Evidence suggests that low birth weight and fetal exposure to extreme maternal undernutrition is associated with cardiovascular disease in adulthood. Hyperemesis gravidarum, a clinical entity characterized by severe nausea and excess vomiting leading to a suboptimal maternal nutritional status during early pregnancy, is associated with an increased risk of adverse pregnancy outcomes. Several studies also showed that different measures related to hyperemesis gravidarum, such as maternal daily vomiting or severe weight loss, are associated with increased risks of adverse fetal pregnancy outcomes. Not much is known about long-term offspring consequences of maternal hyperemesis gravidarum and related measures during pregnancy. We examined the associations of maternal daily vomiting during early pregnancy, as a measure related to hyperemesis gravidarum, with childhood cardiovascular risk factors.In a population-based prospective cohort study from early pregnancy onwards among 4,769 mothers and their children in Rotterdam, the Netherlands, we measured childhood body mass index, total fat mass percentage, android/gynoid fat mass ratio, preperitoneal fat mass area, blood pressure, lipids, and insulin levels. We used multiple regression analyses to assess the associations of maternal vomiting during early pregnancy with childhood cardiovascular outcomes.Compared with the children of mothers without daily vomiting during early pregnancy, the children of mothers with daily vomiting during early pregnancy had a higher childhood total body fat mass , android/gynoid fat mass ratio , and preperitoneal fat mass area . These associations were not explained by birth characteristics but partly explained by higher infant growth. Maternal daily vomiting during early pregnancy was not associated with childhood blood pressure, lipids, and insulin levels.Maternal daily vomiting during early pregnancy is associated with higher childhood total body fat mass and abdominal fat mass levels, but not with other cardiovascular risk factors. Further studies are needed to replicate these findings, to explore the underlying mechanisms and to assess the long-term consequences. Several studies also showed that different measures related to hyperemesis gravidarum, such as maternal daily vomiting or severe maternal weight loss, are associated with increased risks of adverse pregnancy outcomes, including preterm birth and small size for gestational age at birth.18 A previous study among 3,165 Dutch mothers and their children suggested that maternal hyperemesis gravidarum, which was measured as severe weight loss during early pregnancy, was associated with a higher blood pressure in childhood.18 No differences were present in childhood body mass index, glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)-cholesterol. In this study, no information about maternal vomiting during early pregnancy was available.Hyperemesis gravidarum, a clinical entity affecting approximately 0.3–3.6% of all pregnancies, is characterized by severe nausea and excess vomiting leading to a suboptimal maternal nutritional status during early pregnancy and is associated with an increased risk of adverse pregnancy outcomes.3 These fetal developmental adaptations may permanently affect fetal growth, fetal adipocyte development and fat deposition and structure, and the function of cardiovascular organs. This may subsequently lead to an increased risk of adverse birth outcomes and obesity and adverse cardiovascular outcomes in childhood and adulthood.20 Therefore, we examined, in a population-based prospective cohort study among 4,769 mothers and their children, the associations of maternal vomiting during early pregnancy with childhood body mass index, total body fat mass, android/gynoid fat mass ratio, abdominal preperitoneal fat mass area, blood pressure, lipids and insulin levels, measured at 6 years. We also explored whether these associations were explained by maternal, birth, or childhood characteristics.Based on these previous findings, we hypothesized that the children of mothers with daily vomiting during early pregnancy may also have a higher risk of adverse cardiovascular outcomes in childhood. Daily maternal vomiting may lead to suboptimal maternal nutritional status, which may subsequently lead to suboptimal fetal nutrient supply and developmental adaptations in response to a suboptimal fetal environment.22 The study was approved by the local Medical Ethical Committee (MEC 198.782/2001/31). Pregnant women were enrolled between 2001 and 2005. Written informed consent was obtained from all participating mothers. In total, 8,879 mothers were enrolled during pregnancy. From our analyses, we excluded those who enrolled after 22 weeks of gestation (n = 581) and those who did not have information on vomiting during early pregnancy available . Of the included women, 6,778 delivered singleton live born children, of which 4,769 mothers and their children had childhood follow-up measurements available  was calculated.24All children were invited for detailed body fat and cardiovascular follow-up measurements at the age of 6. We measured height and weight without shoes and heavy clothing at a dedicated research facility. Height was measured to the nearest millimeter with a stadiometer . Weight was measured to the nearest gram using an electronic scale , and body mass index  was calculated as a percentage of total body weight (kg) measured by DXA. Android/gynoid fat mass ratio was also calculated, which reflects the waist-to-hip ratio.26Total body fat mass was measured with a dual-energy X-ray absorptiometry (DXA) scanner  and analyzed with the Encore software v.12.6.29 Briefly, a linear (L12-5 MHz) transducer was placed perpendicular to the skin surface on the median upper abdomen. We scanned longitudinally from the xiphoid process to the navel along the midline . Preperitoneal fat mass areas were measured as areas of 2 cm length along the midline starting from the reference point in direction of the navel.27Preperitoneal fat mass was used as a proxy for visceral fat and was measured using abdominal ultrasound examinations with ultrasound LOGIQ E9  and ATL-Philips Model HDI 5000 , as described in detail previously.30 A cuff was selected with a cuff width approximately 40% of the arm circumference and long enough to cover 90% of the arm circumference. We calculated mean systolic and diastolic blood pressure values using the last three blood pressure measurements.We measured systolic and diastolic blood pressure at the right brachial artery, four times with 1-min intervals, using a validated automatic sphygmanometer .We obtained 30-min fasting venous blood samples and measured total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, insulin, and C-peptide levels, using Cobas 8000 analyzer .2) was calculated. We defined gestational weight gain as the difference between weight before pregnancy and weight measured at 30 weeks of gestation .21 Information on maternal educational level, ethnicity, parity, smoking, and folic acid supplement use was obtained via questionnaires. First-trimester nutritional intake was recorded via a food frequency questionnaire.31 Information on pregnancy complications, mode of delivery, gestational age, length and weight at birth, and sex was available from medical records.33 We constructed gestational age-adjusted standard deviation scores (SDS) for birth weight using North European growth standards.34 These gestational age-adjusted SDS for birth characteristics represent the equivalent of z-scores. Infant growth was measured in community health centers according to standardized procedures at 24 months .21 We created age- and sex-adjusted SDS of these infant anthropometrics within our study population using Dutch reference growth charts.35 We obtained information about breastfeeding, TV watching, and timing of introduction of solid foods via questionnaires.21Maternal age and prepregnancy weight were obtained at enrolment. Maternal height (cm) was measured and prepregnancy body mass index  for childhood outcomes to enable a comparison of effect estimates. Since no significant interactions with fetal sex and birth weight were present after taking into account multiple testing, no further stratified analyses were performed. We performed sensitivity analyses by comparing maternal daily vomiting with never vomiting; by assessing a dose–response relationship using maternal vomiting categories as a continuous variable, from category “never” up to category “daily”; and by performing similar analyses using daily nausea during early pregnancy. In order to reduce potential bias associated with missing data and to maintain statistical power, we performed multiple imputations of missing covariates by generating five independent datasets using the Markov Chain Monte Carlo method after which the pooled effect estimates were calculated. All analyses were performed using SPSS version 21.0 for Windows .First, we examined differences in subject characteristics between mothers with and without daily vomiting during early pregnancy with ANOVA and chi-squared tests. Second, to provide further insight into the correlation of measures related to hyperemesis gravidarum with maternal weight, nutritional status, and birth outcomes, we assessed the associations between maternal daily vomiting and nausea during early pregnancy with multiple parameters of maternal nutritional status and birth outcomes using univariate linear regression models. Third, we examined the associations of maternal daily vomiting during early pregnancy with childhood outcomes and the role of potential mediators using linear regression models. For these analyses, we used different linear regression models: (1) A basic model including child’s age and sex. (2) A confounder model, which additionally included covariates selected on their associations with the outcomes of interest or a change in effect estimate >10%. Covariates included were maternal age, educational level, ethnicity, prepregnancy weight, parity, smoking, folic acid supplement use, diet, delivery mode, and pregnancy complications. We included childhood height as a covariate in all models focusing on fat mass outcomes. (3) Intermediate models, which additionally included potential intermediates . (4) A fully adjusted model including all confounders and all potential intermediate covariates. Supplementary Figure p < 0.05). Results of the non-response analysis are shown in Supplemental Table S1. Compared with mothers included in the analyses, those lost to follow-up were more likely to be older, less educated, and more often vomited daily during early pregnancy; their children had a lower gestational age at birth, lower birth weight, and were less frequently breastfed.The characteristics of included participants are shown in Table S2, S3, and S4. Maternal daily vomiting and nausea during early pregnancy was associated with a higher maternal prepregnancy weight and BMI, but with lower total gestational weight gain. No significant differences in trimester-specific weight gain were present. Among the women with daily vomiting and nausea during early pregnancy, total caloric, fat, and protein intakes tended to be lower, compared with women without daily vomiting and nausea during early pregnancy. The children of mothers with daily vomiting during early pregnancy had a lower birth weight and a lower gestational age at birth, compared with the children of mothers without daily vomiting during early pregnancy. These effects were weaker for maternal daily nausea during early pregnancy.Univariate associations of maternal daily vomiting and nausea during early pregnancy with maternal weight status were studied using multiple parameters of maternal nutritional status and birth outcomes shown in Supplementary Tables p < 0.05). The effect estimates were not affected when we restricted the analyses to daily vomiting versus never vomiting only. Supplementary Table S5 shows that in the dose–response analysis, only maternal daily vomiting during early pregnancy was associated with higher childhood body fat mass measures. Also, similar tendencies were observed when using daily nausea during early pregnancy instead of daily vomiting (results not shown).Table S6 shows that in the dose–response analysis, no associations of maternal daily vomiting during early pregnancy with childhood cardiovascular measures were present.Table In this prospective cohort study, we observed that daily maternal vomiting during early pregnancy was associated with a higher childhood total body fat mass, android/gynoid fat mass ratio, and abdominal preperitoneal fat mass. These associations were not explained by birth characteristics, but partly explained by higher infant growth rates. We did not observe associations of maternal daily vomiting during early pregnancy with childhood blood pressure, lipids levels, or insulin levels.23 This indicator is one component related to hyperemesis gravidarum, but does not fully reflect a severe clinical diagnosis of hyperemesis gravidarum, which includes dehydration, hospitalization, electrolyte imbalance, nutritional deficiencies, and maternal weight loss. We observed that mothers with daily vomiting during early pregnancy had a lower total gestational weight gain and a lower total caloric intake. Due to the small number of mothers who vomited daily or had nausea and reported severe weight loss, we were not able to assess the associations of these combined maternal characteristics with childhood outcomes in our study. However, additional adjustment for gestational weight gain did not explain the observed associations of maternal daily vomiting or nausea with childhood outcomes. Further studies are needed to assess the associations of a clinical diagnosis of hyperemesis gravidarum as well as other maternal characteristics relating to hyperemesis gravidarum with long-term offspring consequences. Detailed information about several maternal and childhood sociodemographic and lifestyle-related factors was available in this study. Extensive adjustment for these sociodemographic and lifestyle-related determinants in our analyses did not explain the associations of maternal daily vomiting during early pregnancy with childhood body fat outcomes. However, because of the observational design, residual confounding due to other sociodemographic and lifestyle-related determinants, such as residential area, maternal physical activity or sedentary lifestyle and childhood nutritional intake, might still be an issue.We used a population-based prospective cohort design involving a large number of subjects who we studied from early fetal life onwards. Information on vomiting during early pregnancy was available for 6,960 mothers, of which 4,769 mothers and their children participated in childhood follow-up measurements. Mothers without offspring follow-up data available were more often less educated and from non-European descent, and more often vomited daily during early pregnancy. The observed associations would be biased if the associations of maternal vomiting during early pregnancy with childhood outcomes differed between those included and those exempted in the analyses. This seems unlikely, but cannot be excluded. However, our study group might indicate a selection toward a relatively healthy population, which could affect the generalizability of our results. We used daily maternal vomiting as an indicator of hyperemesis gravidarum. This approach is in line with previous observational studies.1 Low birth weight or fetal exposure to extreme maternal undernutrition is associated with cardiovascular and metabolic diseases in adulthood.3 Less is known about the long-term effects of milder and more common suboptimal maternal nutritional status during pregnancy.An accumulating body of evidence suggests that suboptimal maternal nutrition during early pregnancy leads to an increased risk of cardiovascular disease in later life of the offspring.38 Previous studies have shown that hyperemesis gravidarum might lead to an increased risk of adverse outcomes during pregnancy.15 A study among 71,468 Norwegian mothers showed that children from hyperemetic mothers – defined as enduring nausea and vomiting starting before the 25th gestational week leading to hospitalization – had a lower gestational age at birth, but not a lower birth weight.23 Another study among 156,091 mothers in Canada showed that hyperemesis gravidarum – defined as severe nausea and vomiting occurring before the 24th gestational week leading to hospitalization – was associated with an increased risk of preterm birth, low birth weight, and small size for gestational age.10 Thus, hyperemesis gravidarum may adversely affect fetal development, leading to both preterm delivery and low birth weight.Hyperemesis gravidarum, characterized by severe nausea and excess vomiting during early pregnancy, resulting in electrolyte, fluid, and nutrition imbalances and deficiencies, is a marker of suboptimal maternal nutritional status during early pregnancy.18 Not much is known about the long-term consequences of maternal hyperemesis gravidarum and its related measurements with childhood adiposity and cardiovascular outcomes. We observed that maternal daily vomiting during early pregnancy was not associated with a higher body mass index of the offspring. Our results are in line with a previous cohort study among 3,165 Dutch mothers and their children.18 This study reported that the body mass index at 5 and 6 years of age was similar between the children of mothers with and without hyperemesis gravidarum, which was measured by a severe weight loss during early pregnancy.Hyperemesis gravidarum represents the extreme of the spectrum of maternal excess vomiting and extreme nausea during pregnancy. Several studies have shown that other less extreme measures relating to hyperemesis gravidarum, such as maternal daily vomiting or severe weight loss, are also associated with an increased risk of adverse fetal pregnancy outcomes, including preterm delivery and small size at birth.39 Similarly, among adults, waist circumference, as a proxy for abdominal fat mass, is related to cardiovascular disease and mortality in adulthood, independent of body mass index.42 In the current study, we observed that maternal daily vomiting during early pregnancy was associated with a higher childhood total body fat mass, android/gynoid fat mass ratio, and abdominal preperitoneal fat mass. To the best of our knowledge, no other studies have examined these associations yet. Surprisingly, none of these associations were explained by gestational age or size at birth, even though both characteristics are known to be related to both general and abdominal fat mass in later life.43 Our findings were partly explained by infant growth, a well-known risk factor for adverse body fat distribution.44 Thus, our findings suggest that the children of mothers with daily vomiting during early pregnancy have higher total and abdominal fat mass levels, which may partly be explained by suboptimal nutrition during fetal life and compensatory growth acceleration during the postnatal period.During childhood, body mass index may not be an accurate marker of fat mass, as it provides no information on body fat distribution and cannot distinguish lean mass from fat mass. Previously we showed that detailed general and abdominal fat mass measures are, independent of body mass index, associated with cardiovascular risk factors at school age.18 A study among 78 participants found that insulin sensitivity was 20% lower in the children of mothers with severe hyperemesis gravidarum, defined as intractable vomiting and electrolyte imbalances during early pregnancy leading to hospitalization.5 Differences in results might be due to the use of different measurements relating to hyperemesis gravidarum, differences in study populations, and adjustments for confounders. Altogether, the results of the present and previous studies do not suggest consistent associations of maternal vomiting during early pregnancy with offspring cholesterol, triglycerides, and insulin levels.We did not observe the associations of maternal daily vomiting during early pregnancy with childhood blood pressure and blood lipids and insulin levels. In contrast, a previous study among 3,165 Dutch mothers and their children suggested that maternal hyperemesis gravidarum, defined as a severe maternal weight loss during early pregnancy, was associated with a higher blood pressure in childhood, but not with childhood total cholesterol and triglycerides.47 However, daily maternal vomiting may also lead to suboptimal maternal nutrition, which may subsequently lead to suboptimal fetal nutrition and developmental adaptations.2 These fetal developmental adaptations may permanently affect fetal growth, fetal adipocyte development and function, and the function of cardiovascular organs, predisposing to adverse body composition and an increased risk of obesity in later life. Importantly, none of the observed associations of maternal daily vomiting during early pregnancy with childhood total body and abdominal fat mass levels were explained by birth characteristics. This finding is in line with previous studies that focused on extreme maternal undernutrition, which showed that exposure to extreme maternal undernutrition during early gestation has an increased risk of obesity and cardiovascular disease in later life, independent of size at birth.48 Together, these findings might imply that the effects of maternal daily vomiting on offspring’s body composition may act through other mechanisms than size at birth, such as alterations in the levels of fetal micronutrients leading to epigenetic changes or endocrine alterations in the offspring.18 Further research is needed to obtain more insights into the causality and underlying mechanisms of the observed associations.The underlying pathophysiology of hyperemesis gravidarum is not known yet. From an evolutionary adaptation perspective, mild vomiting during pregnancy may be considered as a defensive strategy to expel harmful foods or spoiled or teratogenic substances.52 A study among 7,723 and 7,252 children aged 7 and 11 years, respectively, showed moderate tracking of fat mass throughout childhood.50 A study among 2,204 participants showed a strong 27-year tracking between childhood and adulthood measurements of body mass index.49 A study among 4,857 children and adolescents, aged 5–20 years, with a median age of 11 years, showed that childhood obesity was associated with increased rates of premature death from endogenous causes.51 Similarly, a study among 276,835 Danish schoolchildren showed that a higher childhood BMI across the full range was associated with an increased risk of coronary heart disease in adulthood.52 Thus, these studies suggest that even subclinical differences in childhood fat mass levels are related to the development of cardiovascular disease in later life. Further studies are needed to identify the long-term consequences of maternal hyperemesis gravidarum on offspring’s body fat and cardiovascular outcomes.The effect estimates of the associations of maternal daily vomiting during early pregnancy with childhood total body and abdominal fat mass levels were small and are mainly of interest from an etiological perspective. However, since these may be important from a cardiovascular developmental perspective, their effects on the risk of cardiovascular disease should be further studied. Previous studies have shown that childhood body composition measures tend to track from childhood into adulthood.Our results suggest that maternal daily vomiting during early pregnancy is associated with higher childhood total and abdominal fat mass levels, independent of size or gestational age at birth. Further studies are needed to replicate these findings, to explore the underlying mechanisms, and to assess the long-term consequences.


In the French Territories in the Americas (FTA), the risk of birth defects possibly associated with Zika virus (ZIKV) infection was 7.0% (95%CI: 5.0 to 9.5) among foetuses/infants of 546 women with symptomatic RT-PCR confirmed ZIKV infection during pregnancy. Many of these defects were isolated measurement-based microcephaly  or mild neurological conditions. We wanted to estimate the proportion of such minor findings among live births of women who were pregnant in the same region during the outbreak period but who were not infected with ZIKV.In Guadeloupe, pregnant women were recruited at the time of delivery and tested for ZIKV infection. The outcomes of live born infants of ZIKV non-infected women were compared to those of ZIKV-exposed live born infants in Guadeloupe, extracted from the FTA prospective cohort.Of 490 live born infants without exposure to ZIKV, 42 infants  had mild abnormalities that have been described as ‘potentially linked to ZIKV infection’; all but one of these was isolated measurement-based microcephaly. Among the 241 live born infants with ZIKV exposure, the proportion of such abnormalities, using the same definition, was similar .Isolated anthropometric abnormalities and mild neurological conditions were as prevalent among infants with and without in-utero ZIKV exposure. If such abnormalities had not been considered as ‘potentially linked to ZIKV’ in the original prospective cohort in Guadeloupe, the overall estimate of the risk of birth defects considered due to the virus would have been significantly lower, at approximately 1.6% (95% CI: 0.4–4.1).NCT02916732)ClinicalTrials.gov ( Microcephaly defined using growth charts occurs naturally in some healthy infants. Nonetheless, in many recent Zika virus studies, such measurement-based microcephaly and other common mild abnormalities have been automatically attributed to viral exposure during pregnancy. For example, in 2016 in Guadeloupe, we did a cohort study and estimated that the risk of Zika virus related birth defects was 7%. However, most of the ‘abnormal’ live born infants that contributed to this risk estimate were considered healthy by pediatricians, but were found to have measurement-based microcephaly during data analysis. In the current study, we did a cross-sectional evaluation of infants live born in 2016 in Guadeloupe, but whose mothers were not infected with Zika virus during pregnancy. We found that Zika virus unexposed live born infants had similar proportions of measurement-based microcephaly and mild neurological abnormalities as exposed live born infants. If only severe and uncommon birth defects were considered linked to Zika virus in our original cohort study, our risk estimate would have been significantly lower. This research shows that it is important to consider baseline risks and control comparisons when establishing key parameters for emerging diseases, such as Zika virus. Since the first evidence surfaced that linked Zika virus (ZIKV) to fetal microcephaly and other brain abnormalities,,2 key reFor the purpose of determining those birth defects that can actually be linked to ZIKV in an exposed population, an estimation of the baseline level of birth defects in an appropriate ZIKV non-infected control group is necessary. There has been minimal use of comparative control groups within prospective studies on this topic.,14,15 InThe ZIKA-DFA-FE study is registered with ClinicalTrials.gov (NCT02916732) and received ethics approval by the Comité de Protection des Personnes Sud-Ouest et Outremer III (CEBH2016/03), in compliance with the Declaration of Helsinki. All women provided written informed consent to have their, and their infants’, data included in this study.The ZIKA-DFA-FE cohort study (an acronym for “Zika in the French Territories in the Americas in Pregnant Women”), which has been described elsewhere, used fouAt the time of hospital admission for labor, each eligible woman was informed of the study and invited to participate; oral consent was obtained before delivery and written informed consent was obtained before delivery whenever possible or within 24 hours after delivery otherwise. A questionnaire exploring socio-demographic characteristics, such as age, ethnicity, education, profession, and lifestyle factors, was administered. Clinical information, including the number of previous pregnancies, history of adverse pregnancy outcomes, medical history, clinical symptoms consistent with ZIKV infection, and any other clinically significant medical event during pregnancy, was also collected. Exactly as within the prospective cohort study, infant clinical data such as gestational age, length, weight, and head circumference, APGAR scores  were collected on the day of birth, and a standardized clinical examination was carried out in the first four days of life. The medical files of participants were reviewed to collect data on clinical and ultrasound examinations that were performed during the pregnancy.All participating women had blood sampled on the day of delivery, from which sera were frozen and stored. These were tested for the presence of anti-ZIKV IgG antibodies, using the Euroimmun ZIKV IgG immunoassay . The results of any other ZIKV tests  or TORCH testing that had been performed on biological samples collected during the pregnancy were also recorded.th to September 19th 2016), and who had a confirmed negative IgG serology test for ZIKV from blood taken at the time of delivery as well as no other positive or indeterminate ZIKV test during pregnancy.For the cross-sectional study, we selected women from the fourth recruitment method of the ZIKA-DFA-FE study who gave birth to a live neonate during or up to 9 months following the 2016 ZIKV epidemic period  for gestational age and sex. Moderate microcephaly was further defined as proportionate or disproportionate depending on whether the neonate was small for gestational age (ie. birth weight less than -1.28 SD).[Anthropometric abnormalities and other birth defects in infants were classified according to the same case definitions as used in the prospective, ZIKV-exposed cohort study. Microcep1.28 SD).,12,16 ClWe compared infant outcomes from the prospective cohort study of women infected with ZIKV during pregnancy, with thoSee Of the 1484 women enrolled in the cross-sectional study in Guadeloupe, 484 had a negative IgG serology test for ZIKV at the time of delivery. Of these women, 395 additionally had ZIKV IgM testing, and one woman had ZIKV PCR testing, on the blood sample taken at the time of delivery—all results were negative. Of these 484 women, 6 had twin pregnancies and therefore the outcomes of 490 live born infants were observed. In the prospective cohort, of the 250 pregnant women in Guadeloupe who had symptomatic, RT-PCR confirmed ZIKV infection at any time during pregnancy, 245 pregnancies were followed up until the time of the pregnancy outcome. Within this group there were eight cases of fetal demise and four sets of twins, and therefore the outcomes of 241 ZIKV exposed live born infants were observed. See Table 1.The mean age of ZIKV non-infected women was 30.7 years (SD = 6.4), and that of ZIKV infected women was 30.0 years (SD = 6.3). There was a higher proportion of reported smoking in the ZIKV-infected cohort during pregnancy compared to the ZIKV non-infected: 4.2% (95%CI: 2.0–7.6%) versus 0.6% (95%CI: 0.4–1.8%). There were also more unemployed women in the ZIKV non-infected group compared to the ZIKV-infected one: 48.1% (95%CI: 43.3–52.4) versus 34.6% (95%CI: 28.6–41.0). See Table 2.Of the 490 live born infants of women without ZIKV infection during pregnancy, 66  were small for gestational age, and 41  had microcephaly. One of these cases of moderate microcephaly had a possible genetic aetiology (Adams Oliver syndrome). None of the 41 cases of microcephaly were identified as such by the attending clinicians (i.e. they were defined later after comparing infant measurements to a standardized growth curve) and for none were there reports of other structural brain or clinical abnormalities. Additionally, one infant  had an abnormality that could be a consequence of CNS dysfunction, which was clubfoot. Five infants  had other abnormalities that are not considered as linked to ZIKV infection, including: supplementary fingers or toes (n = 2), anal imperforation (n = 1), urinary tract abnormalities (n = 2). Besides two infants born to HIV-infected mothers who were small-for-gestational-age, there were no other abnormalities identified in the remaining four infants born to mothers with TORCH infections. See There was no significant difference in risk of anthropometric or other mild birth defects in live born infants whose mothers had a symptomatic PCR-confirmed ZIKV infection during pregnancy compared to those whose mothers had no evidence of prior ZIKV infection at the time of delivery in Guadeloupe . See There was no association  between ZIKV exposure and abnormalities in live born infants in a multiple logistic regression model adjusting for age, occupation, and ethnicity. When compared to women 25–34 years of age, women 18–24, 35–39, and 40 or more years of age were more likely to have a live born infant with abnormalities with ORs of 5.1 (95%CI: 2.5–10.7), 2.3 (95% CI: 1.0–5.0) and 2.9 (95% CI: 1.0–8.0), respectively. There was no association between either occupation or ethnicity and the likelihood of having a live born infant with abnormalities. Table 3), 14 of these were either isolated anthropometric abnormalities  or isolated mild CNS dysfunction defects (i.e. clubfoot), as well as one case of spina bifida . Of these eight cases of fetal demise, there were two instance of miscarriage and three instances of stillbirth without any evidence of fetal anomalies. The other three instances of fetal demise were terminations of pregnancy for fetal anomalies (TOPFA) that were severe and suggestive of Zika Congenital Syndrome. In our original report, all neurological or ocular birth defects from Guadeloupe that were potentially linked to ZIKV infection in Guadeloupe were included in our risk estimate and so this included the 16 abnormalities in live born infants as well as the three cases of TOPFA .[Of the 16 abnormalities seen among 241 live born infants of women with ZIKV infection during pregnancy , andinfection In the o.7–11.7). If, as mst study itself prescribes that approximately 2% and 0.1% of healthy infants should have a head circumference at birth that falls below -2 and -3SD, respectively, on their pooled international growth standard.[st study noted varying levels of fit for individual populations when compared to their pooled standard,[We found no statistically significant difference in the risk of neurological birth defects in live born infants of ZIKV infected and non-infected women in Guadeloupe during the 2016 epidemic period. This can primarily be explained by the fact that most of the abnormalities reported as ‘potentially linked to ZIKV’, for both the exposed and non-exposed pregnancies in this study, represent identification of isolated microcephaly in live births; these cases were defined based only on anthropometric measurements, with known clinical and radiological findings for each infant being normal. This diaThis is the largest study of ZIKV non-infected women from a defined epidemic region that has been used as a comparative control group against ZIKV-infected pregnant women followed up during pregnancy. It was conducted in a resource-rich setting where the standard of care during pregnancy is high. The exposure statuses of each of the two groups included were well defined. The ZIKV infected women from Guadeloupe were confirmed via RT-PCR within days of infection. Relying This study has several limitations. As we had no directly comparable prospective cohort, we used a group of ZIKV non-infected women delivering at the same hospitals and in the same time period. Our aim was not to compare the risk of birth defects from conception to delivery in the two groups, since this information was not available for the ZIKV non-infected group, but to compare the proportion of microcephaly and other birth defects among live born infants, to see if the minor abnormalities found in the ZIKV-exposed group could be confidently related to ZIKV infection. If the ZIKV non-exposed group had been followed prospectively in a similar fashion to the ZIKV exposed pregnancies, this would have allowed for a more complete comparison of the risk of abnormalities and adverse outcomes. There is limited recent baseline data on the incidence of pregnancy loss in Guadeloupe, specifically. One 2004 study from centres across France, including Guadeloupe and Martinique, found an overall TOPFA incidence at any gestational age of 0.7%. In 2005 st standards and found incidences of 2.5% and 3.5%.[This study highlights the importance of a control group for establishing the baseline risk of anthropometric and other birth defects when determining the risk of congenital abnormalities that can be linked to a given infection during pregnancy. This is particularly true for abnormalities defined by anthropometric measurements, where regional variations may exist;,33–35 inand 3.5%.In our case, reassessment of the risk of birth defects from a ZIKV exposed prospective cohort after consideration of mild abnormalities seen in a time and place-matched control group, indicates an initial significant overestimation of the risk of birth defects potentially associated with ZIKV at the time of birth. Our new estimates of approximately 4%, 1%, and 0% for the risk of birth defects following ZIKV infection in first, second, and third trimester, respectively, are comparable to those from a recent meta-analysis of prospective studies on this topic. Still, a


Low birth-weight is a major risk factor for perinatal death in sub-Saharan Africa, but the relative contribution of determinants of birth-weight are difficult to disentangle in low resource settings. We sought to delineate the relationship between birth-weight and maternal pre-eclampsia across gestation in a low-resource obstetric setting.Prospective cohort study in a tertiary referral centre in urban Uganda, including 971 pre-eclampsia cases and 1461 control pregnancies between 28 and 42 weeks gestation.Nonlinear modeling of birth-weight versus maternal pre-eclampsia status across gestation. Models were adjusted for maternal-fetal characteristics including maternal age, parity, HIV status, and socio-economic status. Propensity score matching was used to control for the severity of pre-eclampsia at different gestational ages.Mean birth-weight for pre-eclampsia cases was 2.48 kg (±0.81SD) compared to 3.06 kg (±0.46SD) for controls (p < 0.001). At 28 weeks, the mean birth-weight difference between pre-eclampsia cases and controls was 0.58 kg (p < 0.05), narrowing to 0.17 kg at 39 weeks (p < 0.01). Controlling for pre-eclampsia severity only partially explained this gestational difference in mean birth-weight between pre-eclampsia cases and controls. Holding gestational age constant, pre-eclampsia status predicted 7.1–10.5% of total variation in birth-weight, compared to 0.05–0.7% for all other maternal-fetal characteristics combined.Pre-eclampsia is the dominant predictor of birth-weight in low-resource settings and hence likely to heavily influence perinatal survival. The impact of pre-eclampsia on birth-weight is smaller with advancing gestational age, a difference that is not fully explained by controlling for pre-eclampsia severity. Women were recruited to the study in three waves: July 2009, August 2010–June 2011, and September 2014-Dec 2016 . There w2.2The primary study outcome was birth weight, measured in kilograms. We also converted the birth weights observed in our population into gestational age and sex-adjusted centiles using an international reference standard Gestational age in weeks was calculated for each pregnancy based on last menstrual period or ultrasound scan. We considered only singleton infants born between 28 and 43 weeks estimated gestation. Infant sex was designated as male or female at delivery. We included ‘fresh’ stillborn babies (those judged by the clinician to have died shortly before delivery), but not ‘macerated’ stillbirths. We also included cases of early neonatal death.Maternal-fetal characteristics for each pregnancy were recorded in the study data set, and were either derived directly from the clinical notes or from information provided by the women themselves. Maternal characteristics included maternal age (in years), ethnicity, and parity. Detailed information on the mother’s ethnicity and the family ethnicity  was recorded, and encoded in our regression models as a binary variable for whether the mother belonged to the predominant local Ganda ethnicity. Women were asked whether they had experienced a severe febrile illness during the pregnancy. Given knowledge of local infectious disease prevalence and population susceptibility 2.3In our primary analysis, we assessed the relationship between pre-eclampsia and birth weight across gestation. Regression splines were used to fit birth weight as a nonlinear function of both gestational age and pre-eclampsia status. To isolate the effect of pre-eclampsia, our model also adjusted for several other maternal-fetal characteristics, including stillbirth, infant sex, maternal age, maternal job type, parity, and HIV status. The model—including the location of spline knots and the variables/interactions included—was chosen using a stepwise selection process that is detailed in Our spline model is highly flexible, in that it allows for a time-varying effect of pre-eclampsia on birth weight, i.e. an effect that varies continuously as a function of gestational age. Therefore, to calculate a model-adjusted average effect of pre-eclampsia across multiple gestational ages, we calculated Friedman’s partial-dependence function As a secondary analysis, we investigated whether a time-varying effect of pre-eclampsia on birth weight could be explained by the fact that cases of pre-eclampsia tended to be more severe at earlier gestational ages. To do so, we used propensity score matching to pair observations at high and low gestational ages with similar pre-eclampsia severity markers . We then32387 women were recruited to the study, of whom 971 had confirmed pre-eclampsia and 1416 were normotensive. The cohort characteristics are described in Pre-eclampsia was more severe in pregnancies delivered at earlier gestational ages . For womMean birth weight  was significantly lower (p < 0.001) among pre-eclampsia cases (2.48 kg ± 0.81 SD) compared to normotensive pregnancies (3.06 kg ± 0.46 SD). Compared to international standards adjusted for sex and gestational age After controlling for maternal-fetal covariates in our spline model, the birth weight deficit associated with pre-eclampsia—that is, the model-adjusted difference in mean birth weight between the pre-eclampsia and normotensive groups—persisted throughout gestation. This birth weight deficit was greater at earlier gestational ages , stillbirth (p < 0.05), and having unskilled or no employment (p < 0.05). Factors significantly associated with higher birth weight included higher maternal age (p < 0.05) and primiparity (p < 0.001) . To illuFinally, our analysis of variance showed that at low gestational ages (<34 weeks), pre-eclampsia predicts 7.1% of the variation in birth weight, versus 0.7% for all other maternal-fetal characteristics except gestational age . At high4We show that, in urban Uganda, maternal pre-eclampsia is the dominant influence on birth weight across all gestations. In our population, pre-eclampsia alone accounts for approximately ten times more of the variability in birth weight than all other identified risk-factors combined. We demonstrate that although the effect of pre-eclampsia on birth weight is consistently present, there is substantial reduction in this birth weight deficit at later gestational ages.Although it is clear that mothers with more severe pre-eclampsia tend to deliver at earlier gestations, the higher birth weight deficit at earlier gestations still persists even after controlling for pre-eclampsia severity using matching. The evidence is thus insufficient to conclude definitively that increased growth restriction in pregnancies delivered at lower gestational ages is explained by higher pre-eclampsia severity. Pre-eclampsia severity is defined clinically in terms of maternal symptoms, but may also be considered in terms of direct placental impact. It is important to note that pregnancies delivered earlier may have more adverse placental phenotypes, and this may be better correlated to fetal growth restriction than maternal symptoms. This is an important consideration for future research.The particular setting of our study, in a difficult-to-study maternity population, is a major strength and may be relevant to the observed dominance of pre-eclampsia as a risk factor for fetal growth restriction. The urban Ugandan population is relatively treatment-naïve in the context of pre-eclampsia, due to the high prevalence of late presentation. This provides a rare opportunity to study the natural history of pre-eclampsia, which is often masked by early detection, medical treatment, and careful timing of delivery, yet is evident in this unusual data set. Furthermore, the high prevalence of pre-eclampsia in this setting and contemporaneous data collection strategy has enabled the collection of a large number of cases of pre-eclampsia of high severity. The sophisticated and flexible modelling employed in our study has enabled us to quantify the magnitude of risk associated with pre-eclampsia and other observable characteristics.Our analysis is subject to several limitations. First, there are several degrees of freedom in our process: specification of the spline basis, variable selection, and form of the interaction between gestational age and pre-eclampsia. The need for a choice regarding each of these is inherent in any parametric model specification, and we have tried to minimize researcher degrees of freedom here by using stepwise selection to decide the form of the model see . Second,Our finding that pre-eclampsia severity is not a good correlate of the magnitude of fetal growth restriction is in keeping with the findings of smaller studies of severe pre-eclampsia in higher income settings th centile) babies in the pre-eclampsia group than in the normotensive group. This finding is an important subject for future research, as it may be independently associated with other adverse perinatal outcomes.Although an association between pre-eclampsia and fetal growth restriction is described in other contexts, the magnitude of the growth restriction ascribed to pre-eclampsia is rarely as large as in this cohort In the sub-Saharan African clinical setting, simple heuristics to guide practice are often invaluable. The ability to recognize babies at highest risk of being born at low birth weight maximizes the chance of appropriate interventions. Understanding which factors are the key determinants of birth weight in a particular context also allows the formulation of strategies targeted at reducing the incidence of low birth weight and hence improving perinatal survival. Our results suggest that perinatologists should regard the timing of delivery in the context of pre-eclampsia as a better predictor for associated fetal growth restriction than indices of pre-eclampsia severity.CA is supported by an Isaac Newton Trust[12.21(a)]/Wellcome Trust ISSF [105602/Z/14/Z]/ University of Cambridge Joint Research Grant. This work was funded by the Wellcome Trust (094073/Z/10/B), and a Wellcome Trust Uganda Postdoctoral Fellowship in Infection and Immunity held by AN, funded by a Wellcome Trust Strategic Award, grant number 084344. Supported by NURTURE fellowship to AN, grant number D43TW010132. This work was also supported through the DELTAS Africa Initiative (grant number 107743/Z/15/Z). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (grant number 107743/Z/15/Z) and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government. JES acknowledges the support of a T32 fellowship from the U.S. National Institutes of Health.Annettee Nakimuli: Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing - original draft. Jennifer E. Starling: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing. Sarah Nakubulwa, Imelda Namagembe, Musa Sekikubo, Eve Nakabembe: Investigation, Methodology, Writing - review & editing. James G. Scott: Conceptualization, Formal analysis, Methodology, Supervision, Writing - review & editing. Ashley Moffett: Conceptualization, Supervision, Writing - review & editing. Catherine E Aiken: Conceptualization, Data curation, Formal analysis, Writing - original draft.


Congenital diaphragmatic hernia (CDH) is associated with high mortality rates and significant pulmonary morbidity, mainly due to disrupted lung development related to herniation of abdominal organs into the chest. Pulmonary hypertension is a major contributor to both mortality and morbidity, however, treatment modalities are limited. Novel prenatal and postnatal interventions, such as fetal surgery and medical treatments, are currently under investigation. Until now, the perinatal stabilisation period immediately after birth has been relatively overlooked, although optimising support in these early stages may be vital in improving outcomes. Moreover, physiological parameters obtained from the perinatal stabilisation period could serve as early predictors of adverse outcomes, thereby facilitating both prevention and early treatment of these conditions. In this review, we focus on the perinatal stabilisation period by discussing the current delivery room guidelines in infants born with CDH, the physiological changes occurring during the fetal-to-neonatal transition in CDH, novel delivery room strategies and early predictors of adverse outcomes. The combination of improvements in the perinatal stabilisation period and early prediction of adverse outcomes may mitigate the need for specific postnatal management strategies. Congenital diaphragmatic hernia (CDH) is a rare birth defect that affects around 1 in 2500 live births.The diaphragmatic defect can be surgically closed after birth, but the degree of disrupted lung development aggravated by herniation of abdominal organs into the chest determines survival.In recent decades, researchers have focused on developing antenatal interventions aimed at both improving pulmonary development by increasing the gas exchange area, as well as reducing PH. The benefit of temporary tracheal occlusion using fetoscopy is currently evaluated in a randomised clinical trial and the results are expected this year. In preclinical research antenatal administration of vasodilator drugs, such as sildenafil, resulted in improved pulmonary vessel development; its efficacy is being investigated in a small clinical phase I/IIb trial.In this review, we will focus on the perinatal stabilisation period in the delivery room. We will summarise the available guidelines and discuss the known science on how the sequence of physiological changes is altered in infants with CDH. In addition, we will provide an overview of novel delivery room strategies that aim to improve short and long-term outcomes as well as early predictors of adverse outcomes.Current guidelines regarding the resuscitation of CDH infants are predominantly opinion based, since trials assessing delivery room management are lacking. Several large European centres published a consensus document, which was updated in 2015 (CDH EURO Consortium), and a similar guideline by the Canadian CDH Collaborative was published recently.In both the European and Canadian guidelines, the key principle of stabilisation is to establish adequate perfusion and oxygenation while avoiding high airway pressures.In the fetal circulation , left veAt the time of cord clamping several adaptations in the cardiovascular and pulmonary system occur as the low resistance placental vascular bed is removed from the systemic circulation . The losFor almost all infants born with CDH, transition to the neonatal phenotype is complicated and support from medical professionals is essential for survival. However, our knowledge of how pulmonary hypoplasia affects the cardiopulmonary physiological changes during the transition period is largely based on animal models.Hypoplastic lungs have low compliance, resulting in delayed lung aeration after birth .29 In paPostnatal LV dysfunction—both systolic and diastolic—adds to the haemodynamic instability during transition in these CDH infants.In an ovine diaphragmatic hernia (DH) model, the lambs developed respiratory acidosis and poor cerebral oxygenation immediately after birth, confirming the hypothesised worsening of hypoxia after ICC.Improving outcomes of infants born with CDH requires clinical trials assessing innovative strategies in these infants. Considering it is a rare condition, large trials may not always be feasible as recruitment is not sufficient to achieve statistical and clinical significance within a reasonable time frame. This is also reflected by the fact that all concepts described in this paragraph have a low grade of recommendation (D). We will discuss early predictors of adverse outcomes and innovative treatment strategies during the perinatal stabilisation period that seem promising based on preclinical studies.Several early prediction models and parameters during the immediate postnatal period have been suggested, which could allow initiation of prophylactic therapies in an attempt to individualise healthcare for each neonate. Herein we will discuss two parameters and three prediction models.In a prospective study, tidal volume (Vt) of spontaneous breaths during stabilisation was associated with mortality, chronic lung disease incidence and the need for inhaled nitric oxide.2 value, which is obtained by means of arterial blood gas sampling. However, as most CDH infants have a patent ductus arteriosus at this time and preductal PaO2 values are thus higher than postductal PaO2 values, the site of blood gas sampling could result in a major difference in the calculated OI.The oxygenation index (OI), a value calculated to estimate the severity of respiratory failure, may predict mortality in CDH infants.2:FiO2 ratio, body temperature, diuresis and seizure activity.The Score for Neonatal Acute Physiology-II (SNAP-II Score) was first created in 2001 and is calculated with the use of arterial blood pressure, pH, PaO2[max]−PaCO2[max] and the authors hypothesised that in CDH infants with severe PH, arterial PO2 would be less than PCO2 as result of compromised gas exchange. However, the clinical accuracy of this model was not sufficient in predicting outcomes of individual patients, and a subsequent clinical trial reported poor discrimination in predicting outcomes as well.The Wilford Hall/Santa Rosa prediction formula was developed as an easy-to-use clinical method for predicting outcomes in the first 24 hours after birth.et al developed a third clinical prediction model in 2014.Brindle Many of the severely affected CDH infants are treated with ECMO, although its efficacy in CDH remains debatable. The apparent small gain in survival rates may be offset by increased long-term disability in survivors.2 levels are rapidly increased in response to reduced SpO2 levels without knowledge of cerebral blood flows, leading to a rapid increase in cerebral oxygen delivery.Respiratory support is a major topic in delivery room management for CDH infants, however, it is a double-edged sword as the importance of ventilator-induced lung injury (VILI) and oxygen toxicity is not to be underestimated. ‘Gentle ventilation strategies’ and permissive hypercapnia have been implemented in delivery room management and on intensive care units, as less aggressive ventilator strategies could reduce the risk of VILI and oxygen toxicity.2 lower than 1.0.2 levels may prevent subsequent pulmonary vasoconstriction.2 0.5 vs 1.0 (68 vs 45 patients), no differences in immediate postnatal outcomes were found.2 during stabilisation were more likely to have an adverse outcome.The CDH EURO Consortium also recommends that CDH infants may benefit from starting with FiOIn infants predicted to have mild hypoplasia that are potentially capable of a smooth transition into neonatal life, the initiation of invasive respiratory support may actually cause damage to the lungs. To avoid VILI for this specific group—infants with a left-sided defect, intra-abdominal liver and o/e LHR ≥50%, a trial of spontaneous breathing was postulated in the consensus CDH EURO Consortium guideline.The importance of appropriate timing of cord clamping in optimising perinatal stabilisation of newborns has been evaluated comprehensively over the last decade.2 and preductal SaO2, an almost 20-fold greater PBF at the time of cord clamping (p<0.001), and a significantly lower pulmonary artery pressure at 10 min after ventilation onset (p<0.001).In physiological-based cord clamping (PBCC), the umbilical cord is clamped after the infant has established lung aeration with spontaneous breathing or respiratory support.et al performed cord clamping after colorimetric carbon dioxide detection and as such, it is likely that cord clamping was performed without the infants being ‘stabilised’.As mentioned before, due to the hyper-reactive pulmonary vasculature in CDH infants temporary PH could lead to a cascade ultimately resulting in persistent PH.Based on the promising preliminary results we will initiate a multicentre and international randomised controlled trial evaluating PBCC in CDH infants, the PinC trial (NL69575.078.19), starting in 2020. Left-sided CDH infants will be randomised to either standard approach  or PBCC, meaning the umbilical cord will not be clamped until after the infant is stabilised. A consistent definition for physiological stability has not been established yet, but the goal is to assess adequate lung aeration. We decided to use physiological parameters as heart rate and oxygenation as proxy for aeration, because of its simplicity and is analogous to what is currently used in a trial evaluating the outcomes of PBCC in very preterm neonates.Infants with CDH will face important respiratory challenges immediately after birth and in some patients, deterioration of their clinical situation may be the result of our current management strategies at birth. We propose that this earliest time period of newborn life has the potential to provide both reliable markers for prediction of outcomes, such as PH, and the possibility to adopt innovative management strategies in an attempt to have more children survive. Optimising perinatal stabilisation could protect hypoplastic lungs from damage due to the treatments that we employ to save their lives.


Hepatocellular carcinoma (HCC) is the most common liver cancer and featured with prominent disparity in incidence and mortality rate between male and female. It remains unclear whether alterations of phospholipids (PL) in hepatic tissues contribute to the pathogenesis, progression, and disparity of HCC.Using electrospray ionization mass spectrometry (ESI–MS), PL profiles including 320 individual phospholipid species in 13 PL classes were determined in paired samples from HCC and adjacent benign hepatic tissues (BHT).(1) Concentrations of PLs in most of individual species, in subgroups and in total were decreased in HCC than in BHT in all studied population; (2) the number of individual PL species significantly different between HCC and BHT, and the number of PLs in six subgroups and in total decreased in HCC were more in male population than in female population; (3) panels of PL parameters  were identified as biomarkers in differentiation of HCC from BHT, and in the prediction of pathological grade and clinical stage of HCC with high sensitivity, specificity, and accuracy.It is concluded that alterations of PLs in hepatic tissues play important roles in pathogenesis, progression, and gender disparity of HCC.The online version of this article (10.1007/s12072-020-10056-8) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is a primary malignancy of liver with high morbidity and mortality worldwide . HCC is Phospholipids (PLs) have diverse biological functions in cells. In addition to being building blocks in membranous structures, PLs also play important roles in cellular signaling and cell–cell interactions in tissues. Metabolic alterations of various PLs have been reported to link to cancer proliferation. However, differences in phospholipid profiles have been rarely studied between HCC and benign hepatic tissues (BHT), and not investigated between male and female populations.Lipidomics is a powerful tool available for the analysis and characterization of lipids and their interacting moieties. Since the developments in mass spectrometry, advanced analytical methods are able to identify and quantify a large number of individual species, classes and subgroups of lipids, especially PLs at the same time . Lipid pIn the present study, we performed ESI–MS for 320 phospholipid species in 13 phospholipid classes on paired HCC and BHT tissues from 32 male and female HCC patients. Our aims are to determine differences in PL distribution between HCC and BHT, to reveal gender disparity in PL profiles and their clinic relevance between male and female populations, to identify PL biomarkers in the differentiation of HCC from BHT and in the prediction of progression of HCC, and to seek novel therapeutic targets in PL metabolisms for treatment of HCC.This study was approved by the Institutional Review Board of the University of Mississippi Medical Center. The study cohort comprised 32 patients with clinical HCC. A total of 64 samples, including 32 HCC tissues and their accompanied BHT were serially collected by the Cooperative Human Tissue Network (CHTN). The written consents were obtained from patients prior to the donation of tissue samples. To insure quality and quantity of samples, each sample should be collected within 10 years and weighted > 50 mg. The diagnosis of each HCC and BHT sample was reconfirmed by the pathologists at sites patients were enrolled in. Histological features for BHT samples included normal liver tissues, inflammation/hepatitis, alcoholic fatty liver disease (AFLD), non-alcoholic fatty liver disease (NAFLD), and cirrhosis, but none of BHT samples contains liver cancer cells. Because the numbers in each of the categories with benign hepatic changes were limited, all non-cancer tissues adjacent to HCC were grouped as benign hepatic tissues (BHT). None of the identifiable information, such as patient’s name, date of birth, and contact information was provided. The geographic, clinical, and pathological information is listed in Table Extraction of total lipids from HCC and BHT tissues was performed with chloroform and methanol, following a modified Bligh and Dyer protocol . BrieflyWeighed fresh-frozen HCC or BHT tissues were minced and homogenized in RIPA buffer  with protease inhibitor. The lysates were centrifuged, and the supernatants were collected and stored at − 80 °C. Protein concentration  was determined with the Pierce® BCA Protein Assay Kit  following the manufacturer’s instructions. Briefly, a series of dilutions of bovine serum albumin (BSA) were prepared as a set of protein standards to determine the standard curve. Then the BCA working reagent (WR) was prepared by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B . 25 µL of HCC or BHT protein was pipetted in replicate into a microplate well and 200 µL of the WR was added to each well, and incubated at 37 °C for 30 min. The quality and quantity of the total isolated proteins were measured and assessed at 570 nm with the Benchmark microplate reader .The concentrations of PL species were determined by an automated ESI–MS method. Briefly, 35 µl lipid extract and internal standard mixture were combined with solvents so that the ratio of chloroform/methanol/300 mM ammonium acetate in water was 300/665/35, and the final volume was 1.2 ml. The unfractionated lipid extract in an autosampler vial was introduced by continuous infusion into the ESI source on a triple quadrupole MS/MS , using an autosampler  fitted with the required injection loop for the acquisition time and presented to the ESI needle at 30 µl/min. PLs were detected by 80 cycles of ESI–MS (MS1) scanning from 150 to 400 u. The source temperature (heated nebulizer) was 100 °C, the interface heater was on, − 4.5 kV was applied to the electrospray capillary, the curtain gas was set at 20 (arbitrary units), and the two ion source gases were set at 45 (arbitrary units). The background of each spectrum was subtracted, the data were smoothed, and peak areas integrated using a custom script and Applied Biosystems Analyst software. The data were isotopically deconvoluted, and the PLs were quantified in comparison to a 15:0 internal standard. Peaks corresponding to the target PLs in these spectra were identified and molar amounts calculated in comparison to the internal standards for PLs. Finally, the data were corrected for the fraction of the sample analyzed and normalized to the sample volume to produce data in the unit of nmol/mg wwt HCC or BHT tissues, nmol/mg protein, and percentage of each phospholipid species out of total phospholipids according to the number of nmol/mg wwt.T test. Correlation and/or regression between two series of numerical data was analyzed by bivariate correlation and/or linear regression in Graphpad prism 8 software. Chi-square test was used to compare changes in percentage between two groups. Cluster3 and Treeview software and programs were used to graph heatmap and hierarchical clustering analysis (HCA). The R software  was applied in bioinformatics analyses. The generalized linear model (GLM) with binomial distribution was used to predict disease and control status based on lipid concentration by R function of GLM. The package of ROCR was used to estimate, sensitivity, specificity, recall, precision, F-measure, and area under curve (AUC). Information gain (InfoGain) was determined with simple logistics classification algorithm (a supervised attribute ranking method). The significant p value was set at 0.05 for all results.Statistical analysis was carried out in SPSS software (IBM SPSS Statistics 24 software). The mean concentrations of PL parameters between the two groups were compared by independent samples To monitor the stability of the detection system and to minimize technical errors originating from sample collection and sample preparation , ten repwwt fresh-frozen hepatic tissue. Another was in nmol/mg protein. The correlation and regression analyses were performed on the concentrations of total PLs in 13 classes between two methods. The results indicated that two methods are linearly correlated and this correlation was highly significant  as shown in Fig. To further prevent errors and to quantitate accurately and reliably, the concentration of each PL species in each sample was determined by two methods. One method was to calculate PL concentration in nmol/mg p = 0.00010), female population , and male population . Top ten most abundant individual PL species account for more than 50% of total PLs in both BHT and HCC in all studied populations. In the level of PL class, among 13 PL classes, PC was the most abundant, accounting for more than 50% of total PLs, and PE-Cer was the least abundant, accounting for 0.01% or less of total PLs in both BHT and HCC in all studied populations  for the 62 species shown in Fig. In female population, significant differences in concentrations between BHT and HCC were only seen in seven PL species: PC 30:0), ePS (38:3), LPC (22:5), ePC (34:0), ePE (34:0), PC (36:1), and PI (42:6) as shown in Fig. 0:0, ePS In male population, significant differences in PL concentrations between BHT and HCC were seen in 59 out of 320 individual PL species. Unlike in female population, there were more PL species significantly lower in HCC than in BHT in male population: among 59 individual PL species, 11 PL species were significantly increased, and 48 were statistically decreased in HCC than in BHT. The hierarchical cluster analysis (HCA) for the 59 species as shown in Fig. Taken together, in all population and male population, more PL individual species were significantly decreased in HCC than in BHT, and these decreased PL species more related to younger male HCC patients in late stage, but not to HCC grade. On the contrary, in female population, more PL individual species were significantly increased in HCC than in BHT.p = 0.0157 and p = 0.0156, respectively), and stratified male population . None of PL classes was statistically different between BHT and HCC in female population. While majority of PL classes were lower or unchanged in HCC than BHT, PE-Cer is the only PL class being obviously higher in HCC than in BHT in all studied populations, though the differences were not statistically significant.In the level of PL class, total ePE and total PE were significantly lower in HCC than in BHT in all population (p = 0.0157) and stratified male population (p = 0.0076), but not in stratified female population.PLs in all subgroups were lower in HCC than in BHT in all studied populations. However, significantly decreased PLs in HCC than in BHT was only seen in ethanolamine-containing subgroup in all population  PL concentration was higher than detection limit (0.002 nmol/mg wwt) in at least one of BHT and HCC; (2) the differences in PL concentrations between BHT and HCC were statistically significant, no matter it is higher or lower in HCC; (3) all items of predicting power, especially sensitivity, specificity, and accuracy (AUC) were above 60% simultaneously. The PLs that fulfilled these criteria were all PL individual species, including six in all population: ePC 34:2), ePC (36:2), PC (30:0), PC (34:2), PE (34:2), and SM (24:0); two in female population: PC (30:0) and PS (42:9); six in male population: PA (36:4), PC (30:0), PC (34:2), PE (34:3), PE (38:6), and PI (36:2), as shown in Fig. :2, ePC , which was the only identified PL biomarker that significantly higher in HCC than in BHT in all studied populations. Another one was PC 34:2), the most abundant PL species was lower in HCC than in BHT in all studied populations. Both increased PC (30:0) and decreased PC (34:2) in HCC were of strong diagnostic and prognostic powers for HCC, respectively. To test if the diagnostic and prognostic powers can be synergistically enhanced through the combination of these two PL biomarkers, the ratio of PC (34:2) to PC (30:0) was derived and evaluated in all studied populations. Analyses with methods in statistics and bioinformatics demonstrated that the ratio of PC (34:2) to PC (30:0) had much stronger power in the differentiation of HCC from BHT  to PC (30:0) are not only able to differentiate HCC from BHT with high sensitivity, specificity, and accuracy but also are able to predict the progression of HCC in terms of clinical stage and pathological grade. As compared to female population, male population had more gender-specific PL biomarkers in the differentiation of HCC from BHT and in the prediction of HCC progression.This study first employed ESI–MS techniques in determining global PL profiles in paired BHT and HCC samples from same female and male patients. We found that (1) the concentrations of PLs in total, in most subgroups and classes, and in majority of individual species were lower in HCC than in BHT; (2) such decreases of PLs in HCC were more prominent in male patients than in female patients; (3) a panel of individual PL species can be used as diagnostic and prognostic biomarkers for HCC, either common to all population, or specific to stratified female and male populations.Phospholipids are essential components of all cells. Previous studies suggested PLs increase with cell transformation and tumor progression in many cancers such as breast cancer and colorectal cancer cells , 12. Nonp = 0.017) and stratified male population (p = 0.009). Phosphatidylcholines (PC) accounted > 93% choline-containing PLs (sphingomyelins were not included in this study) in both BHT and HCC in all studied populations. Decrease in PC and other choline-containing PLs in HCC might imply that the metabolism of these PLs favors degradation catalyzed by PC-specific phospholipase D in Kennedy’s pathway. On the other hand, the expression level of choline kinase-α, catalyzing the synthesis of phosphocholine is elevated in HCC [Each PL subgroup contains same head moiety. We found that the concentrations of PLs in all subgroups were lower in HCC. However, among PL subgroups, statistical difference was only seen in choline-containing subgroup between BHT and HCC in all population p = 0.0 and strad in HCC  and othed in HCC .Sphingobacterium spiritivorum ATCC 33861 can induce endonucleolytic DNA cleavage and apoptotic activity in human myeloid leukemia HL-60 cells in vitro [Ceramide phosphorylethanolamines (PE-Cer) are a group of sphingolipids that play critical roles in non-mammalian eukaryotes, similar to that of sphingomyelins in mammals . Althougin vitro . PE-Cer in vitro . PerhapsEffective therapies are feasible with surgical resection if HCC is diagnosed at an early stage . HoweverInterestingly, PC (30:0) was found to be more abundant in breast cancer area as compared to the stroma surrounding the cancer, and considered as abnormal lipid metabolism-generated metabolites that promote cancer metastasis . Kurabe The incidence, morbidity, and mortality rates of HCC are obviously disparate between male and female populations , 29. TheMale gender, onset of HCC at younger age, late clinical stage, and higher tumor grade are all associated with poor prognoses of HCC. In agreement, our study indeed revealed that decreased hepatic PLs were more related to male sex, younger age at onset, and HCC in late stage, but not to HCC grade. This could be due to the fact that only 2 out of 32 cases were classified as Grade III, and the majority of cases were classified grade I and grade II, according to the Edmondson–Steiner Classification of HCC.In conclusion, the findings in this study could provide new evidence in understanding the roles of PLs in oncogenesis, progression and gender disparities, in exploring new PL biomarkers in diagnosis and prognosis, and in discovering novel therapeutic targets for HCC.Supplementary file1 (XLSX 250 kb)Below is the link to the electronic supplementary material.


N = 1,364), as well as from their partners and children. Retrospective childbirth experience was measured 3 months postpartum with a latent factor consisting of five items asking about the feelings that women have about their childbirth. Parental stress was measured at 3 months postpartum for partners and 3 and 12 months postpartum for mothers using the adult domain of the parental stress index (PSI). Finally, parent-child attachment is observed in a subsample of 223 women and children at 12 months postpartum with the Strange Situation Procedure (SSP). Results show that women’s birth experience was significantly related to both mothers’ and their partners’ parenting stress. However, birth experience was not related to child attachment, neither directly nor indirectly via maternal stress. These findings emphasize the long-lasting impact that childbirth may have on both parents. Future research is still needed to further investigate which protective factors may weaken the association between birth experience and parental stress.Women, as well as their partners, can experience childbirth in many different ways. A negative childbirth experience may have adverse effects on the entire family, resulting, for instance, in parental stress symptoms and a weakened parent-child relationship. Parental stress, without sufficient resources to compensate for it, may also in and of itself negatively influence the parent-child relationship. This study contributes to the current knowledge of the psychological effects of childbirth experience by using longitudinal data collected with both self-reports and observational measures, as well as multiple informants . The aim of this study was to investigate whether 1) women’s retrospective birth experiences were related to maternal and paternal parenting stress, 2) birth experience was indirectly associated with child attachment via maternal stress, and 3) birth experience was directly related to child attachment. Data were collected from a mixed sample of community and at-risk primipara women ( Childbirth may be experienced in many different ways. For most women, giving birth is a positive experience, but it can unfortunately also be experienced as negative, sometimes even as traumatic , 2. NegaBased on previous research, the prevalence of women’s negative childbirth experiences ranges from 6.8 to 44% , and up Family stress theory , 17 statThe second factor impacting the level of parental stress is the parents’ evaluation of the stressor . This inThe third factor influencing parental stress levels in response to an event refers to a combination of personal resources, coping resources, and ability to adapt to a new situation . KnowledN = 56; , as well as from their partners and children, who participated in the longitudinal Generations2 project (www.generaties2.nl/) that takes place in the Netherlands, in the Amsterdam area. The ‘large cohort’ consisted of all participants in the study , women in the ‘normative group’ who took part in the more extensive measurements , and women in the ‘at-risk’ group who also took part in these more extensive measurements, and had additionally reported experiences with youth care, psychologists, or psychiatrists before the age of 18. A prenatal diagnosis for a congenital abnormality of the fetus was an exclusion criterion for all three groups. For the second objective of this study, we combined the data of the ‘normative’ and ‘at-risk’ subgroup of women and children from whom the attachment data was collected (N = 223). This was done to obtain a larger and more representative sample regarding both demographics and attachment outcomes. Later in the text we refer to these subgroups as a ‘focus sample’ and to the entire group as a ‘cohort sample’. In the cohort sample, women were on average 29.5 years old . The majority of them were born in the Netherlands (91.3%), were either married or cohabiting (90%), and had completed a tertiary education (88.3%). Of the children, 49.2% were boys and 50.7% girls. For two children, gender was not reported. Descriptive information of both samples are presented in Data were collected from a mixed sample of community  and at-risk primipara women .Parenting stress was assessed using the parent domain of the Dutch version of the Parenting Stress Index PSI , 38], wh, wh38)],Infant-parent attachment was measured with the Strange Situation Procedure , a goldParticipants were recruited via the study website and midwifery practices in Amsterdam and the surrounding area. Women in the ‘at-risk’ group were additionally recruited from youth care facilities and institutes. Data were collected via questionnaires at 3 months  and at 12 months  postpartum, as well as during an assessment at the research facility shortly after the child turned one year . Participants received the questionnaires and returned them to the research team. If questionnaires were not returned, participants received a reminder email and, if needed, were contacted by phone. All participants signed a written informed consent, and for participants under the age of 18 years, a written informed consent was obtained also from parents and/or other legal guardians.An online power calculator developed specifically for structural equation models  was usedp-values, which suggests that the missing scores were missing completely at random. Second, the Spearman correlation test was used to investigate whether the five items on women’s birth experiences were related with each other. All items showed significant (p <.01), though not strong correlations with the other four items both in the cohort (r = 0.18–0.51) and the focus sample (r = 0.17–0.45).The preliminary data exploration was done using IBM SPSS Statistics (version 26.0). First, Little’s MCAR test was used to analyze the patterns of the missing scores. The test resulted in non-significant Further analyses were conducted with the R package 'lavaan' , which iPrior to the main analyses, we tested whether the five dichotomous items on birth experience could be combined into one latent variable. This is an important step in order to estimate whether the five items assess the same underlying construct. In lavaan, the factor loading of the first indicator of a latent variable  is fixed to 1 by default. Factor loadings for the other variables were left free. In addition, residual variances were added automatically to all variables. This analysis was done separately within the cohort and the focus sample (subgroups normative and at-risk).The first study objective was to investigate whether the latent variable on birth experience was associated with continuous variables on paternal and maternal stress. The latent variable on birth experience resulting from the previous step was used. First, the association between birth experience and maternal stress was assessed. In a second step, paternal stress was added to the model. Paternal stress at 3 months after birth and maternal stress at 12 months after birth had a covariance between them. Finally, residual variances were added automatically. These analyses were performed with the cohort sample. To ease comparability with research questions 2 and 3, the analyses were repeated with the focus sample, although these analyses were underpowered.r = .73). We decided to use the latent variable on maternal stress instead of maternal stress at 3 or 12months in order to resemble more trait-like  stress instead of state-like  stress. Residual variances were added automatically. This analysis was done with the focus sample, as attachment data were collected for this subgroup only.The second study objective investigated whether birth experience was indirectly associated with attachment. First, we tested whether the latent variable on birth experience was indirectly associated with the dichotomized (secure-insecure) indicator of child attachment via a latent variable on overall maternal stress, and whether the latent variable on birth experience was directly associated with child attachment. Maternal stress loaded on the variables maternal stress at 3 months after birth and maternal stress at 12 months after birth. Mean maternal stress scores at both time points were almost similar, respectively M = 119.2 (SD = 31.8) at 3 months and M = 119.9 (SD = 30.7) at 12 months. Further, the correlation between maternal stress scores at both time points was very high . This analysis was also done with the focus sample.First, structural equation modeling was used to test whether the latent variable on birth experience had a good fit in the cohort and the focus sample. The fit was comparable in both samples: CFI was .98 in the cohort and in the focus sample, TLI was .96 in the cohort and .97 in the focus sample, RMSEA was .09 in the cohort and .07 in the focus sample, and SRMR was .07 in the cohort and .07 in the focus sample. Adding a covariance between the thematically linked fourth and fifth items improved model fit. This modification can be explained and understood theoretically, as these two items focus specifically on the fear that a woman may have had for her own or her baby’s life, while the other three items assess more general emotions (both positive and negative) related to the birth experience. Model fit was excellent in both samples: CFI was 1.00 in the cohort and in the focus sample, TLI was 1.00 in the cohort and 1.02 in the focus sample, RMSEA was .00 in the cohort and in the focus sample, and SRMR was .01 in the cohort and .03 in the focus sample. Results are presented in p <.04), whereas maternal age, marital status, and child gender were not . We therefore performed the analyses for the first research question with and without education level and non-Dutch origin as potential confounders in the model. Structural equation modeling was used to test the first study objective: whether birth experience was associated with maternal and paternal stress. Analyses were repeated with four slightly different models  in the cohort sample, which all had good model fit  and the model with maternal and paternal stress . Results showed slightly lower effect sizes for maternal stress (β = .12) and paternal stress (β = .14), which failed to reach significance. However, given that these analyses were underpowered in the focus sample and effects were in the same direction, results remain inconclusive.β = .01, p = .70) nor directly .For the second study objective, structural equation modeling was used to test whether birth experience was indirectly associated with child attachment via overall maternal stress . Additionally, it was tested whether birth experience was directly associated with child attachment. For the first analysis, four-way attachment classifications were dichotomized into secure (51%) and insecure (49%). The underlying distribution of the insecure group was 12% avoidant, 14% resistant, and 23% disorganized. The results showed a good model fit . Yet, birth experience was neither associated with child attachment indirectly via maternal stress  nor directly .For the second analysis, the continuous scale for attachment disorganization was used. The results showed a good model fit . Similar to the previous analysis, birth experience was neither associated with child attachment indirectly via maternal stress  and direct effect of birth experience on parent-child attachment. Our first hypothesis regarding the association between negative birth experience and parenting stress was confirmed. We found that mother’s negative birth experience was indeed positively associated with both the mother’s and partner’s parental stress. However, birth experience was neither associated with child attachment indirectly via maternal stress nor directly.N = 56; , which, in turn, may lead to more sensitive parenting practices . FurtherFinally, the relatively low power in the analyses in which child attachment was included may explain why we did not find a significant indirect or direct effect of birth experience on child attachment. Collecting observational attachment data requires intensive measures, which makes it difficult to gather large sample sizes. The current sample size would have been large enough to find a medium effect, but not to detect a small effect. The effect sizes were as small as .01 for the indirect effect and .04–.07 for the direct effect. Thus, power was not sufficient to detect this effect. However, it is debatable whether effect sizes this small would have any clinical relevance, should they be significant.The current study has several strengths that ought to be mentioned. First, a longitudinal study design has its benefits over a cross-sectional design, as it may give indications for causal relationships. Second, previous research on birth experience and child attachment have been strongly mother-focused, whereas the current study included also parenting stress data from partners. Third, unlike in a majority of the previous studies, we used a gold standard observational measure to assess child attachment, which adds new information on the attachment relationship to the literature on mother-reported bonding and sensitivity. Finally, in the past years, the number of studies focusing on psychological aspects of childbirth has been increasing and so has the number of available instruments assessing childbirth experiences. However, up to date, there is no gold standard assessment for birth experience . In the However, this study also has limitations that need to be considered when generalizing the findings to other samples. First, although previous studies included women with both negative and traumatic birth experiences, it is possible that, in the current sample, even the most negative birth experiences were not necessarily traumatic. For instance, as can be seen in This longitudinal study, combining both self-reported and observational data, as well as multiple informants , investigated the effects that a mother’s birth experience may have on maternal and paternal stress. Furthermore, this study investigated whether birth experience is indirectly associated with child attachment via maternal stress, and whether birth experience is directly associated with child attachment. Both self-reported maternal and paternal parenting stress were found to be associated with a mother’s self-reported birth experience, even until a year after birth. However, self-reported birth experience was neither indirectly nor directly associated with observed infant-parent attachment. These findings emphasize the impact that childbirth may have on both parents, and this way on the family functioning. Thus, after a family has went through a negative birth experience, care professionals ought to support both parents and the couple’s relationship, instead of focusing only on the new mother. Furthermore, the long-lasting effect of a negative birth experience on parental stress emphasizes the need for a strong collaboration between various care sectors that are involved in a family’s life during the first year postpartum . Future research is needed to further investigate which protective factors may weaken the association between birth experience and parental stress, as this will hopefully enable better support for families who have experienced a negative or traumatic childbirth experience.10.34894/IETFLC.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found here: doi: The studies involving human participants were reviewed and approved by Medical Ethical committee of the VU Medical Centre (METc), registration number NL24319.029.08. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors contributed to the presented idea  and the theoretical framework. MV and MO were involved in data collection for the large longitudinal study that offered data for the current study. MV initiated and verified the analytical methods. AH conducted the analyses and all authors discussed the results. AH designed the figures and tables. AH wrote the manuscript with critical feedback and input from MV and MO.This study was supported by a grant from the foundation Stichting tot Steun Netherlands and a grant from the Netherlands Orgqanization for Scientific Research .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: To study factors that influence the age of discharge of very low birth weight (VLBW) preterm neonates from the Neonatal Intensive Care Unit (NICU).Setting: This cohort study was conducted in the NICU, Department of Pediatrics, Kalinga Institute of Medical Sciences, Bhubaneswar, India, from April 2019 to December 2019.Patients: Neonates whose birth weight was <1500 g and gestation <37 weeks were enrolled. Those having major congenital malformation were excluded.Outcome: Age at discharge (in days).Results: Out of 114 neonates enrolled, 84 neonates completed the study and discharged, 29 neonates died during the study, and one patient left against medical advice. Demographic, antenatal, neonatal factors, and mother’s participation in care of the baby were compared with respect to age at discharge in univariate analysis. Those found significant on univariate analysis were subjected to multivariate analysis. In the multivariate analysis model two independent predictors were observed, birth weight and gestation, which were inversely related to age at discharge.Conclusion: Birth weight and gestation are the two most important factors which significantly influence the age at discharge from hospital of VLBW preterm neonates. India has a large burden of very low birth weight (VLBW) babies, i.e. those who are <1500 g, account for only about 5% of all live births [Provision of prolonged neonatal intensive care consumes both financial and medical resources and also increases the risk of nosocomial infections, disturbances in biological rhythm, poor parent-infant bonding, and possible failure to thrive after discharge. The current availability of NICU beds in India at present is far less than the required number. In order to meet the demands, there has been an increasing trend towards early discharge of neonates from NICUs.A study from South Africa demonstrated that it was possible to discharge VLBW infants when they achieved weights of 1650 g or more . There iThe study was conducted in the NICU, Department of Pediatrics, Kalinga Institute of Medical Sciences, Bhubaneswar from April 2019 to December 2019. This was a cohort study that comprised a birth cohort of preterm neonates weighing <1500 g followed until discharge from the hospital. The neonates included in the study were inborn neonates who fulfilled the following criteria: birth weight <1500 g amd gestation <37 weeks. Amongst the neonates who fulfilled the above criteria, those having major congenital malformation were excluded. Primary outcome of the study was length of hospital stay (in days) as determined from the date of birth until the date of discharge. The study used a standardized discharge criteria for all neonates enrolled in the study. The newborn had to fulfill all the following criteria to be eligible for discharge from the hospital neonatal intensive care unit: 1) infant feeding completely from breast and/or spoon/cup, 2) weight gain of at least 15 g/day over three consecutive days, 3) clinically well , 4) maintaining normal body temperature on own, and 5) not on any injectable medication.Since length of stay was likely to be influenced by birth weight of the newborn, the sample size was calculated to detect a difference in the mean length of stay between neonates with birth weight <1250 g and those with birth weight 1250-1499 g. In order to detect a difference of 14 days in the mean length of stay between neonates in the two weight categories with an absolute precision of five days with a confidence of 95%, it was calculated that 45 subjects needed to be enrolled. It was decided a priori that the study would enroll at least 50 VLBW preterm newborns.Data collectionIn all enrolled infants the following data were collected. All data were entered into a structured pretested performa. Data collection at enrollment were: 1) demographic data included maternal age, parental education, occupation, income and housing condition, 2) antenatal and intranatal data , and 3) neonatal data- at enrollment of neonate birth weight, gestational age  software. A univariate analysis was used to determine significant risk factors for length of stay. Those found significant on univariate analysis were analysed for independent prediction of length of hospital stay using a multivariate linear regression model. A subset analysis of risk factors for early and late discharge was also done. Continuous normally distributed data were analysed using student t-test and nonnormal data by the Mann-Whitney test. Proportions were compared by Chi-square or Fisher exact test. For all comparisons a probability of 5% was considered significant. Ethical approvalThe institutional ethics committee approved the study protocol.One hundred thirty preterm newborns weighing <1500 g were screened, of whom 114 were enrolled in the study. Reasons for non-enrollment are detailed in Figure There were 84 (73.6%) newborns that completed the study. Table Early vs. late dischargeIt was observed that infants in the early discharge group had significantly higher birth weight, higher gestation, had less resuscitation need, achieved enteral feed volume earlier, and transited to cup and breastfeeding earlier Table . The latAlmost all the mothers were available for care of their infants, except for two in the late discharge group who never participated in baby care because of illnesses . There was no significant difference between the groups with regard to the percentage of time mothers were available for infant care or the age at which mother began to get involved in care of her infant in the NICU.Length of hospital stay (determinants)Multiple linear regression was carried out using length of hospital stay (in days) as the dependant variables and all other variables as independent predictors. The two independent predictors observed were birth weight and gestation, which were inversely related to length of hospital stay Table .VLBW preterm care is still a huge challenge in developing countries like India because of financial constraints and limited medical resources. Our study focuses on several maternal, social, and neonatal factors that are likely to determine the length of stay of VLBW preterm neonates in the NICU. Out of the factors studied, birth weight and gestation were found to be most significant which influence the timing of discharge of these babies. In a large population-based cohort, Altman et al.  observedIn the Kotgal study , the meaIn a more recent study from under-resourced settings in Southern Africa, Mokhchane et al.  noted thThe study by Altman et al.  looked aPulver et al.  observedHintz et al.  had lookAltman et al.  considerIt may be concluded from the present study that birth weight and gestation have independent correlation with length of stay especially when minimum fixed criteria for early discharge using multiple parameters is utilized. Therefore units which handle infants with smaller gestation and smaller weight tend to have longer length of stay of these infants.