We aimed to investigate whether gestation at birth, birth weight, and head circumference at birth are still associated with brain volume and white matter microstructure at 9–10 years in children born late-preterm and at term. One hundred and eleven children born at ≥ 36 weeks gestation from the CHYLD Study cohort underwent brain magnetic resonance imaging at 9 to 10 years. Images were analysed using FreeSurfer for volumetric data and tract-based spatial statistics for diffusion data. Of the cohort, 101 children were included for volumetric analysis . Shorter gestation at birth, lower birthweight, and smaller birth head circumference were associated with smaller brain volumes at 9 to 10 years, both globally and regionally. Amongst the perinatal factors studied, head circumference at birth was the strongest predictor of later brain volumes. Gestation at birth and absolute birthweight were not associated with diffusion metrics of white matter skeleton. However, lower birthweight z-score was associated with higher fractional anisotropy and lower radial diffusivity. Our findings suggest that even in children born late preterm and at term, growth before birth and timing of birth are still associated with brain development in mid-childhood. Preterm birth interrupts this pattern of normal brain development in the in-utero environment, and is associated with smaller brain volumes and altered white matter microstructure during early childhood and adolescence3. For example, children born late preterm (34 to 36 weeks) had smaller volumes of the total tissue  and thalamus compared with their term-born peers at 6 to 13 years2. Size at birth is also related to brain volumes throughout childhood and adolescence in children born preterm4. However, to our knowledge, few studies have explored these relationships in those born at or near term6. Furthermore, it is unclear whether slower growth before birth, leading to lower birth weight, the timing of birth, or both, is important for later brain growth, and little is known about the relationships between gestation or size at birth and white matter microstructure during childhood.Fetal brain development is a complex process that occurs continuously throughout pregnancy. During the third trimester, brain size increases four-fold, accompanied by the formation of tertiary sulci and gyri7. One study has reported a positive association between head circumference at birth and total brain volume at ten years in children born at term8.Head circumference is a commonly used indicator of brain size during infancy and early childhood, and there is a weak association between head circumference during childhood and concurrent brain volumes9. The impact of preterm birth on later brain growth is also greater in boys than girls; but it is not known whether there are any sex differences in the relationship between size at birth and later brain development.Boys are at increased risk of preterm birth and have larger head circumference than girls from the second trimester onwardsIn this study, we aimed to investigate the relationships between gestation at birth, birthweight, and head circumference at birth, and brain volume and white matter microstructure in school-aged children born late-preterm and at term using magnetic resonance imaging (MRI). We hypothesised that children born at shorter gestation, at lower birth weight, and with a smaller head circumference at birth would have smaller brain volumes and also would have altered white matter maturation at 9 to 10 years. Additionally, we investigated whether these relationships differ in boys and girls.11. Infants with serious congenital malformations or terminal conditions were excluded11.The Children with HYpoglycaemia and their Later Development (CHYLD) study is a prospective cohort investigation of moderate to late preterm and term neonates born from December 2006 to November 2010 at Waikato Hospital, Hamilton, New Zealand at ≥ 32 weeks’ gestation and with one or more risk factors for neonatal hypoglycaemia 12.All surviving children who had not previously withdrawn were invited to undergo a neurodevelopmental assessment at 9 to 10 years’ corrected age between 2018 and 2020. Details can be found elsewhere13. Up to 75 eligible children in each hypoglycaemia group were randomly selected using random number tables by a staff member who was not involved in the MRI study. Parents of these randomly selected children were asked for consent to be approached about the MRI study at the time of seeking consent for the neurodevelopmental assessment. Those who consented were then approached in order of the children’s dates of birth until there were no further families who had agreed to be approached in the group, or until 30 MRI studies had been completed in each group , mild (1 to 2 hypoglycaemic events 2.0 to 2.6 mM), severe (any hypoglycaemia events < 2 mM or ≥ 3 hypoglycaemic events), or clinically undetected hypoglycaemia 13, and are not explored in this paper. The complete study was approved by the Health and Disability Ethics Committee (16/NTB/208/AM02), the parents or legal guardian gave written informed consent, and the child gave verbal assent. All methods were carried out in accordance with relevant guidelines and regulations.The effects of neonatal hypoglycaemia on brain outcomes and white matter microstructure are described elsewhere3 isotropic voxel resolution, slice thickness = 0.85 mm, repetition time (TR) = 2000 ms, echo time (TE) = 3.51 ms, field of view (FoV) = 210 mm, 176 slices, and flip angle 9°) and diffusion-weighted images  were acquired between 2018 and 2020 using 3 T scanner  equipped with a 20-channel head coil.The T1-weighted structural (0.8mmhttp://surfer.nmr.mgh.harvard.edu/). The complete cortical and subcortical reconstructions of the T1-weighted images were performed using the ‘recon all’ script, as described elsewhere15. Briefly, the procedure includes the removal of non-brain tissue using the watershed algorithm, automated Talaraich transformation, segmentation of the subcortical white and deep grey matter-volumetric structures, intensity normalisation, tessellation of the grey/white matter boundary, automated topology correction, and surface deformation. Then, the images were registered to a spherical atlas based on individual cortical folding patterns to match cortical geometry across participants, and the cerebral cortex was parcellated into 34 regions per hemisphere based on the sulci and gyri16. For the volumetric measures, global ; regional , cerebellum, and brainstem were considered. For regional and cerebellum measures, both the left and right sides were combined together.All structural imaging data were processed using the software tool: FreeSurfer 17. Images were corrected for eddy currents and head motion, and non-brain tissue was stripped. Following quality control of the diffusion-weighted images, the diffusion toolbox was used to fit the tensor model at each voxel and compute fractional anisotropy (FA), axial diffusivity (AD), mean diffusivity (MD), and radial diffusivity (RD) maps.The diffusion-weighted images were quality controlled and processed with the FSL diffusion toolbox  in FSL19. The socio-economic decile was derived from the New Zealand deprivation index (NZDPI), where 10 indicates the most and 1 the least deprived20. Baseline demographic characteristics and clinical measurements were compared between groups using the Students’ t-test and one-way analysis of variance for continuous data and Pearson’s Chi-square or Fisher’s exact tests for categorical data. Linear regression analyses were used to identify the relationships between gestation at birth, birth weight, head circumference at birth and global and regional brain volumes. As this was an exploratory study, we did not perform any adjustments for multiple comparisons.Birth weight and absolute head circumference at birth were obtained from clinical records. Birth weight was measured naked using calibrated electronic scales to the nearest 10 g. Birth head circumference was measured at the maximal occipitofrontal circumference with a non-stretchable tape to the nearest 0.1 cm. Z-scores were calculated using New Zealand population reference rangesAdditionally, to investigate the independent effects of perinatal factors on brain volumes, we created two models: Model 1 where both gestation at birth and birthweight z-score were included, and Model 2 where both gestation at birth and head circumference z-score were included.p ≤ 0.05, corrected for multiple comparisons using family-wise error, and thresholded with threshold-free cluster enhancement option18. All statistical analyses were performed using JMP version 14.0 , except for diffusion data which was analysed using FSL.For the diffusion data, generalised linear models were used to assess the relationships between gestation at birth, birth weight, and diffusion metrics  in TBSS using FSL’s ‘randomise’ permutation tool. The results were considered significant if Of the 248 eligible children, the parents of 170 agreed to be approached about MRI, and 111 children underwent an MRI scan. Of these, 10 were excluded due to excessive motion artefacts, processing errors or missing sequences, 101 children (49 (49%) boys; median age, 9.5 (range: 8.9 to 12.4) years) were included in the volumetric analysis and 98 in the diffusion analysis  , absolute birthweight , birthweight z-score , absolute head circumference at birth , and head circumference at birth z-score  were positively associated with total brain volume  = 25,329 mm2 0.05 to 0.20).However, the correlation coefficients for all these associations were weak .All partial coefficients for these associations were weak  of white matter skeleton overall or in boys and girls separately, and the sex interaction terms were not significant. However, the birthweight z-score was negatively associated with FA and positively associated with RD in the right superior longitudinal fasciculus and right temporal part of the superior longitudinal fasciculus Fig. . BirthweWe further investigated the relationship between birthweight z-score and FA of white matter skeleton separately in children of diabetic mothers and children with other risk factors for hypoglycaemia . We found that a lower birthweight z-score was associated with FA of white matter skeleton in the left inferior longitudinal fasciculus and inferior fronto-occipital fasciculus in children with other risk factors for hypoglycaemia but not in children of diabetic mothers, but the interaction terms were not significant .In this prospective cohort study of children born at 36 to 42 weeks’ gestation, we found that shorter gestation at birth, lower birthweight, and smaller head circumference at birth were associated with smaller brain volumes both globally and regionally at 9 to 10 years. Interestingly, both birthweight and head circumference at birth were associated with later brain volumes in these children, even after taking gestation at birth into consideration. Although gestation at birth was not associated with any of the diffusion metrics of the white matter skeleton, lower birthweight z-score was associated with higher FA, particularly in long association fibres. These associations were similar in girls and boys. Overall, these findings indicate that, even amongst those born late preterm and at term, size at birth has a persisting influence on later brain development.6. Our findings support the likely benefit of longer gestation, even across the last few weeks of pregnancy, for the fetal central nervous system, where much of the maturation takes place during the third trimester, including dendritic arborization, synaptic development, axonal outgrowth, glial formation, neuronal circuitry, and connectivity formation1. Fetal MRI studies have also shown a positive association between gestational weeks and brain volumes over the period from 18 to 39 weeks21. Taken together, our findings likely reflect the fact that intrauterine influences, such as oxygenation, circulation, maternal and placental hormones, and growth factors22, which are important for fetal brain growth during the third trimester, are abruptly interrupted at birth, even in children born at 36 to 42 weeks’ gestation.We found an association between gestation at birth and brain volumes at 9 to 10 years, consistent with previous studies reporting a similar association between gestation at birth and brain volumes during mid-childhood4. Interestingly, these relationships remained significant, although weak, even when gestation at birth was taken into consideration. Previous studies have hypothesised numerous factors, such as placental factors, maternal nutritional status, stress, ethnicity, age, body weight, parity, and environmental exposures during pregnancy24, that could be associated with poor growth of the fetus in the in-utero environment. Our findings suggest that growth before birth might have a small but persisting influence on later brain volumes at 9 to 10 years in addition to that of gestation at birth.We also found an association between birthweight and brain volumes at 9 to 10 years, which is consistent with previous studies reporting an association between lower birthweight and smaller brain volumes during childhood and adolescence8. Interestingly, when we combined gestation at birth and head circumference at birth z-score in the same model, only head circumference at birth z-score remained significant and independently associated with total and most of the regional brain volumes studied at 9 to 10 years of age, suggesting that brain growth before birth, in particular, has a small but persisting influence on later brain volumes. Additionally, our findings suggest that head circumference is the strongest predictor at birth for later brain volumes in children.Head circumference at birth is considered an indicator of brain development and is widely considered an index of brain weight over the entire course of pregnancy. Our results demonstrated a positive association between head circumference at birth and brain volumes at 9 to 10 years, consistent with earlier studies showing a strong relationship between head circumference at birth and brain volumes during infancy and early childhood25. Although the reason for the finding is unclear; it might reflect the postnatal acceleration of white matter after birth. Earlier studies have shown that white matter structures are immature at birth and are surrounded only by premyelin sheaths. Myelination of white matter begins from 54 postconceptional weeks onwards and increases rapidly during the first postnatal year, then continues until late adolescence27. Overall, our results suggest that white matter development that takes place postnatally may not be disrupted by being born late preterm or early term.We expected a positive association between the gestation at birth and birthweight and FA of white matter skeleton, as measured by diffusion imaging at 9 to 10 years. However, we found no association between gestation at birth and diffusion metrics of the white matter skeleton, consistent with an earlier study showing no association between gestational age at birth and FA of the white matter skeleton in 8-year-old children born at term28. In normal brain development, FA generally increases with age from childhood to adulthood in most of the white matter fibres, whereas RD generally decreases with age, because the water content in the extracellular space decreases, resulting in a decrease in RD, while the relative increase in the intracellular water content is restricted perpendicular to the myelin membranes, but not parallel to them, causing an increase in FA29. Previous studies in adults born small for gestation age at term, and in adults born preterm and very low birthweight, have reported lower FA in short-and long-association fibres compared to healthy controls born at term3. Therefore, we expected lower birthweight z-score to be associated with lower FA and higher RD of white matter skeleton. However, in our cohort, most of the large babies were born to diabetic mothers. A study in children of diabetic mothers has reported abnormal white matter microstructure at 33 and 36 weeks of gestational age, especially in the deep white matter regions, compared to children of non-diabetic mothers30. For this reason, we carried out a post-hoc analysis and found a negative association between birthweight and FA of white matter skeleton only in children with other risk factors but not in children of diabetic mothers. Therefore, any potential effects of maternal diabetes on offspring white matter microstructure are unlikely to explain our findings. Alternatively, given the number of analyses, our results could be due to type-1 error.Contrary to our hypothesis, lower birthweight z-score was associated with higher FA and lower RD of the white matter skeleton at 9 to 10 years in long association fibres . To our knowledge, none of the earlier studies have studied this association between birthweight z-score and white matter maturation in mid-childhood in children born at ≥ 36 weeks. FA is a measure of the intravoxel directionality of white matter anisotropy, whereas RD measures diffusion perpendicular to the white matter fibres and is associated with myelin injury and/or decreased myelination31. Our findings suggest that this “brain sparing” effect is incomplete and that even mild degrees of intrauterine growth restriction are associated with smaller brain size to mid-childhood. Lastly, head circumference at birth is the strongest predictor of later brain volumes in children, at least to mid-childhood.The findings of this study have some clinical significance. Firstly, longer gestation, even amongst those born at term, has potential benefits for later brain development. Secondly, this study emphasises the importance of prenatal growth for later brain development, both globally and regionally. Earlier studies have reported fetal ‘brain-sparing’ and rapid postnatal catch-up growth, particularly in head size, in children with intrauterine growth restrictionThis is one of the first studies to investigate the association between specific perinatal factors and brain development and maturation at 9 to 10 years in children born late-preterm and at term. We included a large sample of children who had standardised anthropometric measurements at birth. However, this analysis has some potential shortcomings. It was an observational study on a subset of children from a large prospective cohort study, and few data were available regarding the many potential confounding prenatal and postnatal influences on brain development such as nutritional status, educational training, home environment, sleep, physical activity, etc. Although we were unable to take these factors into account in our analysis, they generally might be expected to weaken the relationships between perinatal events and later brain development. Thus our findings that these relationships are weak but still detectable at 9 to 10 years of age emphasise the importance of prenatal events for later brain development. In addition, all our included children were born at risk of neonatal hypoglycaemia and therefore, our findings cannot be generalised to other children born late preterm and at term.Gestational age and size at birth are both associated with later brain development, even across late preterm and term gestations, suggesting that both growth before birth and duration of gestation are important for later brain development.Supplementary Information.


Preterm birth is a leading cause of infant morbidity and mortality; its multifactorial causes are an obstacle to understanding etiology and pathogenesis. The importance of cytokines and inflammation in its etiology and association with the short cervix is nowadays well-proven. To date, there are no reliable biological or biochemical markers to predict preterm birth; even though the length of the cervix has high specificity, its sensitivity with the cervix below 2.5 cm is low.We study the association of plasma cytokine levels and cervical length in search of predictors of preterm birth.We evaluated a total of 1400 pregnant women carrying a single fetus between 20 and 25 weeks of gestation, and 1370 of them after childbirth in a nested case–control study of a prenatal cohort. Eligible pregnant women were interviewed and submitted to obstetric morphological and transvaginal ultrasound with cervical length measurement, gynecological examination, and blood collection. Preterm birth occurred in 133 women, 129 included in the study, and a control group randomly selected at a 2:1 ratio. A total of 41 cytokines with a higher probability of being associated with preterm birth or being of significance during labor were determined.Cytokine and cervical length analysis by multivariate analysis of the conditional interference tree revealed that growth-related oncogene values of less than 2293 pg/mL were significantly associated with a cervical length of less than 2.5 cm.As well as a cervical length shorter than 2.5 cm, growth-related oncogene levels of less than 2293 pg/ml may be associated with an increased risk of PB. Analysis based on the association of biomarkers and of the interaction between cytokines is a promising pathway in search of a predictor of preterm birth. A biochemical marker associated with a short cervix that allows earlier and more specific selection of patients at risk of preterm birth is relevant.Preterm birth (PB) is a challenge in obstetrical practice. An estimated 15 million babies worldwide are born too early every year . In the The current consensus in the literature is that the mechanisms involved in PB are multifactorial. The change from quiescent myometrium to a contractile one involves a change in signaling between the anti-inflammatory and proinflammatory pathways, with the activation of chemokines, cytokines, and metalloproteinases. A single inflammatory pathway is probably activated both in term and preterm births via an innate immune system. During the acute phase, this process is due to the activation of receptors that recognize pathogen-associated molecular patterns (PAMPs). During the chronic phase, it based on the activation of the receptor for advanced glycation end products (RAGE)  recognizSeveral studies have demonstrated a strong association between a high concentration of inflammation biomarkers and a short cervix  and may Evaluation of the cervix by transvaginal ultrasound is today considered with predictive value for the detection of pregnant women at high risk of PB . The cerRozenberg (2017) pointed out that the predictive value of a short cervix is 17.8% when considering a PB prevalence of 4.3%, which means that if this cut-off point is applied, most women with a short cervix and no history of PB will give birth at term. Thus, it is justified to look for a biomarker that will improve the predictive value of a short cervix for PB .On this basis, the objective of the present study was to look for cytokines and chemokines’ associations with a short cervix that could be candidates for biomarkers and improve the predictive value of the measurement of cervix length alone.The present study was a case–control nested in a convenience cohort approved by the Research Ethics Committee of The University Hospital, Faculty of Medicine of Riberão Preto, University of São Paulo . The pregnant women recruited from two Brazilian cities, São Luís, state of Maranhão, and Ribeirão Preto, state of São Paulo, between February 2010 and February 2011. The present analysis conducted on the Ribeirão Preto cohort and recruitment performed in hospitals and health units of the municipal health network. A total of 1400 pregnant women evaluated during the second trimester of gestation, and 1370 evaluated postpartum in the maternities of Ribeirão Preto at the time of childbirth.Inclusion criteria were healthy pregnant women with a single fetus, between 20 to 25 weeks of gestation. The last menstrual period (LMP) established the gestational age when accordant with the ultrasound done before 20 weeks. If discordant we used ultrasound gestational age. Exclusion criteria were fetuses with major congenital malformations or chromosome syndromes diagnosed before 20 weeks of gestation. All patients signed written informed consent to participate in the study.The women selected were interviewed and submitted to exams, including obstetric ultrasound, gynecological examination, and blood collection between 20 and 25 weeks of gestation. The patient’s selection is summarized in a flowchart Fig. .Fig. 1FlWe calculated the sample size and the power of the study based on the reported prevalence of explanatory variables. Due to the fact that this was a case–control study nested in a broader cohort with a convenience sample, the sample size was calculated in the base project .The ultrasound exams were carried out by two trained observers advised about the methods always using the same equipment , with a 5–9 MHz transvaginal transducer. The cervix length and its biophysical aspects were assessed according to the criteria described by TO et al. in 2001 . The cerThe gynecological examination made for the detection of bacterial vaginosis used the NUGENT > 7 criterion or presence of clue cells for the diagnosis.Venous blood collected for the determination of plasma cytokines and chemokines used a high sensitivity kit . Plasma centrifuged at 10,000 rpm for 10 min, and the technique was carried out according to manufacturer recommendations, with analyte quantitation using a Luminex 200 instrument . We selected 41 cytokines whose presence in maternal blood was frequently associated with PB or to be important for labor in previous studies , 17. ThePregnant women who gave birth before 37 weeks of gestational age were considered cases. We monitored confounding variables that might influence the number of PB and maternal inflammation in the cohort. The following data were obtained at childbirth: hospital and date of birth, gestational age at birth in days, birth weight, and time of maternal amenorrhea.The continuous quantitative variables, such as maternal age and parity, were divided into ranges. Maternal age divided into three groups: < 19, 19–35 and > 35 years, a division justified by the fact that adolescent pregnant women and older pregnant women face specific risks and peculiarities. The parity was also divided into three groups: primiparae, 2 to 3 childbirths, and multiparae. Again, the specific risks and peculiarities of each group justify the stratification.We defined discrete qualitative variables, such as smoking habits and the history of PB, as that associated with any quantity or frequency of smoking during the various gestational periods, any type of smoking cigarette. The occurrence of previous PB before 37 weeks of gestation was not stratified and established as a PB.We performed exploratory data analysis by measurements of central position and dispersal, and the qualitative variables described in terms of absolute and relative frequencies. Univariate analysis was carried out to determine the factors associated with the outcome (PB), and the results analyzed by the nonparametric Mann–Whitney test (or Wilcoxon test for independent samples) and the chi-square test.We used the conditional inference tree to determine the factors associated with PB. The analysis used the R software version 3.3.3. The univariate relationship between cytokines and outcome was first determined, followed by the construction of two additional models, one of them considering as covariables the cytokines that were relevant in univariate analysis and cervical length, and the other involving all cytokines plus the cervical length variable.PB occurred in 133 of the 1370 pregnant women included in the study. Due to the loss of 4 women to follow-up, the case group consisted of 129 women, with a 9.4% prevalence of PB. The case and control groups did not differ significantly in parity, maternal age, or mean BMI, as shown in Table There was an association between PB and maternal smoking history during the present gestation, with a more significant proportion of PB, as expected. However, the occurrence of genitourinary infection  during pregnancy was not associated with the outcome of our study Table .Table 2AWhen the dependent variables were related to the PB outcome, the univariate logistic regression model’s application revealed that 14 cytokines, cervical length, and newborn age differed significantly between the PB and the control groups. Five of these 14 cytokines classified as inflammatory , five as an anti-inflammatory , and four as having both functions . The mean values detected were surprising lower in the case group than in the control group , and cervical length showed a relevant difference between the case group and control. Three of these six cytokines are inflammatory, and three have both pro and anti-inflammatory functions.However, multivariate analysis of the conditional inference tree considering these six cytokines as covariables and cervical length revealed that only GRO was significantly associated with a cervical length of less than 2.5 cm and PB when its value was below 2293 pg/ml (Fig. We had similar results when considering all the cytokines understudy as covariables plus cervical length. Again, a GRO value below 2293 pg/ml is associated with a cervical length of less than 2.5 and PB (Fig. The proinflammatory cytokines classically associated with PB in the literature, such as IL-6 and IL-8, did not associate with PB or with short cervical length in this study. This finding in serum cytokines may have biological plausibility. Simhan et al., 2003 studying cervical proinflammatory cytokines and the risk of clinical chorioamnionitis in pregnant women, finding lower cervical levels of IL-1b, IL-6, and IL-8 associated with clinical chorioamnionitis knowingly related with PB . Mean cyThe present data showed that total GRO serous levels lower than 2293 pg/ml was significantly associated with a cervical length of ≤ 2.5 cm and PB. Six cytokines showed association with short cervix and PB in the univariate analysis of the conditional inference tree. Of these, five showed association with PB in the literature. One, IFN.A2, although involved in the inflammatory response, has not been reported to be related to PB. Out of these five cytokines, only GRO showed association with PB in multivariate analysis using the conditional inference tree. This fact deserves a more in-depth analysis, along with literature data, regarding this association with PB.The literature has shown an association between increased serum proinflammatory cytokines and PB . In studStudies on animal models of IL-6 deficiency have shown an average 24 h delay in the triggering of parturition in deficient mice compared to mice without IL-6 deficiency and an increased resistance to PB induced by lipopolysaccharides . StudiesThe CXC family is one of the two major subfamilies of chemokines and initially described as an endogenous growth factor of tumor cells in melanomas. It is produced by different cell types, such as synovial cells, monocytes, fibroblasts, and endothelial cells, and its production is directly influenced by IL-8, which is responsible for activating neutrophils and chemotaxis of inflammatory response cells .GRO is a chemokine which consists of three subunits: GROα/CXCL1, GROβ/ CXCL2, and GROγ/CXCL3 produced by different cell types such as synovial cells, monocytes, fibroblasts, and endothelial cells its production are directly influenced by IL-8, which is responsible for neutrophil activation and the chemotaxis of cells of the inflammatory response . Some stChemokines appear to act in a network of interaction, guaranteeing a robust defense system whose protection continues functioning even when one of the links of the network is compromised . ChemokiMost of the studies on cytokines and chemokines analyzed on amniotic fluid, and few assessed the association of chemokines of the CXC family, mainly CXCL1/CXCL2/CXCL3, in maternal serum with PB. Aminzadeh et al. determined CXCL10 and CXL12 in the cord blood of newborns and maternal serum, and detected a significantly increased CXCL10 concentration in both the mothers and newborns of the PB group, while the concentration of CXL12 was significantly increased only in the cord blood of preterm newborns . LaudansThe serum value of total GRO detected in the present study was less than 2293 pg/ml in association with a short cervix and with PB. There is no obvious explanation in the literature about the reason for the reduction of some chemokine levels in pregnant women who progress to PB. The lower chemokine concentrations may predispose to chorioamnionitis, which is a possible cause of PB .It is positive that the present study is nested in a prospective cohort, confounding variables evaluated in terms of the outcome with the reduction of information bias. The excluded pregnant women carrying twin fetuses permitted greater control of the confounding variables. Data collection between 20 and 25 weeks of gestation it is the ideal time for the detection of biomarkers of PB and measurement of cervical length .The single determination of cytokines between the 20th and 25th week of gestation limited the conclusions; a longitudinal assessment of the interaction of biomarkers might have provided more relevant data. The late release of cytokines in the inflammatory pathway of labor and their association mainly with acute inflammation may also be a limitation for their use as a PB predictor.Inclusion of all preterm deliveries, spontaneous or provider-initiated preterm deliveries, has a possible association with the occurrence of an error type II that can justify not finding a significant variation of cytokines between the control and case groups.Several studies have shown that multiple factors influence the inflammatory load that, based on multiple and redundant biological clocks, will influence the triggering of labor and PB. The understanding of their interactions and associations in the search for biomarkers has already led to some progress in the prediction of PB. However, their detection is still late and does not permit the establishment of appropriate therapies for prematurity prevention.The present study shows a GRO value below 2293 pg/ml associated with a cervical length of less than 2.5 and PB. Our data suggest that the evaluation of GRO during the second trimester with cervical length represents a promising pathway in search of a predictor of PB.


Knowledge on the association between offspring birth weight and long-term risk of maternal cardiovascular disease (CVD) mortality is often based on firstborn infants without consideration of women’s consecutive births. We studied long-term CVD mortality according to offspring birth weight patterns among women with spontaneous and iatrogenic term deliveries in Norway (1967–2020). We constructed birth weight quartiles (Qs) by combining standardized birth weight with gestational age in quartiles  for the women’s first 2 births. Mortality was estimated using Cox regression and expressed as hazard ratios (HRs) with 95% confidence intervals (CIs). Changes in offspring birth weight quartiles were associated with long-term maternal CVD mortality. Compared with women who had 2 term infants in Q2/Q3, women with a first offspring in Q2/Q3 and a second in Q1 had higher mortality risk , while risk was lower if the second offspring was in Q4 . The risk increase associated with having a first infant in Q1 was eliminated if the second offspring was in Q4 . These patterns were similar for women with iatrogenic and spontaneous deliveries. Inclusion of information from subsequent births revealed heterogeneity in maternal CVD mortality which was not captured when using only information based on the first offspring. BMIbody mass indexCIconfidence intervalCVDcardiovascular diseaseHRhazard ratioICDInternational Classification of DiseasesMBRNMedical Birth Registry of NorwayQquartileLow infant birth weight is associated with increased risk of maternal cardiovascular disease (CVD) mortality . HoweverWhen studying the relationship between offspring birth weight and future maternal health, measures that account for birth weight variation by gestational age may be more informative than absolute birth weight , especiaIn the present study, we wanted to evaluate heterogeneity in maternal CVD mortality risk according to change in offspring birth weight by gestational age among women with 2 term births. Using linked data from population-based registries in Norway, we tested the hypothesis that changes in offspring birth weight quartiles from the first pregnancy to the second influence long-term risk of maternal CVD mortality. We also wanted to evaluate whether associations differ by type of delivery (spontaneous vs. iatrogenic delivery).This was a population-based cohort study using data from the Medical Birth Registry of Norway (MBRN), which has been based on mandatory notification of all births taking place in the country from 16 gestational weeks onward since 1967 . Data arhttps://www.strobe-statement.org/) for cohort studies .Our study complied with the Declaration of Helsinki and was approved by the Regional Committee for Medical and Health Research Ethics. Informed consent was not required, since the data were deidentified, and the researchers did not have any contact with participants. We followed the STROBE checklist  less than −5 or greater than 5, and pregnancies with a gestational age greater than 46 weeks.We included women with 2 or more births whose first birth was registered between 1967 and 2013, providing women with at least 7 years to have finished their reproduction. About 95% of Norwegian women have their second child within 7 years of the first . We focuz score were calculated using mean values and standard deviations from the Norwegian population . Birth weight was registered in grams. The validity of registered gestational age and birth weight data in the MBRN is high  was available from 1999 onwards and 2006 onwards, respectively.Information on cigarette smoking (no (referent) or yes (daily/sometimes)) and body mass index /height (m)International Classification of Diseases (ICD) codes. For our main analyses, we combined deaths due to ischemic heart disease  and International Classification of Diseases, Ninth Revision (ICD-9) codes 410–414; International Classification of Diseases, Tenth Revision (ICD-10) codes I20–I25) and cerebrovascular disease/stroke  into one group (“cardiovascular deaths”). We also examined all-cause mortality, circulatory system diseases , and noncirculatory diseases  mortality.Maternal mortality was registered in the Cause of Death Registry using Frequency and contingency tables were used when constructing parity-specific cutoff points for all first and second births (Web Table 2). Categorical variables were summarized using proportions, while continuous variables were summarized with mean values and standard deviations. Mortality was estimated using Cox proportional hazards models providing hazard ratios (HRs) and 95% confidence intervals (CIs), with woman’s age as the underlying time variable. Women were considered at risk of CVD mortality from their last pregnancy to either death or censoring, whatever came first. In our data there seemed to be no excess maternal CVD mortality by pregnancy complications after the age of 70 years. As a result, we right-censored all observations at the age of 70 years . Schoenfeld residuals were checked for any evidence of deviation from the proportional hazards assumption. In addition to the cause-specific hazard models, we also fitted a subdistribution hazard model to account for competing risk .We performed 2 main analyses when estimating maternal CVD mortality risk. First, we used only information from women’s first birth. Women with spontaneous first deliveries in Q2/3 were designated the reference group. Second, we calculated mortality risks by combining standardized birth weight data from first and second births. Women with both offspring in Q2/3 and spontaneous delivery were the reference group in these analyses. Estimates were adjusted for maternal age at first birth , year of last delivery, maternal education (<11 years (low) vs. ≥11 years ), and pregnancy complications.Several sensitivity analyses were performed. We excluded women with known risk factors for CVD (in both pregnancies), including pregnancy complications , placental abruption and preeclampsia , offspriDue to missing information on maternal smoking and prepregnancy BMI, we also performed E-value–based sensitivity analysis to determine the extent to which unmeasured confounding may have influenced the observed association . The E-vSTATA, version 17 , was used for all statistical analyses.After exclusions , the stuAmong the 735,244 included women, 32,129 died, with 3,037 deaths being from cardiovascular causes. In A total of 518,961 women (70.6%) had spontaneous deliveries in both pregnancies, while 216,283 women (29.4%) had an iatrogenic delivery in the first and/or second pregnancy. Among women with a spontaneous first delivery, 11.8% had an iatrogenic delivery in the second pregnancy, while 5.6% of the women had an iatrogenic delivery in both pregnancies. Sensitivity analysis excluding women with pregnancy complications, offspring congenital anomalies, and conception by in vitro fertilization did not change the CVD mortality pattern but attenuated risk (Web Table 6). Similarly, restricting the analysis to women born in Nordic countries who had children with the same partner or to births with a gestational age of 39–41 weeks did not change the mortality pattern. Adjusting for all of these factors, including interpregnancy interval, did not change the mortality patterns.E-values ranged from 1.11 to 2.71, implying that unmeasured confounding of this extent was required to explain the observed associations. We also observed similar patterns between change in offspring birth weight quartiles from the first pregnancy to the second pregnancy and maternal risk of dying from all causes, circulatory causes, and noncirculatory causes (Web Table 7). Mortality estimates were similar in both hazard models (cause-specific and subdistribution) (Web Table 8). Finally, mortality patterns were similar for women with 3 term births (Web Table 9).n = 79,289) had missing data on either offspring gestational age or birth weight for the first 2 births. These women were younger and had higher proportions of persons with low education, pregnancy complications, and iatrogenic deliveries than the study population (data not shown). For those who gave birth after 1998, women with missing data were also more often smokers.Out of 1,088,309 women, 7.3%  had up to 66% increased CVD mortality risk compared with women with 2 births in Q2/3. On the other hand, giving birth to a term second offspring in the highest quartile (Q4) was associated with similar or lower long-term maternal mortality, independent of the first offspring’s birth weight quartile. This was unexpected, as fetal growth acceleration is associated with reduced glucose tolerance , 24. OneChanges in offspring birth weight quartiles from the first birth to the subsequent birth seem to capture heterogeneity in maternal CVD mortality risk and illustrate that moving from one birth weight quartile to another between the first birth and the second adds valuable information with regard to a woman’s future risk of CVD death: Within all birth weight quartiles of first offspring, maternal relative risk of CVD death decreased by increasing second offspring quartile. Moving from higher quartiles of offspring birth weight to lower quartiles in consecutive births was, in most cases, associated with a higher mortality risk than was found for women with both infants in the middle birth weight quartiles. However, moving from a lower birth weight quartile to a higher quartile was only associated with reduced mortality risk when the first offspring was in Q2/Q3 and the second was in Q4, indicating that having a first infant in the lowest birth weight quartile is a relatively stable marker of future mortality risk. This heterogeneity in CVD risk according to change in offspring birth weight quartiles might be masked if only the first infant’s birth weight information is used, as previous studies have done , 10.CVD mortality has been found to be higher in iatrogenic deliveries than in spontaneous preterm deliveries , 26. TheStrengths of this study include its population-based design, the large sample size, prospectively collected data, and low proportions of missing data. Long-term mortality risk was assessed using information from women’s 2 subsequent births (both live births and stillbirths). We had follow-up for maternal deaths occurring up to 53 years after women’s first birth, median follow-up being 24 years. By using standardized offspring birth weight and parity-specific cutoff values when grouping infants into quartiles, we minimized the possibility of exposure misclassification. The use of observed birth weight–by–gestational age charts in the term population is likely to have been reasonably valid, with little variation and bias . The majThere were some limitations in our study, however, including lack of data on CVD risk factors such as nutritional intake, physical activity, and other environmental factors. Pregnant women were not routinely screened for gestational diabetes before the mid-1980s in Norway . SimilarCurrent guidelines  recommenChanges in offspring birth weight quartile from the first pregnancy to the second may offer important information on heterogeneity in women’s future risk of CVD death. Within all birth weight quartiles of first offspring, maternal relative risk of dying from CVD decreased by increasing quartile of the second offspring, with a similar pattern being observed among spontaneous and iatrogenic deliveries. Women with a first offspring in the lowest birth weight quartile seem to have more consistently increased CVD mortality risk and may benefit from intervention aimed at preventing and reducing future risk of CVD. Our findings highlight the importance of including information from women’s subsequent births for identification of high-risk subgroups for specific follow-up.Web_Material_kwad075Click here for additional data file.


Cardiac hydatidosis is a relatively rare complication of echinococcosis. Understanding the atypical manifestations, potential associated risk factors, and epidemiology leads to optimal and timely management.Cardiac hydatidosis is a relatively rare complication of echinococcosis, with a potentially life‐threatening condition. Here, we reported a large interventricular septal hydatid cyst bulging in the left ventricle accompanied by a huge cervical lamp with recurrent hepatic cysts that underwent cardiac surgery to excise the cyst uneventfully. Together, a good understanding of the atypical manifestations, potential associated risk factors, and epidemiology leads to the optimal and timely management of such patients to minimize worse outcomes.2A 68‐year‐old woman presented to a health care center with a clinical manifestation of a slow‐growing and painless lump on the right side of the cervical region over several weeks. She had no cardiac symptoms. History‐taking revealed working in a sheep farming area in her twenties. Past medical and surgical history included hypertension and hepatic hydatidectomy 2 years ago.Ultrasound imaging revealed a 47 mm × 75 mm cervical cyst expanded to the superior mediastinum with neither inflammatory response nor spasm of the cervical muscles. The cervical cyst consisted of a bilayer membrane with several membrane‐attached scolices, indicating an active hydatid cyst . The lesion was lateral to the common carotid artery and posterior to the internal jugular vein with no cervical lymphadenopathy. Besides, the abdominal ultrasound examination showed multiple active, recurrent hepatic cysts in both the right and left lobes (stage 1), encompassing all liver segments. There was no evidence of biliary dilatation as well.Electrocardiography revealed normal sinus rhythm, and right axis deviation, with inverted T waves in inferior and V2‐V3 leads. There is no evidence in favor of an atrioventricular block Figure .E. granulosus IgG antibodies confirmed the echinococcosis. Treatment with antiprotozoal medication was started. After 2 weeks, the patient underwent coronary angiography, which was normal. Cardiac surgery using cardiopulmonary bypass (CPB) for cystectomy was persuaded to minimize the risk of cyst contents spillage. The CPB technique was established by cannula inserting into the ascending aorta, superior vena cava (SVC), and inferior vena cava (IVC) after the routine median sternotomy.In the transthoracic echocardiographic (TTE), a bulging and well‐defined echo‐lucent cystic mass in the interventricular septum measuring 33 × 42 mm was detected  was injected into the cyst, and after several minutes, the exposed cyst was evacuated completely Figure . The sur3According to the WHO Informal Working Group on Echinococcosis (WHO‐IWGE) ultrasound classification, hydatid cyst consists of three stages, including active , transitional (CE3), and inactive or calcified cysts .Peripheral embolization, anaphylactic reactions, and sudden deathAlthough most cardiac hydatic cysts in the literature are reported in young patients, we reported an old lady (68 y/o) with a recurrent hepatic hydatidosis accompanied by a huge homogenous cystic mass in the cervical and intracardiac regions. In our case, the cystic lesions near the portal venous confluence and the left portal vein may be the leading cause of extrahepatic hydatidosis. It is worth noting that the multiplicity and dispersion of the lesions, typical imaging findings, a history of husbandry procedures, a history of hepatic cysts, geographic location, and positive result of serologic test strongly established the hydatid cyst diagnosis. A previous study represented a 70 y/o female with no history of being in a sheep rising area with signs in favor of right heart failure and cardiac hydatidosis complicated hydatid cyst and pretamponade.In conclusion, a good understanding of the atypical manifestations, potential associated risk factors, and epidemiology leads to the optimal and timely management of patients with rare echinococcosis to minimize worse outcomes.Maryam Faramarzpour: Project administration; resources; writing – original draft. Sirous Jafari: Project administration; resources. Mehrzad Rahmanian: Resources. Akram Sardari: Writing – original draft. Farnoosh Larti: Conceptualization; resources; writing – review and editing.No funding was received for this study.No conflict of interest was present to be disclosed.Written informed consent was obtained from the patient regarding this research process.


To analyse placental changes in infants’ gestational age < 34 weeks and its correlation to short-term respiratory outcomes or death until hospital discharge.Information regarding all in-house born preterm infants born before 34 weeks gestation and born from January 2009 until December 2014 were collected and included among others, placental pathology and relevant data on demographics and outcomes of infants.Placental abnormalities was found in 157/253 (65.05%) cases. Acute placental inflammation was found to be the most common in both groups of premature neonates, followed by maternal vascular underperfusion. Maternal vascular underperfusion was significantly more common in GA ≤ 27 weeks compared to infants GA 28–33 weeks . Similarly, chronic placental inflammation was more common in infants GA ≤ 27 weeks compared to infants GA 28–33 weeks . Infants with placental pathology had a lower median birth weight . Infants with any placental disease had higher rates of death until hospital discharge  and higher rates of any stage of bronchopulmonary dysplasia . There were no significant differences in mechanical ventilation rates, duration of mechanical ventilation and duration of supplemental oxygen therapy.Identifiable placental abnormalities were found in most infants born < 34 weeks gestation. Placental pathology is associated with increased rates of bronchopulmonary dysplasia and death until hospital discharge. The fact that placental pathology recently became more commonly associated with neonatal morbidity and mortality  seems, nMVU caused by the spiral artery pathology is manifested by villous agglutination, increased perivillous fibrin, retroplacental hematoma, infarction and placental hypoplasia . This paFVU is caused by thrombotic occlusion of umbilical, stem and chorionic vessels, which is manifested by the presence of avascular villi. This group of placental lesions has been associated with stillbirth , asphyxiAPI represents the response to the chemotactic gradient in the amniotic cavity, with acute chorioamnionitis corresponding to maternal and funisitis along with chorionic vasculitis, corresponding to fetal inflammatory host response . API is CPI represents the infiltration of the placenta by lymphocytes, plasma cells or macrophages, usually as a result of infections, and includes chronic villitis, chorioamnionitis and deciduitis . CPI is Lesions affecting the placenta are often part of only one of the aforementioned groups; however, the simultaneous occurrence of malperfusion and inflammation pathologies in preterm births may lead to diverse neonatal complications and impaired fetal growth . It woulIn this study, we analyse placental changes in infants gestational age (GA) < 34 weeks and their correlation to short-term respiratory outcomes or death until hospital discharge.This retrospective cohort study was conducted at a regional university hospital with a level III Neonatal Intensive Care Unit. Information regarding all preterm infants born before 34 weeks gestation and born from January 2009 until December 2014 were collected and included, among others, placental abnormalities and relevant data on demographics and outcomes of infants.Clinical data of these infants included GA, birth weight and AS at 1 and 5 minutes after birth, in addition to the duration of oxygen support therapy. Duration of hospitalisation until, either discharge, transfer to a central specialised medical facility, or death, was also taken into account.The placentas of all infants are routinely examined by a gynecologist-perinatal pathologist; in addition, any suspicious placentas and placentas of all neonates  were delivered to the pathology department, stored at temperature of 4–6 °C , macroscopically examined for any visible abnormalities by an independent clinical pathologist blinded to patient health information and eventual clinical outcomes. Placental weight was obtained after the removal of the umbilical cord, fetal membranes and non-adherent blood clots. Any abnormality of pathological material seen on gross examination were then submitted for microscopical-histological analysis. All samples are stained with eosin and hematoxylin as standard.Microscopic analysis is performed on placental tissue blocks; all observed gross pathology of the placenta, the transverse section of the umbilical cord, the free part of the membrane section and at least two parts of the parenchyma from the centre and parts of the edge of the placenta (villi and intervillous space) are analysed. All observed macroscopic and microscopic abnormalities are described, and the percentage prevalence is calculated and presented.Observed placental pathologies were defined in accordance with the reports by Redline et al. ,4,5,6 anThe presence of any pathology belonging to the four major categories was considered significant placental pathology. Study patients were divided into three groups based on placental pathology: those with no placental pathology, those with a single significant placental pathology from one of the major groups, or multiple placental pathologic lesions (presence of ≥ 2 significant lesions).BPD was defined according to severity (mild/moderate/severe). For infants born < 32 weeks gestation, the disease was diagnosed at 36 weeks GA or at discharge from the hospital, whichever occurred earlier; the diagnostic criteria included supplementary oxygen for at least 28 days, and breathing ambient air (mild BPD), < 30% oxygen (moderate BPD), > 30% oxygen and/or the use of respiratory support (severe BPD). For infants born ≥ 32 weeks gestation, the diagnostic criteria included supplementary oxygen for at least 28 days, breathing ambient air (mild BPD), < 30% oxygen (moderate BPD), > 30% oxygen and/or the use of respiratory support (severe BPD) at 56 postnatal days or discharge, whichever came first . GA was The study was approved by the hospital's research ethics committee under protocol number R2-13170/2020 .T test, Mann–Whitney's U test or Kruskal–Wallis test (post hoc Conover). All p values are two-sided. The significance level was set to Alpha = 0.05. Statistical analysis was performed using MedCalc® Statistical Software version 20.009  and SPSS 23 .Categorical data are presented in absolute and relative frequency. Continuous variables are described by the arithmetic mean and standard deviation in the case of distribution following the normal, and other cases by the median and interquartile range (IQR). Differences in category variables were examined by the Chi-square test and Fisher's exact test. The normality of the distribution of numerical variables was examined by the Shapiro–Wilk test. Differences between continuous variables were tested by Student's The final cohort included 253 singleton neonates born before 34 weeks gestation and their placentas (40 infants with GA ≤ 27 weeks and 213 infants GA 28–33 weeks). The demographic and clinical data of the analysed groups are shown in Placental abnormalities were found in 157/253 (65.05%) cases. API was found to be the most common in both groups of premature neonates, followed by MVU .p = 0.018). Similarly, CPI was more common in infants GA ≤ 27 weeks compared to infants GA 28–33 weeks .MVU was significantly more common in GA ≤ 27 weeks compared to infants GA 28–33 weeks . Placentas with any pathology were more often SGA (36.7%). No placenta without pathology was found to be SGA. Infants with any placental disorders had higher rates of death until hospital discharge  and higher rates of any stage of BPD . There were no significant differences in mechanical ventilation rates, duration of mechanical ventilation and duration of supplemental oxygen therapy . In a paper by Mestan et al., MVU was reported in 121/283 (42.75%) infants of GA 26.8 ± 1.5 weeks (9), while a study by Tugrul et al. shows an incidence of over 51% (394/772) . SGA placentas have previously been found to be associated with prematurity, fetal malformations or trisomy, small for date fetus, neonatal high hemoglobin and several maternal factors such as low pregnancy weight gain, low maternal pregravid body weight, high maternal hemoglobin during pregnancy, gestational hypertension, paid employment during pregnancy, low parity, maternal diabetes, Cytomegalovirus (CMV), Herpes simplex virus (HSV) or other chronic infections and other causes of reduced uteroplacental blood flow   vs. 25 (26.0); = 0.013] . This isIn our study, placental pathological changes are also associated with death until hospital discharge . A largeThe placenta plays a crucial role in influencing fetal and neonatal mortality, morbidity and overall outcomes; however, its condition is often neglected in routine neonatology work . When weNonetheless, a notable correlation emerged between placental pathology and both GA and birth weight. CPI was identified as the predominant factor linked to reduced birth weight and shorter gestation periods at birth . The resThe difference from our study lies partly in the type of inflammatory changes. In our study, the placental changes were more of a chronic character. Similarly, other authors have found that no correlation between premature birth and the type of placental pathology, for example, Suresh et al. , 49. In Identifiable placental abnormalities were found in most infants born < 34 weeks gestation. Placental pathology is associated with increased rates of BPD and death until hospital discharge.


Assisted reproductive technologies (ART) have increased the incidence of multiple births, which can have a negative impact on maternal and offspring health. The study aimed to investigate the association between genetically predicted multiple birth and the risk of 42 common diseases of the nervous, psychiatric, cardiovascular, respiratory, digestive, and endocrine systems.The study utilized two-sample Mendelian randomization (MR) analysis to explore the potential causal relationship between genetically predicted multiple birth and the genetically predicted risk of diseases. The study used the FinnGen and UK Biobank datasets for analysis.The study found no significant causal relationship between multiple birth and psychiatric disorders. However, the lower limits of the 95% confidence intervals for bipolar affective disorder and anxiety disorders were not robust, indicating a need for further investigation. The study found that multiple birth may be a strong risk factor for infantile cerebral palsy, and caution is necessary in both natural and ART multiple births. The study revealed a potential causal relationship between multiple birth and coronary heart disease, ischemic heart disease, and deep vein thrombosis, which may be related to abnormal intrauterine environments in multiple pregnancies. Surprisingly, multiple birth appears to have a protective effect against some respiratory diseases, such as chronic obstructive pulmonary disease and asthma.The study highlights the need for caution regarding the risk of infantile cerebral palsy, cardiovascular diseases, and psychiatric disorders in multiple birth. Our study can lead to the development of preventive strategies and improved clinical management for affected infants.The online version contains supplementary material available at 10.1186/s12967-023-04423-w. The world is currently facing a critical issue of population decline, with several countries experiencing a decline in birth rates . This trMothers with multiple pregnancies may be more vulnerable to a range of complications during pregnancy , includiHowever, it is not clear whether the susceptibility of twin offspring to certain diseases is due to innate genetic factors or acquired environmental factors, such as epigenetic changes. Furthermore, it is unclear whether twin pregnancy itself or complications caused by twin pregnancy cause the adverse long-term outcomes in offspring. This field is largely unexplored due to the length of follow-up required for such studies and the difficulty of intervention.Mendelian randomization (MR), as an emerging epidemiological method in recent years, utilizes genetic variants as instrumental variables (IVs) to assess the causal effects of exposure factors on outcomes . Here, "This study aims to estimate the association between genetically predicted part of a multiple birth and the genetically predicted risk of several diseases. Using the FinnGen and UK Biobank databases, we assessed the susceptibility of offspring of multiple birth to 42 common diseases of the nervous, psychiatric, cardiovascular, respiratory, digestive, and endocrine systems.The study design is presented in Fig. In this study, we conducted a two-sample MR analysis  using si–6) were considered as IVs, and we applied LD pruning to ensure that the selected SNPs were independent within a 10 Mb window with an r2 < 0.001. We also searched for secondary phenotypes in PhenoScanner [2) and F-statistics to assess the extent of weak instrument bias [2 was calculated using the formula of 2 × MAF × (1-MAF) × β2, and F was calculated using the formula of R2 × (N − K − 1)/(1 − R2). If F > 10, we considered the correlation between IVs and exposure to be strong enough, and the MR analysis results would be less affected by weak instrument bias.In this study, we employed stringent criteria to select IVs for our two-sample MR analysis. All SNPs that were significantly associated with multiple birth  , MR-EggeTo ensure the robustness of our findings, we employed various sensitivity analyses. To assess the heterogeneity of the IVs, we conducted Cochran’s Q test, where a p value < 0.05 indicates heterogeneity . We usedAll statistical analyses were conducted using TwoSampleMR  packagesIn this part, we explored the association between multiple birth and nine common psychiatric disorders, including attention deficit hyperactivity disorder (ADHD), depression, autism, bipolar disorder, cognitive impairment, schizophrenia, mood disorders, anxiety disorders, and suicide or self-inflicted injury, using MR analysis. Figure Additional file Additional file In this section, we conducted an MR analysis to explore the association between multiple birth and eight neurological disorders, including stroke, subarachnoid hemorrhage, transient ischemic attack, epilepsy, migraine, Alzheimer's disease, adult and infantile cerebral palsy, and mental retardation. The IVW results, as shown in Fig. However, the MR Egger analysis results, presented in Additional file Despite the sensitivity analysis revealing heterogeneity in the results for epilepsy and cerebral palsy in the FinnGen database, MR-PRESSO did not detect any outliers. In addition, the association between multiple birth and transient ischemic attack from the FinnGen database was not reliable due to significant pleiotropy.Additional file Overall, these findings suggest that multiple birth is not significantly associated with any of the neurological disorders investigated, except for a potential strong association with infantile cerebral palsy that requires further investigation.As shown in Fig. Additional file Although MR analysis found significant heterogeneity between multiple birth and arterial hypertension (FinnGen and UKBB), ischemic heart disease (FinnGen), and myocardial infarction (FinnGen), MR-PRESSO did not detect any single outlier. There seems to be a potential causal relationship between multiple birth and deep vein thrombosis, whether the data for deep vein thrombosis is from FinnGen  or UKBB . The results of the one-leave-out test also suggest that both of these results are susceptible to the influence of a single SNP, and the estimated causal relationship becomes more significant after removing a certain SNP . As shown in Fig. Additional file As shown in Fig. Assisted reproductive technologies have raised multiple birth rates, potentially heightening health risks for both mothers and offspring. This study aimed to investigate the association between genetically predicted part of a multiple birth and the genetically predicted risk of 42 common diseases of the nervous, psychiatric, cardiovascular, respiratory, digestive, and endocrine systems.Regarding psychiatric disorders, we found no significant causal relationship between multiple birth and any investigated psychiatric disorders, whether the data came from FinnGen or UK Biobank. This result may differ from some clinical studies , 38. PosIVW analysis found no significant causal relationship between multiple birth and several common neurological diseases. However, according to MR Egger, multiple birth may be a strong risk factor for infantile cerebral palsy . Unlike the IVW method, which assumes that all genetic variants affecting the outcome variable come from the same causal pathway, the MR Egger method is more flexible and can draw causal conclusions from non-causal genetic correlations as long as a weak bias assumption is met . TherefoAfter excluding outliers, IVW analysis revealed a potential causal relationship between multiple birth and coronary heart disease . Simple mode also suggested an increased risk of ischemic heart disease in multiple births . In addition, GWAS data from FinnGen  and UK Biobank  suggested a potential causal relationship between multiple birth and deep vein thrombosis, and both results could become significant after the exclusion of a single SNP. Previous studies have suggested that abnormalities in the early intrauterine environment may lead to adverse long-term effects in offspring, recognized as metabolic memory (MM). Multiple clinical and experimental studies have shown that MM may cause persistent endothelial dysfunction in offspring, amplifying the vulnerability to long-term cardiovascular complications , 15. OurSurprisingly, multiple birth appears to shield against some respiratory diseases, such as COPD  and asthma , excluding single SNP. One possible explanation is that twins could acquire early-life immune safeguards via shared placenta and fetal membranes, potentially curbing future asthma and COPD risk. Furthermore, in the Finnish population, multiple births also exhibit reduced susceptibility thyroiditis . Non-identical twins sharing the uterine environment are exposed to non-identical antigens from each other, which may induce a higher level of tolerance. This partially elucidates multiple births’ conceivable role as a protective factor against asthma and thyroiditis, necessitating validation through subsequent clinical and basic research.MR analysis regarding the link between multiple birth and duodenal ulcer, based on data from FinnGen and UK Biobank, exhibited divergent trends. FinnGen’s data suggests that multiple birth may be a protective factor for duodenal ulcer , while UK Biobank’s data suggests a potential causal relationship between multiple birth and increased risk of duodenal ulcer . Notably, most results obtained from the two databases do not match completely, possibly stemming from differences in population and age. UK Biobank focused on individuals aged 40–69 within the UK, while FinnGen's study population may include any Finnish residents. In addition, FinnGen's data also indicates an increased risk of chronic gastritis in multiple birth . These results need to be interpreted with caution, and studies on the impact of twins on disease incidence rates among different populations are necessary.Although multiple births can ameliorate negative population growth, they may increase the health risks of mothers and offspring. Pregnancy complications can cause adverse outcomes for mother and child, and even have long-term adverse effects on offspring. Thus, as the use of ART gain prominence, it is crucial to investigate the health prospects of offspring from multiple pregnancies, safeguarding their long-term well-being and guiding strategies to counterbalance the adverse consequences of population decline. Moreover, ART-conceived and naturally conceived multiple births may exhibit some differences in the genetic component. ART, encompassing in vitro fertilization and cultivation, involves additional laboratory procedures and interventions that could potentially influence genetic component. Processes like embryo screening within ART might lead to differences in the frequency of certain gene types or variants in ART-conceived individuals. Similarly, the process of embryo cultivation and implantation could also impact their genetic composition. However, it should be noted that in most genetic characteristics, ART-conceived and naturally conceived children should be similar since the genetic information primarily originates from the genetic contributions of their parents. The genetic information of ART-conceived infants still derives from their biological parents' genetic material, and the in vitro fertilization process does not involve modifications to the genome.The study’s results have implications for clinicians and policymakers. They highlight the potential for varied disease risk among offspring of multiple pregnancies. Vigilant monitoring of the long-term health trajectories of these offspring, particularly those born from ART, becomes imperative. Further research is needed to investigate the potential associations between multiple birth and other diseases and conditions.The study's strengths include its large sample size, utilization of MR analysis, and the availability of data from two large databases. By using "a more realistic p value", Smith et al. suggest that compared to conventional observational epidemiology, Mendelian randomization studies can capitalize on the limited associations between a specific genetic variant and other genetic variants, as well as between genetic and nongenetic variables. This enables them to provide an unbiased estimation of the relationship between the factors directly influenced by the genetic variant and disease outcomes, making them less susceptible to confounding .However, this study has several limitations. Firstly, the absence of pertinent data hindered the ability to perform separate analyses for monozygotic and dizygotic multiple births. Specifically, in dizygotic multiple births, there may be significant differences in genotypes among individual fetuses. Similarly, comparing distinctions between ART and non-ART twins proved challenging, given potential genetic variations introduced by the ART process. Secondly, the study population included only individuals of European ancestry, and the results need to be validated in more diverse populations. Thirdly, while two-sample Mendelian randomization is a powerful discovery tool, one-sample MR would confirm these findings and enable a direct evaluation of potential confounding factors and covariates that may contribute to pleiotropic effects. Lastly, the results obtained through Mendelian randomization in this study lack direct support from relevant clinical randomized controlled trials, warranting cautious interpretation. Despite these limitations, the study provides valuable insights into the potential health risks associated with multiple births.In summary, this research adds to the growing body of evidence on the health risks associated with multiple birth. Although some of the findings lack robustness, the study suggests that individuals who are born from multiple pregnancies should be aware of the increased lifetime risk for bipolar affective disorder, anxiety disorders, infantile cerebral palsy, coronary heart disease, ischemic heart disease, deep vein thrombosis, and chronic gastritis. This study emphasizes the importance of investigating the health outcomes of individuals who are born from multiple pregnancies and highlights the need for further research on the long-term health consequences of various types of pregnancies. The study's findings have important clinical implications for managing multiple pregnancies and the long-term health outcomes of offspring.Additional file 1: Table S1. SNPs that were used as instrumental variables met the conditions: p value < 5 × 10–6 and F > 10. * The secondary phenotypes of rs605765 obtained by PhenoScanner include waist circumference, weight and number of operations, some of which are associated with BMI, and BMI was identified as a confounder for lots of diseases. In the subsequent one-leave-out test, we focused on whether the MR Results were stable after removing this SNP.Additional file 2: Table S2. Characteristics of each method used in this study.Additional file 3: Table S3. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of mental illness.Additional file 4: Table S4. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of nervous system disease.Additional file 5: Table S5. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of cardiovascular system disease.Additional file 6: Table S6. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of respiratory system disease.Additional file 7: Table S7. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of digestive system disease.Additional file 8: Table S8. Two-sample Mendelian randomization estimations showing the effects, heterogeneity and horizontal pleiotropy of multiple birth on the risk of endocrine system disease.Additional file 9: Material S1. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and mental illness.Additional file 10: Material S2. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and nervous system disease.Additional file 11: Material S3. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and cardiovascular system disease.Additional file 12: Material S4. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and respiratory system disease.Additional file 13: Material S5. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and digestive system disease.Additional file 14: Material S6. The scatter plot, funnel plot and leave-one-out plot for the MR analysis of multiple birth and endocrine system disease.


Length of hospital stay (LOS) is one of the essential indicators for evaluating the efficiency and the quality-of-care service delivered. predicting LOS is critical for resource allocation, decision-making, lowering neonatal morbidity and death, enhancing clinical outcomes and parent counseling. In addition, extended hospital stays (long LOS_NICU) place a burden on the healthcare systems decreasing bed turnover rates as well as their financial stand and the mental stress on families. In Ethiopia, there is limited evidence on the determinant factors that influence on LOS.To determine factors affecting neonatal intensive care unit length of stay for all preterm newborns who were discharged alive.The study used a secondary data source, was collected for the Study of Illness in Preterm (SIP) infants project. The research study was a multicenter, cross-sectional, observational clinical study that took place in five Ethiopia hospitals from July 1, 2016, to May 31, 2018. The predictors of LOS were determined using Fine-Gray's competing risk analysis.For this study 3,511 preterm infants admitted to the NICU were analyzed. About 28.8% of the preterm infants died during their time in neonatal care while 66.6% were discharged alive. At the end of the study 4.6% babies were still in the NICU. The overall median LOS (death or discharge) was 7 days, with an interquartile range of 8 days. The cumulative incidence of discharge rose with increasing in gestational age and birth weight, on the contrary, the rate of discharge was decreased by 45.7% with the development of RDS (SDH ratio: 0.543), by 75.9% with the development of apnea (SDH ratio: 0.241), by 36.2% with sepsis, and by 43.6% with pneumonia (SDH ratio: 0.564).Preterm newborns with a low gestational age and birth weight have a greater probability of having a prolonged LOS. Complications of the medical conditions RDS, apnea, sepsis, pneumonia, anemia, asphyxia, and NEC substantially raise LOS considerably. Preterm birth, defined as giving birth before 37 weeks of gestation, is one of the leading causes of infant disease and mortality. It has been identified as a global concern, accounting for an estimated 11% of all live births globally . AccordiAccess to quality care after delivery for admitted infants will have a greater role in improving their lives. Preterm newborns require special neonatal care due to their high risk of longer stays in the NICU. consequently, Predicting LOS in the NICU has gained attention in recent years. Length of hospital stay (LOS) is an essential index for assessing the efficiency of patient quality of care after delivery. Lowering the duration of hospital stay reduces the risk of nosocomial infection and medication adverse effects while additionally enhancing treatment quality. With Increasing LOS, the neonate can take a long duration of IV medication, a broad spectrum of antibiotics and other interventions that can increase the probability of having severe drug side effects and antibiotic resistance .Predictions of the duration of stay are also required to support discussions between parents and physicians regarding the expected length of stay for a newborn . The capA secondary data source which was collected for the Study of Illness in Preterm (SIP) infants project. The SIP study was a prospective, multicenter, cross-sectional, observational clinical study conducted in five hospitals in Ethiopia from July 1, 2016, to May 31, 2018. Gondar University Hospital, Jimma University Hospital, and three Addis Abeba institutions  or physical examination using the New Ballard Score (NBS).If the difference between GA assessed by NBS and that calculated from LMP was not greater than 2 weeks, the last menstrual period GA was assumed to be correct and would be considered for this study. On the other hand, GA assessed by Ballard would be taken for this study if the difference between the two measurements was greater than 2 weeks . PretermAnemia is defined here as hematocrit (HCT) that is more than 2 standard deviations below the age or less than the normal range for postnatal age and birth weight. For clinical purposes HCT < 45% in the 1st week of life .Neonatal sepsis is defined as a clinical syndrome of bacteremia with systemic signs and symptoms of infection in the first 4 weeks of life .RDS defined accordingly, respiratory distress syndrome occurs primarily in premature infants; its incidence is inversely related to gestational age and birth weight. Surfactant deficiency (decreased production and secretion) is the primary cause of RDS. clinical presented with, tachypnea, prominent (often audible) grunting, intercostal and subcostal retractions, nasal flaring, and cyanosis .Jaundice (Hyperbilirubinemia) is defined as a newborn presents with yellowish discoloration of sclera, skin, mucus membranes and In newborns, jaundice appears when total bilirubin (TB) is more than 7 mg /dl. Neonatal jaundice can be classified as either physiologic or pathologic .The diagnosis of hypoglycemia depends on the clinical setting but not solely on specific blood glucose level. For intervention or further evaluation, hypoglycemia could be defined as a blood glucose level less than 40 mg/dl .Perinatal asphyxia is an insult to the fetus or newborn due to lack of oxygen (hypoxia) and/or a lack of perfusion (ischemia) to various organs .The outcome variable of interest was the LOS of preterm infants who were discharged from the NICU. However, preterm infants who died in the NICU were considered as a competing event. Preterm infants who were still in the unit at the end of the follow-up were considered censored events. The number of days from the time of admission to the time of discharge was used to calculate the LOS.Both demographic and clinical characteristics of the preterm newborn were considered as candidate predictors for the LOS of preterm infants. The demographic characteristics tested in the analyses were gestational age , sex  and birthplace . Clinical characteristics included birth weight , maternal mode of delivery , gestation type (“Multiple/Single”), respiratory distress syndrome (RDS), apnea, early onset neonatal sepsis, pneumonia, anemia, hyperbilirubinemia, feeding problems, hypoglycemia, asphyxia, necrotizing enterocolitis (NEC), and hypothermia. Maternal characteristics such as maternal age , pre-eclampsia, antenatal hemorrhage, maternal fever, and antenatal care received were also evaluated for candidacy.At the beginning of the analysis, the data was analysed in various ways  to obtain details that could help make decisions about the next steps of the analysis. Histograms were used to explore the characteristics of the time until discharged alive and the time to death. The plot of cumulative incidence of follow-up time was used to illustrate the probabilities of in-hospital death and being discharged alive over time.To determine the predictors of LOS, a competing risk analysis proposed by Fine-Gray was used . The proThe analysis is performed using the “crr” function under the “cmprsk” package in the R statistical software. As variable selection criteria for the Fine and Gray model, the stepwise regression procedure (forward and backward) was implemented using the R package “crrstep”.In the study period, a total of 3,852 preterm infants were admitted to the neonatal intensive care units of the study hospitals. Among these admitted preterm infants, 341 preterm infants were ineligible by the exclusion criteria as shown in Summary characteristics of the preterm babies were analyzed associated with the outcome of interest is provided in The remaining 4.6% of the babies were still in the unit at the end of the study follow-up. Around 27% of babies were born at or below 32 weeks gestational age, of which 570 died, 307 were discharged alive and 79 were still in the unit. More than 69% of preterm babies weigh less than 2,000 grams. There were 1,077 babies born weighing less than 1,500 grams, of which 592 died, 377 were discharged alive and 108 were still in the unit.Out of 1,882 male preterm infants 67.1% and 29.45% were discharged alive and died before discharge respectively. Among 1,629 female preterm infants 65.9% and 28.1% were discharged alive and died before discharge respectively. About 87.1% of preterm infants below 28 weeks of GA died before discharge from the hospital and 6.9% were still in NICU. About 12.8% of preterm infants between 35 and 36 weeks of GA died prior to discharge from the hospital and 85.7% were discharged alive. 69% of women with multiple births discharged alive while 26% of the women were died prior to discharge.67.8% of mothers who gave birth by the C-section were discharged alive while 26.8% were died prior to discharge from the hospital.The median LOS (death or discharge) was 7 days, with an interquartile range of eight days. The cumulative incidence function (CIF) was used to depict the likelihood of death in the hospital and discharge alive over time With a median duration of stay of 7 days, only 14% of the newborns diagnosed with apnea were discharged alive before the end of the study's follow-up. The majority of the preterm infants admitted with the clinical conditions hyperbilirubinemia and hypoglycemia were discharged alive with an approximate median duration of stay of 11.5 days.P-values resulting from the final Fine and Gray model with all candidate predictors are shown in For variable selection, the stepwise regression procedure in the forward or backward direction using the R package “crrstep” was used. All the aforementioned demographic and clinical characteristics listed in The cumulative incidence of discharge increased with an increase in gestational age and birth weight. In relation to preterm infants with a gestational age lower than 28 weeks, the sub-distribution hazard of discharge was 3.4 times higher for those infants with 28–31 weeks of gestation, 5.5 times higher for infants with 32–34 weeks of gestation, and 6.1 times higher for infants with 35–36 weeks of gestation. The discharge sub-distribution hazards for preterm neonate with birth weights (in grams) of 1,000 to less than 1,500, 1,500 to less than 2,000, and 2,000 and above were 2.8, 5.7, and 8.0 times higher, respectively, than for preterm neonate with birth weights of 1,000 and less. The rate of discharge decreased by 45.7% with the development of RDS (SDH ratio: 0.543), by 75.9% with the development of apnea (SDH ratio: 0.241), by 36.2% with sepsis (SDH ratio: 0.638), and by 43.6% with pneumonia (SDH ratio: 0.564). Similarly, the sub-distribution hazard of discharge decreased by 28.5% for infants diagnosed with anemia (SDH ratio: 0.715), by 64.9% for infants diagnosed with asphyxia (SDH ratio: 0.351) and by 55.8% for those infants diagnosed with necrotizing enterocolitis (SDH ratio: 0.442).The capacity to accurately estimate LOS in the neonatal ICU has become more essential in recent years. As newborn survival rates have improved, so has the number of babies who require extended hospitalizations in the neonatal unit. However, there has been limited study on how to estimate LOS and what variables are necessary to take into account. The current study conducted a systematic review of the literature to determine what factors should be addressed in future LOS assessments.Numerous studies have looked at predicting LOS in preterm newborns admitted to the NICU. In this study, the occurrence of Respiratory Distress Syndrome, Apnoea, Neonatal Sepsis, Pneumonia, Anaemia, Asphyxia, low birth weight, lower gestational age, and Necrotizing Enterocolitis were significant predictors of prolonged hospital LOS among preterm newborns in the NICU.In our study, 66.6% of preterm babies survived and were discharged alive; the deathrate was 28.8%, in comparison 10.1% in the Saudi study and 12.8% in the Indian study. These disparities can be explained by the fact that newborn survival varies with healthcare quality.Low birth weight and gestational age were the most important variables associated with longer LOS among neonates. The median LOS was 7 days for babies delivered between 23and36 weeks of gestation, which is same with a study conducted in Ethiopia on Children Admitted to the NICU . HoweverBirth weight influences the duration of hospital stay with an SDH ratio of 2.8, 5.7, and 8 times higher for preterm infants weighing 1kg−1.5 kg, 1.5–2 kg, and ≥2 kg as compared to preterm newborn weighing below1kg grams, respectively. The inverse association of our finding between gestational age and LOS was similar to the study in Nigeria which concluded the LOS increased with decreasing birth weight and gestational age . SeveralPreterm babies with complicated clinical conditions will prolong their length of stay in the hospital. In the present study, preterm infants with respiratory distress syndrome (RDS) and sepsis had greater incidence of LOS which is not unexpected finding in our analysis as it is already known that RDS and sepsis increase LOS among hospitalized preterm infants . A similThe study has limitations in analyzing the clinical conditions which are a different clinical staging, disease severity, and complication, such as NEC, RDS, and anemia which have an impact on LOS. The readmission information and maternal antenatal steroids treatment not assessed due to limited information sources.For planning to improve service efficiency, the cumulative incidence of being discharged alive should be assessed by incorporating time to death. LOS in the NICU for preterm infants is influenced by both demographic and clinical characteristics. The cumulative incidence of discharge was increased for every increase in gestational age and birth weight. Having complications from the clinical conditions of RDS, apnea, sepsis, pneumonia, anaemia, asphyxia, and NEC in preterm infants significantly increases their hospital LOS.