0 days to 24 weeks 6 days. Among the women enrolled in the trial, 16.5% had a history of prior preterm birth. Women in both treatment groups who had a cervical length ≤15 mm were prescribed 200 mg vaginal progesterone nightly . No reduction in spontaneous preterm birth was seen at <34 weeks , and no difference was noted in adverse neonatal outcomes  with pessary placement compared to usual care.The results of randomized trials investigating whether placement of an Arabin pessary in women with short cervix reduces the risk of preterm birth have been conflicting.55 However, subsequent to the publication of that meta-analysis, the report of a single-center trial from Italy was published. In that trial, 300 women with no history of preterm birth and a cervical length ≤25 mm at 18 weeks 0 days to 23 weeks 6 days were randomized to pessary or usual care.54 Women with a cervical length ≤20 mm, nearly 90% of the women in the trial, were also treated with 200 mg vaginal progesterone. Treatment with pessary was associated with a 52% reduction in spontaneous preterm birth at <34 weeks , as well as a reduction in adverse neonatal outcomes . Additional studies investigating the cervical pessary are currently ongoing in the United States and elsewhere, but in 2020, use of a pessary for prevention of preterm birth in women with short cervix remains investigational.A 2017 meta-analysis of study-level data on more than 1,400 women, many of whom were included in the previously discussed trials, concluded that for singleton pregnancies in women with a cervical length <25 mm, placement of an Arabin pessary does not reduce the rate of preterm birth at <34 weeks or improve neonatal outcomes.34 A total of 413 women were treated from 24 to 34 weeks’ gestation. The incidence of delivery prior to 34 weeks was reduced to 19.2% in the group that received vaginal progesterone vs 34.4% in the placebo group . Eighty-five percent of the women included in the Fonseca et al study had no prior history of preterm birth. In a subgroup analysis of these women without a history of preterm birth, a significant reduction in preterm birth rate at <34 weeks was noted in women with short cervix (≤15 mm) who received progesterone .The efficacy of vaginal progesterone in women with a sonographic diagnosis of short cervix has been demonstrated by 2 multicenter, randomized controlled trials and by independent patient-level meta-analyses that included data from these studies and several smaller trials. Fonseca and colleagues conducted a double-blind trial that randomized women with a cervical length ≤15 mm to 200 mg vaginal progesterone or placebo.37 Moreover, the study demonstrated a neonatal benefit, with a significant reduction in respiratory distress syndrome . Only 16% of the study population had a history of prior preterm birth, and even after excluding these subjects, progesterone was still associated with a significant benefit in the setting of isolated short cervix .In the PREGNANT trial, Hassan and colleagues reported that administration of vaginal progesterone gel (90 mg) to women with a cervical length of 10 to 20 mm identified at 19 weeks 0 days to 23 weeks 6 days of gestation resulted in a significant reduction in the rate of preterm birth at <33 weeks of gestation  and at <35 and <32 weeks of gestation.38 Two of the 5 studies included in the meta-analysis specifically enrolled women with short cervix, while the women from the other studies represent the short cervix subgroup enrolled in the total study patient population.58 The meta-analysis reported a reduction in the risk of preterm birth at <32 weeks of gestation  with vaginal progesterone treatment; preterm births at <28, <34, and <37 weeks of gestation were significantly reduced as well. In addition, the meta-analysis showed a reduction in composite neonatal morbidity and mortality , as well as a reduction in birthweight <2,500 g and <1,500 g.38 This meta-analysis included data on 128 women with a cervical length of 21 to 25 mm; in this subgroup, the reduction in preterm birth was not statistically significant .A 2018 meta-analysis incorporated data on 974 singleton gestations with a cervical length ≤25 mm.33 In addition, with evidence of the benefits of vaginal progesterone administration in the setting of short cervix and its cost-effectiveness, some authors have recommended universal cervical length screening for asymptomatic women without a prior preterm birth.61 The cost-effectiveness of such recommendations, however, is founded on a single, not serial, cervical length measurement at the time of the anatomy ultrasound examination. While treatment with vaginal progesterone is not associated with any significant risks, its use for the indication of short cervix is off-label in 2020.While these data are compelling, treatment with vaginal progesterone following the diagnosis of short cervix and the threshold of cervical length at which to initiate treatment remain areas of debate. The US Food and Drug Administration (FDA) did not approve vaginal progesterone for the indication of preterm birth prevention in the setting of short cervix, in part because data from the PREGNANT trial failed to demonstrate a benefit when only US patients were analyzed. In addition, the FDA declined approval because vaginal progesterone did not appear to be effective in Black or obese women. Despite debate about the clinical utility in all subgroups, given the data on the potential benefit and lack of harm, the ACOG and SMFM have recommended vaginal progesterone as a management option for women with short cervix.62 Vaginal gel formulations do not contain peanut oil and can be used by women with peanut allergies.63 Other contraindications to vaginal progesterone treatment include the typical contraindications to hormonal therapy, such as hormone receptor–positive breast cancer.Many formulations of micronized progesterone contain peanut oil in the excipients . Women with severe peanut allergies, such as anaphylaxis, should not receive micronized progesterone capsules.64 In February 2011, the FDA approved 17-P for the prevention of recurrent preterm birth, and use of this agent for prevention of recurrent preterm birth became the standard of care in the United States.The National Institute of Child Health and Human Development (NICHD) Maternal-Fetal Medicine Units Network conducted a multicenter double-blind randomized controlled trial of 463 women with a singleton pregnancy and prior spontaneous preterm birth between 16 and 36 weeks who received 17-P or placebo. Treatment with 17-P was associated with a 34% reduction in recurrent preterm birth at <37 weeks of gestation , as well as significant reductions in early preterm birth at <32 and <35 weeks and significant reductions in infant complications .65 A total of 1,708 women with a prior spontaneous preterm birth were enrolled and randomized to either 17-P or placebo. The study showed that 17-P administration did not reduce the rate of preterm birth at <37 weeks of gestation , nor did it reduce neonatal morbidity .66 This study could not recruit well in the United States because 17-P was already on the market and incorporated into the standard care for women with prior preterm birth, resulting in significant demographic and risk differences between the PROLONG trial and the NICHD study. Only 22% of patients enrolled in PROLONG were from the United States; 61% were from Russia and Ukraine. In addition, only 1.1% of patients had a cervical length <25 mm and only 7% of the patients were Black—two of the greatest risk factors for preterm birth. These differences in study populations and the conflicting results of the 2 trials have introduced considerable uncertainty and controversy into the management of patients with prior preterm birth. In October 2019, the FDA convened an advisory panel to review the data on 17-P. The panel voted 9-7 to recommend that the drug be removed from the market.67 In October 2020, the FDA Center for Drug Evaluation and Research proposed withdrawal of 17-P from the market. The final decision is subject to potential additional public hearings and ruling from the FDA Commissioner.68As one of the conditions for market approval, the FDA required the manufacturer of 17-P to conduct a replication trial. The multisite international PROLONG  trial used the same study design, inclusion criteria, and exclusion criteria as the previously noted NICHD trial.69 Given the preponderance of data in a US population, the SMFM states that women with a singleton gestation and a history of prior spontaneous preterm birth between 20 and 36 weeks 6 days of gestation may be prescribed intramuscular administration of 250 mg 17-P weekly, starting at 16 to 20 weeks of gestation until 36 weeks of gestation or delivery. If 17-P is not available or the patient declines the medication, vaginal progesterone may be a reasonable alternative.69The SMFM has advocated that critical differences between the study populations in the original NICHD trial and the PROLONG study account to some extent for the disparate results. A 2020 statement from SMFM concludes that providers can reasonably continue to use 17-P in women with a risk profile similar to that of the enrollees in the NICHD study.70 The incidence of preterm birth was reduced at <37 weeks  and at <34 weeks of gestation . While subsequent studies have produced mixed results, a 2019 meta-analysis readdressed the question of the optimal intervention for women with a prior preterm birth and concluded that vaginal progesterone treatment was associated with a reduction in recurrent preterm birth at <34 weeks  and <37 weeks . 17-P also reduced preterm birth at <37 weeks  and neonatal death .71 Many of the studies in this meta-analysis were conducted outside of the United States, so these findings may not be generalizable to the US population.The utility of vaginal progesterone has been investigated in women with prior spontaneous preterm birth. In 2003, da Fonseca and colleagues reported the results of a double-blind randomized controlled trial of 142 women at high risk for preterm birth (94% had a prior preterm birth) who were treated with either 100 mg vaginal progesterone per day or placebo.73 Traditional history-based cerclages are placed at 12 to 14 weeks’ gestation. Ultrasound-indicated cerclage should be considered for women with a prior spontaneous preterm birth if the cervical length shortens to <25 mm in the midtrimester (16 weeks to 23 weeks 6 days).74 Although some controversy exists regarding optimal cervical length screening regimens, based on the regimen used in the largest randomized trial that demonstrated benefit, women with a prior spontaneous preterm birth at <34 weeks should begin transvaginal cervical length assessment at 16 weeks, with serial measurement every 2 weeks until 23 weeks.74 If cervical length shortens to ≤30 mm during surveillance, the screening interval should be reduced to weekly.44Cervical length is inversely related to risk of recurrent preterm birth and gestational age at delivery, and the transvaginal approach offers better sensitivity to detect cervical shortening than the transabdominal approach.75 of patient-level data derived from 5 randomized trials enrolling a total of 504 women (n=250 cerclage procedures).48 Among women with singleton gestations, a prior preterm birth, and sonographic diagnosis of short cervix, cerclage reduced preterm birth at <37 weeks by 30% and reduced composite perinatal morbidity and mortality by 36%. The number of cerclage procedures needed to prevent 1 perinatal death in this group was estimated to be 20.75The strongest evidence for benefit of cerclage in the setting of short cervix comes from a meta-analysis76 For women who do not develop cervical shortening, additional procedures such as cerclage placement may be avoided. Sonographic monitoring with subsequent ultrasound-indicated cerclage in women with a prior preterm birth has equivalent obstetric outcomes to history-indicated cerclage, but only a fraction (36% to 42%) of the monitoring cohort ultimately requires cerclage.76Furthermore, for women with a history of preterm birth, especially if the diagnosis of cervical insufficiency is questionable, data indicate that they can be safely monitored with transvaginal ultrasound.78 In a secondary analysis of the Owen et al cerclage trial44 that evaluated the subgroup of women with short cervix who were also using 17-P, investigators found a trend toward benefit in the cerclage group.79 This study was not, however, designed or powered to examine the combination therapy or the therapies’ interactions. Thus, although no additional benefit to cerclage has been reported when cervical shortening occurs in women on 17-P,80 the SMFM recommends continuation of 17-P in women who receive an ultrasound-indicated cerclage.81Data regarding the efficacy of combining therapies, such as intramuscular progesterone with or without vaginal progesterone and with or without cervical cerclage, are limited. The utility of combined treatment with history-based cerclage and intramuscular 17-P is unclear, and small retrospective studies have reported mixed results.Preterm birth remains a major public health problem. Strategies including cervical length screening, 17-P administration, vaginal progesterone administration, and cervical cerclage placement may help reduce the rates of preterm birth in specific patient populations. Development of and adherence to protocols for patient evaluation and risk stratification will help identify patients at highest risk for preterm birth and the optimal targets for therapeutic interventions.


Lactobacillus species to be associated with full-term birth whereas an unknown Prevotella species was more abundant in the spontaneous preterm group. Although we studied a very miscegenated population from Brazil, our findings were similar to evidence pointed by other studies in different countries. The role of Lactobacillus species as a protector in the vaginal microbiome is demonstrated to be also a protector of spontaneous preterm outcome whereas the presence of pathogenic species, such as Prevotella spp., is endorsed as a factor of risk for spontaneous preterm delivery.Preterm birth remains the main contributor to early childhood mortality. The vaginal environment, including microbiota composition, might contribute to the risk of preterm delivery. Alterations in the vaginal microbial community structure might represent a risk factor for preterm birth. Here, we aimed to (a) investigate the association between preterm birth and the vaginal microbial community and (b) identify microbial biomarkers for risk of preterm birth. Microbial DNA was isolated from vaginal swabs in a cohort of 69 women enrolled at hospital admission for their delivery. Microbiota was analyzed by high-throughput 16S rRNA sequencing. While no differences in microbial diversity measures appeared associated with the spontaneous preterm and full-term outcomes, the microbial composition was distinct for these groups. Differential abundance analysis showed  According to the World Health Organization, every birth before 37 weeks of pregnancy is considered preterm. Each year, about 15 million babies are born prematurely in the world . PretermThe epidemiological and clinical natures of preterm birth are not yet fully understood , 10, 11.Different microbes also have been correlated with preterm delivery , 22, butThis study was carried out with samples collected from women attending the Hospital de Clínicas de Porto Alegre (HCPA). Experimentation used a convenience sampling strategy. Expecting mothers were enrolled at hospital admission for their delivery between May 2014 and March 2016. All women provided written informed consent to allow their samples to be used in the study. The ethics committee of HCPA approved the study protocol. Exclusion criteria were: (1) HIV or congenital infections,( 2) drug user or alcoholic, (3) urinary tract infections or (4) antibiotic usage in the third trimester of gestation, (5) urogenital infection in the last 3 months, and (6) gestational diabetes.Streptococcus, intrapartum penicillin administration, and delivery mode.Sixty-nine pregnant women were analyzed in this study. Twenty-three of them had spontaneous preterm labor (before 33 weeks of gestation), whereas 29 had spontaneous term labor. Another 17 women had non-spontaneous labor but, due to medical reasons affecting pregnancy, had cesarean delivery before 33 gestational weeks. Those subjects called hereinafter “non-spontaneous preterm” group, were used as a second control because they present a microbial community that might not be associated with spontaneous preterm delivery but have a better match in terms of gestational age with the spontaneous preterm group. All pregnant women sampled on this work had vaginal swab (Sterile Specimen Collection Swabs to collect specimens from soft tissue surfaces-Labor swab®) collected up to 4 hours before labor begins, as described by Roesch and colleagues . Collect4, 0.2 mM dNTP’s, 0.1 μM of both the 806R barcoded primer and the 515F primer, 25 μg of Ultrapure BSA  and approximately 50 ng of DNA template. PCR conditions used were: 95 °C for 5 min; 30 cycles of 94 °C per 45 s denaturation, 56 °C per 45 s annealing, and 72 °C per 1 min extension; followed by 72 °C per 10 min for final extension. Fragments presenting around 400 base pairs from resulting PCR products were purified with the Agencourt® AMPure® XP Reagent , and final concentration of the PCR products was quantified by using the Qubit Fluorometer kit  following manufacturer’s instructions. Finally, reactions were combined in equimolar concentrations to create a mixture composed of amplicon fragments of each sample. This composite sample was used for library preparation with Ion OneTouch™ 2 System with the Ion PGM™ Template OT2 400 Kit Template . The sequencing was performed using Ion PGM™ Sequencing 400 on Ion PGM™ System using Ion 318™ Chip v2 with a maximum of 40 samples per microchip.Microbial DNA was extracted from frozen swab samples as previously described by Roesch et al. . All DNARaw reads were analyzed according to the pipeline proposed by the Brazilian Microbiome Project . A tableMaternal variables were analyzed into the R environment . NumericThe 16S rRNA database was analyzed through the phyloseq  and the Initial insights about general microbial structure were provided by analyses of relative abundance  of the most frequent genera and alpha diversity tests.The differential abundance analysis, applied to find microbial biomarkers of preterm birth, was performed by using the DESeq2  with theStreptococcus (GBS). Although the GBS infection rate was higher in the non-spontaneous preterm group, the incidence of GBS infection was not statistically different between spontaneous preterm and non-spontaneous preterm groups. Most cases of non-spontaneous preterm labor presented preeclampsia, whereas there were no cases in the term group and only three cases in the spontaneous preterm labor group. The intrapartum penicillin was used in 15 preterm cases. The term and non-spontaneous preterm groups did not receive prophylactic antibiotics. Intravenous penicillin was administered approximately 4 h before labor in many preterm samples due to a positive test for GBS or due a suspicion of infection in absence of a test. Finally, the three groups differed in terms of vaginal or cesarean delivery. The preterm condition was the major driver of cesarean, especially in the non-spontaneous preterm labor group.The characteristics of the three groups are shown in Table R2 was smaller than 1% and the p value was greater than 0.05. Fifteen out of 23 women from the spontaneous preterm labor group received prophylactic antibiotics during labor, whereas no women from the term group received antibiotics. The R2 for antibiotic usage was 0.034 and the p value was 0.043 indicating that about 3.4% of the variation in the microbial community between groups was explained by the prophylactic use of antibiotics during labor. Intrapartum antibiotics were administrated only in cases with preterm labor. For this reason, this factor could not be used in a multi-factor design with interactions. All OTUs under intrapartum antibiotics influence were removed from the dataset prior to diversity and differential abundance analysis.Permutational analysis of variance was applied to test the effect of confounding variables on the microbiota analyses Table . As signLactobacillus among its samples jointly with a tendency to a high mean of OTUs with best hit to Prevotella and Pseudomonas when compared with terms and non-spontaneous preterm  was found involving either Lactobacillus or Prevotella.To outline the main microbes associated with the term and preterm labor we performed a differential abundance analysis. Pairwise comparisons among spontaneous term and spontaneous preterm groups revealed the abundance of OTUs closest related to two species of th Table . They weapproach . No signLactobacillus, particularly as Lactobacillus iners and Lactobacillus jensenii, might be the main difference between the vaginal microbial community of pregnant women following term or spontaneous preterm labor.The results indicated that the absence of high numbers of OTUs classified as In this work, we attempted to detect biomarkers for preterm labor on the vaginal microbiota of pregnant women. Several studies have described the vaginal microbiota of pregnant women; however, most of them were based in the USA, Canada, Europe, or Mexico , 41–43 aHere, we used next-generation sequencing to analyze a Brazilian cohort composed of 69 pregnant women. The unique feature of this work is the high miscegenation rates of the Brazilian population. The aforementioned studies suggest that women from different ethnic backgrounds have different vaginal microbial communities. Therefore, investigations using cohorts with different ethnic backgrounds are important to better understand the etiology of preterm labor and its relationship with microbes.Vaginal microorganisms possess a known key role in states of health and disease acting as both generators/stimulators and protectors from diseases , 44, 45.Lactobacillus. The second and most strong evidence was obtained by a differential abundance analysis  were associated with the full-term outcome. Non-spontaneous preterm labor presented similar microbial communities composition to those subjects with spontaneous term labor. Indeed, the healthy vaginal microbiota in the Brazilian pregnant woman has low microbial diversity and is dominated by Lactobacillus species [Lactobacillus species are very often correlated to states of health in the vaginal environment [2, formate, or succinate as products [In this context, we presented multiple lines that lead to the presence of different vaginal microbial communities associated with the full-term and spontaneous preterm labor. The first evidence was provided by overall abundance analysis Fig. . Pretermis Table . Lower n species . Besidesironment , 47–50. products . These cproducts , 52, 53.Prevotella in association with the spontaneous preterm labor. In fact, many microorganisms, just like Prevotella species, can produce pro-inflammatory substances that can also lead to a preterm birth [Prevotella strains at different niches. The report by Gupta and colleagues, for example, showed 83% of the Prevotella genome may contribute to singletons and flexible sequences and this condition performs a key role in the adaptation to many body sites [Prevotella strains related dysbiosis in states of disease in highly different body parts, i.e., asthma and bacterial vaginosis [Prevotella is still correlated with inflammatory processes by the activation of Toll-like receptor 2, which leads to production of T helper type 17 cells (Th17) and increase of interleukin 8 (IL-8), interleukin 6 (IL-6), and chemokine (C-C motif) ligand 20 (CCL20) [Mollicutes  [Prevotella [On the other hand, we were able to detect the presence of OTUs closest related to the genus rm birth . Studiesdy sites . Indeed,aginosis , 57. Pre (CCL20) . In addi (CCL20) . The mosexample) , 61, butevotella –64. Thosevotella . The metevotella , 66.L. crispatus was significantly associated with the preterm birth in both cohorts. In line with our results, the cohort from Birmingham, AL, presented decreased abundance of L. jensenii associated with the preterm birth. But contrary to our results, no significant association was detected for L. iners. Moreover, among the women with lower levels of Lactobacillus, a higher abundance of Gardnerella and Ureaplasma was associated with the increased risk of preterm labor. According to Baldwin et al. [Lactobacillus spp. were markedly decreased when compared with vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. As observed in our dataset, the authors also observed deficiency of Lactobacillus and persistence of known pathogenic species, such as Prevotella sp., as a risk factor for preterm birth.Callahan et al.  recentlyn et al. , LactobaPrevotella spp. [Prevotella as a microbial biomarker for preterm labor in the vaginal microbiota.In short, reports from the aforementioned studies as well as from this one converge to a pattern of bacteria either pathogenic or related to stress conditions as increased in preterm cases. Considering this fact and the niche adaptation performed by lla spp. , we are Lactobacillus and the occurrence of other pathogenic taxa  as a possible risk factor for preterm labor.The relationship between the vaginal microbes and the spontaneous preterm labor was already described in racially distinct cohorts. In spite of this, to the best of our knowledge, this is the first study to describe and correlate the vaginal microbiota with the spontaneous preterm labor in a Brazilian cohort. This is especially important because: (i) preterm-microbiota associations are population dependent  and (ii)


To predict spontaneous preterm birth among pregnant women in an African American population using first trimester peripheral blood maternal immune cell microRNA.2; 3) blood sample drawn between 6 weeks to 12 weeks 6 days gestation; 4) live born neonate with no detectable birth defects. Using these entry criteria, 486 samples were selected for study inclusion. After sample quality was confirmed, 139 term deliveries (38–42 weeks) and 18 spontaneous preterm deliveries (<35 weeks) were selected for analysis. Samples were divided into training and validation sets. Real time reverse transcription quantitative polymerase chain reaction (rt-qPCR) was performed on each sample for 45 microRNAs. MicroRNA Risk Scores were calculated on the training set and area-under-the-curve receiver-operating-characteristic (AUC-ROC) curves were derived from the validation set.This was a retrospective nested case-control study in pregnant patients enrolled between March 2006 and October 2016. For initial study inclusion, samples were selected that met the following criteria: 1) singleton pregnancy; 2) maternal body mass index (BMI) <30 kg/mThe AUC-ROC for the validation set delivering preterm was 0.80 , sensitivity 0.89, specificity of 0.71 and a mean gestational age of 10.0 ±1.8 weeks (range: 6.6–12.9 weeks). When the validation population was divided by gestational age at the time of venipuncture into early first trimester  and late first trimester , the AUC-ROC scores for early and late first trimester were 0.79 (95% CI: 0.63 to 0.91) and 0.81 (95% CI: 0.66 to 0.92), respectively.Quantification of first trimester peripheral blood MicroRNA identifies risk of spontaneous preterm birth in samples obtained early and late first trimester of pregnancy in an African American population. The World Health Organization (WHO) has defined preterm birth as those occurring before 37 weeks of gestation . PretermClinically-defined risk factors for preterm birth include a prior history of preterm birth, obesity, hypertension, smoking, diabetes, infection and extremes of maternal age (under 17 or over 40 years old). Laboratory interrogation of urine, cervical mucus, vaginal secretions, serum, plasma and amniotic fluid has been used to supplement clinically defined risk factors to improve prediction of preterm birth. However, the majority of preterm births have no clear risk factor. An individual patient data meta-analysis of 4.1 million singleton births in five high-income countries reported that approximately 65% of all preterm births exhibit none of the 21 pre-specified risk factors  –25. ThesPrevious studies by Winger and Reed unexpectedly identified differentially expressed microRNAs within peripheral blood cells between patients who experienced late pregnancy disorders and those that experienced healthy term births , 33. TheEunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, U.S. Department of Health and Human Services at Hutzel Women’s Hospital, affiliated with the Wayne State University (WSU) School of Medicine and the Detroit Medical Center, Detroit, Michigan. Blood samples were drawn from patients between 6 weeks and 12 weeks 6 days gestational age, prepared as buffy coat by visually-guided pipette aspiration and frozen immediately at -80°C without an RNA preservative. Specimens were maintained frozen for up to eleven years. Specimens were shipped from Wayne State University Detroit, Michigan, USA) directly to the Stanford Human Immune Monitoring Center  on dry ice where they remained blinded as to clinical outcome through testing, identified only by a unique identification number. All patients provided written informed consent, and the use of biological specimens, as well as clinical and ultrasound data for research purposes, were approved by the Wayne State University Human Investigation Committee.This was a retrospective nested case-control study that included samples selected from 11,961 pregnant patients enrolled between March 2006 and October 2016 at the Center for Advanced Obstetrical Care and Research of the Perinatology Research Branch, th percentile and < 90th percentile for gestational age  using the standard proposed by Alexander et al. . ThoughUsing the sample entry criteria described in the Methods section, 486 samples were initially selected for retrospective analysis: 264 healthy, full term deliveries (38.0–42.0 weeks), 69 near term deliveries (37.0–37.9 weeks), 67 spontaneous preterm deliveries without preeclampsia (24.0–36.9 weeks), 77 preeclampsia deliveries (5 were spontaneous preterm deliveries) and 9 medically-indicated preterm deliveries. See To further focus the analysis on preterm birth versus control differences, we included only preterm birth cases < 35 weeks of gestation. After elimination of samples failing to meet these criteria, 299 samples remained for analysis of the original 486. To reduce the number of co-morbidities present in the final analysis, all “non-spontaneous” preterm deliveries (elective C-section), “near term” (37–38 week) or “late preterm” (35–37 week) deliveries and “preeclampsia” deliveries were removed, resulting in 139 control (38–42 week) and 18 spontaneous preterm (<35 week) delivery samples remaining for final study .The 157 patient samples that quantified for final analysis were randomized to two groups. The first group designated as the “training set” included 79 first trimester samples from pregnant patients (70 full term 9 preterm). The second group designated as the “validation set” included 78 first trimester patient samples from pregnant patients (69 full term and 9 preterm); . To helpUsing the 79-sample training set, an AUC-ROC was calculated for each of the 45 microRNAs using Medcalc® Statistical software  . Only miMean gestational age of samples in the validation set was 10.0 ± 1.8 weeks gestation (range: 6.6–12.9 weeks). When the validation population was divided into early first trimester  and late first trimester  groups, the AUC-ROC scores for the early and late groups were 0.79 (95% CI: 0.63 to 0.91) and 0.81 (95% CI: 0.66 to 0.92), respectively.  was 0.80  in specimens collected at a mean of 10.1 ±1.8 weeks gestation (range: 6.6–12.9 weeks).The results of the current research are consistent with previous studies published by our group that have demonstrated that peripheral blood cell microRNA obtained during the first trimester of pregnancy predicts risk of preterm delivery , spontanA clinically useful tool for predicting the risk of preterm birth should demonstrate adequate sensitivity and specificity at a time point when successful intervention can be initiated. Low dose aspirin and/or antiplatelet therapies  have demonstrated efficacy at reducing preterm birth rates and related pregnancy complications, but only when started early in pregnancy , 46. LowPreterm birth rates in the USA are highest in African American infants . The reaIn addition to genetic/epigenetic factors, other covariates may be associated with the predictive value of the 12-microRNA panel. When other covariates  were considered in our current study , most weThough our current study focused on mid to early preterm birth prediction (< 35 weeks gestation), preliminary data suggest that microRNA markers can also predict late preterm birth (> 35 weeks). When our nested study population was expanded (for inquiry purposes) to include preterm birth defined at <36 weeks for both the training and validation sets, the AUC-ROC still remained significant at 0.77,  . The topStrengths of this study include the following: 1) all specimens were blinded to the lab performing the microRNA quantification; 2) all specimens were run in duplicate. Only well-matched duplicates were included in the final calculations; 3) all results were confirmed using training and validation sets; 4) a large number of samples were used given the number of microRNAs tested, increasing statistical power; 5) both early and late first trimester time-frame subgroups yielded similar, yet independent, powerful AUC ROCs, and 6) eleven of the twelve microRNAs most able to identify pregnancy outcome signaled in a common direction, indicating the presence of common (non-random) underlying biology.Limitations of this study include the following: 1) the study was retrospective rather than prospective, 2) frozen samples were used, not fresh; 3) a single patient ethnic group was used (African American) which may limit the generalizability of the results; 4) this was a nested case-control study where disease “prevalence,” is artificially high so the resulting positive/negative and AUC values might not reflect those seen in a natural population; 5) there was no external validation of results; 6) there was a loss of specimens due to (a) elimination of PCR plates that performed poorly and (b) elimination of duplicate specimens that did not match. However, because these performance issues were present in both the preterm and healthy subgroups equally, they should not alter the final conclusions.Quantification of first trimester peripheral blood microRNA predicts risk of spontaneous preterm birth in samples drawn in the early and late first trimester in an African American population. Immune cell microRNA quantification may permit the clinician to institute early intervention at a time when therapy is most effective.S1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.