Background: Perinatal asphyxia is a significant contributing factor for neonatal morbidity and mortality. The aim of this study was to investigate the clinical factors associated with umbilical artery pH variability and fetal acidosis at birth.Methods: This is a single center cross-sectional study in a public regional hospital in southeastern Spain from January to December 2019. The reference population was 1.655 newborns, final sample of 312 experimental units with validated values of umbilical cord blood pH.Results: Factors such as gestational age at term , primiparity (p: 0.01), induced labor (p: 0.02), vaginal delivery (p: 0.01), and prolonged dilation duration (p: 0.00), expulsion duration (p: 0.01), and total labor duration (p: 0.00) are associated with a decrease in umbilical artery pH at birth. However, only three factors are associated with acidosis pH (<7.20) of the umbilical artery at birth: the induction of labor , vaginal delivery , and total duration of labor .Conclusions: Although several factors may affect the variability of umbilical artery pH at birth by decreasing their mean values , only induction of labor, vaginal delivery and total duration of labor are associated with an acidosis (<7.20) of same. The term asphyxia can be defined as an alteration in gas exchange that leads to progressive hypoxia, hypercapnia, and acidosis . In the Mortality and morbidity associated with intrapartum hypoxia persists as a global health problem , being oNeonatal asphyxia alters the passage from intrauterine to extrauterine life, a transit that requires well-orchestrated measures to ensure neonatal survival . In thisThis is a cross-sectional study. The reference population was the 1.655 newborn during the year 2019 between January and December in a hospital, in the southeast of Spain , belonging to the National Health System. The Hospital Rafael Méndez Lorca is a regional public health hospital within the Servicio Murciano de Salud  hospital network; it provides health care to residents of Murcia Health Area III (Spain), numbering 172.630 inhabitants in total. The hospital has its own Maternal and Child Service, with a specialized Obstetrics–Gynecology Department.in utero; births where the umbilical gas analysis was performed after 15 min, since in such cases, fetal acidosis is less precise as a diagnostic method .The sample population comprises the births that occurred in the reference hospital and met the following criteria: hospital births during the study period for which gasometry data and documentation of umbilical cord blood samples, both arterial and venous, were recorded; home births were not included due to the absence of arterial and venous cord blood gasometry records. Exclusion criteria: fetuses that died c method ; and hosc method , 10  of the reference hospital. SELENE, the clinical station of the Murcia Health System's Corporate Hospital Information System, affords multiple ways to access a patient's clinical history. Each clinical history is divided into healthcare processes, each of which contains information in the form of clinical records .In order to analyze the collected information, the data provided through the Hospital database (SELENE) was converted and coded into an Excel spreadsheet, in order to facilitate its subsequent loading into the SPSS 26.0 program for data analysis.The measurement technique and instrumentation employed was the biochemical analysis of umbilical cord blood  via the GEM Premier 4000 approved pH meter .The umbilical cord blood collection technique involves placing one clamp close to the newborn's navel and a second at the end of the umbilical cord, without interfering with the recommended umbilical cord clamping delay of at least 30–60 s post-birth  and withThe blood is extracted, with the least amount of air possible, into two separate 1- or 2-ml syringes pretreated with heparin. After the blood has been extracted, any air bubbles present in the syringes must be eliminated, and the blood gas analysis is performed in a calibrated apparatus, in this case, the GEM Premier 4000 approved pH meter. The analysis must be performed within the first 15 min.The main outcome measures were the variability and acidosis of the umbilical artery pH at birth, identifying as such the arterial pH range <7.20 , 14. Acc2) > 60 mmHg, base excess within normal limits (more than −12 mEq/L)], and mixed acidosis   and nationality (Spanish/foreign).Gestational age , parity (primiparity/multiparity).via the use of mechanical or pharmacological means that produce contractions). According to reasons for induction: retarded intrauterine growth (IUGR): growth percentile <3/growth percentile between 3 and 10 with Doppler hemodynamic alteration; risk of loss of fetal well-being (RLFW): pathological or non-reactive cardiotocographic trace that after performing a biophysical profile <8/10, or a persistently non-reactive cardiotocographic trace at 12–24 h despite having obtained a biophysical profile of 8/10; prolonged pregnancy (PP):(>42 weeks gestation); premature rupture of membranes (PROM): premature rupture of amniotic membranes before birth onset; oligohydramnios: maximum amniotic fluid column <2 cm; maternal disease unrelated to the pregnancy: maternal disease indicating end of gestation and pregnancy-induced hypertension (PIH): hypertension that appears after 20 weeks of gestation, subdivided into gestational hypertension and pre-eclampsia.Type of onset of labor /cesarean ].Type of amniorexis [spontaneous: rupture of the fetal membranes without external action/artificial: rupture of the fetal membranes effected by the health professional through performance of an amniotomy ], presence or absence of episiotomy (no episiotomy/yes episiotomy), sex of the newborn , presence or absence of nuchal cord at birth (no presence/yes presence).Presence or absence of analgesia .Neonatal weight at birth [normal  and abnormal ].Time elapsed since the rupture of the amniotic sac [below the study sample average (<443 min)/above the study sample average (>443 min)].Duration of the dilation stage [below the study sample average (<261 min)/above the study sample average (>261 min)].Duration of the expulsion stage [below the study sample average (<44 min)/above the study sample average (>44 min)].Duration of delivery [below the study sample average (<312 min)/above the study sample average (>312 min)].t-student test in the cases where the independent variables were dichotomous and an analysis of variance (one-way ANOVA) when they had more than two categories. Subsequently, in order to distinguish the factors associated with fetal acidosis at birth, a bivariate analysis was performed with the dependent variable grouped into two categories , applying Pearson's Chi-square test and the Yates correction when the observed frequencies were <5. Finally, in order to analyze the magnitude of the adjusted association of the dependent variable with the independent variables, the variables for colinearity were tested before entering them in the model. Gestational age, parity, labor onset, type of delivery or prolonged durations of dilatation, expulsion, and total labor are intrapartum variables that can be determinant in the variations of blood gases at birth . In the different hypothesis tests, the results obtained for a value of Of the 312 infants constituting the sample, 30.7% (96) presented acidotic pH of the umbilical artery at birth, 79.2% (76) showed respiratory acidosis, and 20.8% (20) showed mixed acidosis .There is a statistically significant association between the decrease in umbilical artery pH mean values and variables such as gestational age at term, primiparity, induced labor, vaginal delivery, and the duration of dilation, duration of expulsion, and total duration of labor above average. No significant association is observed with other variables such as maternal age, maternal nationality, amniotic sac rupture type, presence or absence of episiotomy, presence or absence of analgesia, sex of the newborn, neonate weight, presence of nuchal cords, or above-average duration of rupture of the amniotic sac .F(6): 0.74, p: 0.61], despite the fact that the lowest mean values of umbilical artery pH are found in births whose induction was retarded intrauterine growth (IUGR) (t(100):−3.92, p:0.00), an association that is not found in other types of delivery such as eutocic delivery(t(223): −1.56, p:0.12) or intrapartum cesarean section (t(106): −1.64, p:0.10), even though an instrumented delivery has lower mean values of umbilical artery pH than these.The results do not show significant differences according to reasons for induction [The bivariate analysis with the clustered dependent variable shows a statistically significant association between the presence of umbilical artery pH at birth and factors such as gestational age at term [OR: 6.37 (95% CI: 0.85–47.52)], primiparity [OR: 1.32 (95% CI: 1.04–1.68)], onset of induced labor [OR: 1.76 (95% CI: 1.04–2.96)],vaginal delivery [OR: 2.06 (95% CI: 1.13–3.74)] and duration of dilation [OR: 2.63 (95% CI: 1.55–4.44)], duration of expulsion [OR: 1.74 (95% CI: 1.00–2.99)], and total duration of labor [OR: 2.34 (95% CI: 1.43–3.99)] above average .No significant association was observed with other variables such as maternal age, maternal nationality, amniotic sac rupture type, presence or absence of episiotomy, presence or absence of analgesia, sex of the newborn, neonate weight, presence of nuchal cords, or above-average duration of rupture of the amniotic sac .However, once we introduced into the same matrix those variables that were significant in the bivariate analysis and after performing the collinearity analysis, in order to analyze the adjusted association of the independent variables with the response variable, a colinearity was found between the variables duration of dilation, duration of expulsion, and total duration of labor . After eliminating from the matrix dilation time and duration of expulsión , it is observed that there is no colinearity between the variables , with a variance inflation factor (VIF) <10 (1), condition index <30 (3.16), and proportion of variance <0.5.p: 0.04], completion of vaginal delivery  and total duration of labor  ;  p:0.01] .Factors such as gestational age, primiparity, labor induction, vaginal delivery, or prolonged durations of dilatation, expulsion, and total labor decrease the umbilical artery pH at birth, but only induction of labor, vaginal delivery, and total duration of labor are associated with an acidosis of the same. The inductions of retarded intrauterine growth and instrumented delivery have the lowest mean umbilical artery pH at birth according to induction reason and type of labor completion.Our research reflects worse average values in the biochemical parameters of newborns at birth, and a higher percentage of acidosis in the normal gestational age range  compared with the abnormal gestational age range (<37 or >41 weeks). This aligns with authors such as Parikh et al.  and SandRegarding parity, it has been shown that perinatal asphyxia is more common in newborns of primiparous mothers , 23. In p <0.001) between induced birth and low 5-min Apgar test scores in comparison with spontaneous-onset birth. Yi et al. .Induction of labor has also been related to intrapartum fetal acidosis –32. Gudai et al.  found sen et al.  concludeAccording to induction reason, intrauterine growth restriction is one of the main risk factors for intrapartum fetal acidosis in our results and in those of Kehl et al. , Pinton The reasons for induction detected in the sample: pregnancy indications , maternal indications , and fetal indications   show simUterine contractions in labor result in a 60% reduction in uteroplacental perfusion, causing transient fetal and placental hypoxia. A healthy fetus with a normally developed placenta is able to accommodate this transient hypoxia by activation of the peripheral chemoreflex, resulting in a reduction in oxygen consumption and a centralization of oxygenated blood to critical organs, namely, the heart, brain, and adrenals. Providing there is adequate time for placental and fetal reperfusion between contractions, these fetuses will be able to withstand prolonged periods of intermittent hipoxia and avoid severe hypoxic injury . In indu2, pO2, and sO2 in mothers delivering vaginally in comparison with planned cesarean sections. The significant differences in acid–base values according to mode of delivery is based on processes such as during vaginal delivery, the sheer force of uterine contractions and bearing down results in impaired oxygenation and anaerobic glycolysis with lactate accumulation in both the mother and fetus , thus, controlling interobserver variability with uniform collection criteria; confounding bias: for the main outcome variables, the corresponding multivariate adjustment was made considering that these variables could act as confounders; selection bias: before starting data collection, the sample size was calculated based on the incidence of the study phenomenon.Several factors, such as gestational age, primiparity, labor induction, vaginal delivery, and above-average duration of dilation, expulsion, and total labor duration have a significant association with the decrease in the averages of umbilical artery pH at birth; however, only induction of labor, vaginal delivery, and total duration of labor are associated with acidosis (<7.20) of the same.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comité de Investigación del Área III de Salud. . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.JH, MP, and PP: conceptualization, methodology, investigation, writing—original draft preparation, visualization, supervision, and project administration. JH and MP: software, validation, formal analysis, resources, data curation, and writing—review and editing. All authors contributed to the article and approved the submitted version.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.


Morphological variability in canines is associated with the mother’s size and weight, which likely affects the birth weight of the puppies and their metabolic status. Identifying physio-metabolic alterations in the blood from the umbilical vein to evaluate the concentration of gases, glucose, lactate, calcium, hematocrit levels, and blood pH of newborn puppies will make it possible to determine the risk of complications due to intrauterine asphyxia. The objective of this study is to evaluate the effect of the mother’s weight on the weight of liveborn and stillborn puppies during spontaneous births and the neonates’ blood physiological alterations during the first minute of life. The above allowed us to identify the physio-metabolic maladjustments that newborn puppies suffer from and to determine the risk of asphyxia according to the weight category of the mothers. Results suggest that if the weight of the bitch is >16.1 kg in eutocic births, there is a higher risk of intrapartum physiological alterations and death. The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth, though there is a wide range in birth weights due to the ample morphological variability characteristics of this species.p < 0.05). These signs indicate a process of transitory asphyxiation. The stillborn pups in all four categories had higher weights than their liveborn littermates. C3 and C4 had the highest mean weights  and mortality rates . Results suggest that if the weight of the bitch is >16.1 kg in eutocic births, there is a higher risk of intrapartum physiological alterations and death. The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth.This study aims to determine the effect of the weight of bitches on liveborn and stillbirth puppies from eutocic births, and physiological blood alterations during the first minute postpartum. A total of 52 female dogs were evaluated and distributed in four categories: C1 , C2 , C3 , and C4 . The dams produced 225 liveborn puppies and 47 were classified as stillbirth type II. Blood samples were taken from the umbilical vein to evaluate the concentration of gases, glucose, lactate, calcium, hematocrit levels, and blood pH. The liveborn puppies in C2, C3, and C4 had more evident physiological alterations  than those in C1 ( Deaths blood pH ,34,35,36blood pH ,33,37,38blood pH ,40 assocblood pH ,41, highblood pH . IdentifA network of 10 veterinary clinics in Mexico City was organized to recruit pregnant dogs. Prenatal control was performed from day 25 of pregnancy to 24 h postpartum.A total of 52 young multiparous bitches (2–4 births) were recruited. The inclusion criteria were: (i) clinically healthy dogs; (ii) valid vaccination/deworming record; (iii) fed a commercial formula; (iv) no history of reproductive problems; (v) radiographic and ultrasonographic evaluations to show they were apt for natural births. The exclusion criteria were: (i) primiparous females; (ii) bitches with a history of dystocia or pyometra; (iii) previous type I SB puppies; (iv) malformed fetuses; (v) use of birth inducers or accelerators; (vi) bitches with body condition 8 or 9 (obese) as per the WSAVA scale ; (vii) e® Ed. Professional 2016 database for veterinary clinics.The clinical history of the dogs was compiled, including age, weight, alimentation, preventive medicine status, and a description of the environment where they lived. All information was recorded in the Q.vet® vital sign monitor to evaluate the mothers’ physiological parameters. Clinical signs observed in the peripartum interval included anorexia, anxiety, and nesting behaviors.Diagnoses were confirmed between days 24 and 28 post-service for each dam. Fetal structures and cardiac activity were detected in the gestational sacs using a LOGIQ 400 MD ultrasound machine  equipped with a 3.5 MHz convex transducer to establish probable due dates. Monitoring of fetal maturation and vitality was performed on days 40–50 of gestation. The fetal structure was defined completely to permit the early identification of pyometra cases, type I SB puppies, and malformations. X-rays of the dams’ abdomens were taken on day 45 of pregnancy once bone calcification of the fetuses was achieved to discard early stages of maternal–fetal dystocia and evidence of cephalopelvic disproportion, conditions that would make caesarean sections necessary ; thus, eThe number of LP and SB was recorded by category.(a)Liveborn: A total of 225 LP puppies were recorded for the four categories: C1: 63; C2: 71; C3: 54; C4: 37. The neonates, who had a heartbeat and were breathing during the first minute of life, were considered liveborn puppies. The puppies that died once the birth was over, were considered dead during lactation.(b)Stillbirth: The 47 SB, by the weight categories of their mothers, occurred as follows: C1: 9; C2: 12; C3: 14; C4: 12. The following cases were classified as dead antepartum : fetuses that died after the birth process began but before the expulsion; those with hemorrhagic and edematous appearance; those with grayish-brown discoloration due to an initial state of mummification; more advanced cases, a clear state of dehydration and fur loss. Those fetuses were excluded. The fetuses classified as dead intrapartum (Type II SB) presented the same appearance as the rest of the litter, except for the absence of breathing and heartbeat.® critical blood variable analyzer  to obtain values for the metabolite’s glucose (mg/dL) and lactate (mg/dL), blood gases pCO2 (mmHg) and pO2 (mmHg), the acid–base balance pH, HCO3− (mmol/L), EB (mEq/L), Ca2+ (mmol/L), and hematocrit (Htc %). All profiles were tested for each LP and Type II SB pup.A trained veterinarian took blood samples immediately after birth in less than 10 s. An assistant held the puppy in a supine position and exposed the abdominal region; the pup’s umbilical cord was carefully grasped to insert the needle (26G) of a tuberculin syringe impregnated with lithium heparin to avoid coagulation and alterations of blood values and immediately obtain 0.3 mL samples of venous blood. All samples were processed by a GEM PremierThe weight of each LP and Type II SB puppy was recorded using a digital scale  after drawing the blood samples.® 9 statistical package procedure. Normality tests were performed for all dependent variables to determine differences among the 4 categories (the dam’s weight was the independent variable) regarding the parameters of the blood physio-metabolic profile: pH, pO2 (mmHg), pCO2 (mmHg), glucose (mg/dL), Ca2+ (mmol/L), lactate (mg/dL), hematocrit (Htc %), HCO3− (mmol/L), and EB (mEq/L), as well as the weight of the LP pups (dependent variable). An analysis of variance (ANOVA) was performed with a contrast of means using a Tukey test (p < 0.05).Descriptive statistics were obtained for the variables tested following the Origin Versionp < 0.05.Metabolites = pH, pCO2, pO2, glucose, Ca2+, lactate, hematocrit, HCO3−, EB;µ = general mean;iT = fixed effect;iCN = 1,2,3,4; for the case of SB Type II;iP = birth weight;e = error.Values were considered as significant at The same statistical model was used to analyze the SB Type II neonates.The studies were performed with privately owned dogs. Each owner gave her/his informed consent before data were gathered. During the study, all dogs were treated following the directives and guidelines of Mexico’s Official Norm NOM-062-ZOO-1999 on technical specifications for the production, care, and use of laboratory animals and those related to the field of applied etiological studies . The expp = 0.01), two , and three , with those in C1 having lower weights than those in C2 and C3 (76.99–184.72 g). There were no significant differences in the weight of the LP pups in C3 (374.57 ± 48.18 g) and C4 (381.02 ± 20.24) (p = 0.70).Significant statistical differences were observed (see p = 0.01) weighed less than those in C2 , C3 , and C4 , but there were no significant differences between the weight of the SB pups in C3 (419.86 ± 4.57 g) and C4 (433.75 ± 12.98 g) (p = 0.40).The lowest number of SB occurred in C1 with nine (12.5%), compared to C2, C3, and C4, which had 14.45–24.48%.The effect of the mother’s weight on the blood physio-metabolic profiles of LP and SB are shown in p = 0.001) and those in C2 , C3 , and C4 . The LP in C4 had the largest increase in blood lactate concentrations compared to C1 and C2 (p < 0.05), though there was no significant difference in lactate values between C2 and C3  (p = 0.07)  .p = 0.02), but there were no significant differences between C2 and C3  (p = 0.99) (The Type II SB pups in C4 (13.08 ± 0.41 mg/dL) showed a larger increase in lactate than C1 (11.44 ± 0.52) ( = 0.99) .p > 0.05) (p = 0.04). There were no significant differences between C2 and C4  (p = 0.99)  . In cont = 0.99) .2+, a statistically significant decrease was found in the values of the LP in C1 (1.43 ± 0.01 mmol/L) compared to C2 , C3 , and C4 , but no significant differences in blood Ca2+ concentrations were seen among C2, C3, and C4 (p > 0.05) (2+ levels showed no significant differences among the categories (p > 0.05) (Regarding the metabolite Ca > 0.05) . For the > 0.05) . p = 0.001), C3 , and C4  dams all had higher percentages of hematocrit than those in C1 (44.84 ± 0.55) (p < 0.05). However, no significant differences were seen among the LP in C2, C3, and C4 (p > 0.05) (p > 0.05)  . Regardi > 0.05) .p < 0.05), compared to C3  and C4 . While pH clearly decreased, there were no significant differences in pH values among categories C2, C3, and C4 (p < 0.05) (p > 0.05)  . Similar > 0.05) . 2, results showed that the LP from three groups—C2 , C3 , and C4 —had significant decreases compared to C1 (17.09 ± 0.44 mmHg) (p < 0.05), but there were no significant differences in the pO2 values among C2, C3, and C4 (p > 0.05)  had a significant decrease compared to C1 (9 ± 0.91 mmHg) (p = 0.02), but the pO2 values between C2 and C3 did not differ significantly  (Regarding pO > 0.05) . For bloctively) . 2, a statistically significant increase was observed in the LP in C2 , C3 , and C4 , compared to C1 (47.69 ± 1.01 mmHg). In contrast, no significant differences were found in the pCO2 values of C2, C3, and C4 (p > 0.05) (p = 0.01), C3 , and C4  compared to C1 (1.66 ± 2.08 mmHg), but no significant differences were found in the pCO2 values from groups C2, C3, and C4 (p > 0.05) (Regarding pCO > 0.05) . Upon ob > 0.05) .3− levels was seen in the LP in C2 , C3 , and C4  compared to C1 (21.94 ± 0.26 mmol/L) (p < 0.05), but upon comparing the metabolite values for C2, C3, and C4, no significant differences were found (p > 0.05) (p > 0.05) (A significant decrease in HCO > 0.05) . Regardi > 0.05) . p = 0.01), C2 , and C3 . A similar result emerged when we compared the values for C1 and C3 (p = 0.001), though no significant differences were found between C2 and C3 (p = 0.11) (p > 0.05) (Observations showed that EB increased significantly in the LP in C4 (−8.82 ± 0.43 mEq/L) compared to the values for C1  . Regardi > 0.05) .It was proposed that the weight of puppies at birth can be influenced by diverse factors, as occurs in other mammals. These include the duration of pregnancy ,45,46, rOne important finding involves the weight of the SB, as this was always higher than that of the LP in all four cases (C1–C4). These differences mean 29.16–52.77 g, but categories C3 and C4 had both the highest mean weights  and the highest mortality rates . On one side, and such as other species, including humans, swine, and bovines, low birth weight is considered an important risk factor for neonatal mortality ,53,54. R2 and lactate of 17.38% and 51.45%, respectively, and decreases in blood pH of 1.22%, and levels of pO2 (15.27%) and bicarbonate HCO3− (9.48%), with high EB (45.91%), all due to hypercapnia (an indicator of respiratory acidosis) as the main factor. The most evident alterations occurred in the LP groups with the heaviest dams . These alterations in gases and blood metabolites indicated respiratory and metabolic acidosis (mixed acidosis) resulting from intermittent asphyxia in utero during natural birth. Compensatory alkalosis began as a response to the respiratory and metabolic acidosis in the LP in all groups (C1–C4) due to hypoxia. This explains why pH did not decrease drastically [Fetuses commonly suffer intermittent periods of light hypoxia due to uterine contractions and the mechanical pressure inherent to the birth process . Vassalostically ,63. Hypostically ,65,66. Tstically .Regarding the metabolite glucose, no significant differences were observed in the LP during the first minute of life in any of the four categories, since measurements were in a range of 94.92–103.91 mg/dL. Mila et al.  reported2 levels up to double those registered in the LP (94.66 vs. 47.69 mmHg), lactate values as many as four times higher than in the LP (13.08 vs. 4.80 mg/dL), EB in a range of −14.26 to −15.33, and a decrease in pO2 (6.22%) and bicarbonate HCO3− levels (6.97%). We also observed that the Type II SB showed a significant decrease in plasma glucose concentrations in every group (C1–C4), with a range of 38.78–51.11 mg/dL. This result coincides with the values < 40 mg/dL reported by Lawler [Birth weight was reported as a risk factor for intrapartum hypoxia because newborns with a low birth weight are more likely to suffer oxygen restriction and the secondary effects of hypoxemia ,77. Presy Lawler . We, they Lawler ,79,80, hy Lawler , foals [y Lawler , and domy Lawler ,84. Thesy Lawler .Fetal asphyxia, defined as a condition of hypoxemia with hypercapnia and acidosis, caused the death of the pups at birth in this study. Fetal asphyxia caused the death of 17.27% of the puppies in this study, but the highest percentages of intrapartum deaths were seen in C3 and C4, which together represented 55.31% of the mortality of the pups from natural births.2 indicates a process of metabolic acidosis [2 [In LP, a pH below the reference values was observed (7.35–7.45) in C2, C3, and C4, but this parameter on its own does not permit measuring the accumulated exposure to hypoxia because it is expressed logarithmically . It doesacidosis . In addidosis [2 . 2 and the increase in pCO2 combined to lower the pH, but bicarbonate in the plasma mitigated this imbalance. As a result, the high concentration of bicarbonate generated a mixed acidosis that exacerbated the increase in ionized calcium and the decrease in protein-bonded calcium [The intense, constant uterine contractions necessary for fetal expulsion can compress the umbilical cord, drastically decreasing both placental and umbilical blood circulation . The dec calcium , produci calcium  indicateDuring pregnancy, fetuses depend on their mother for gas exchange and correct oxygenation through the placenta. This exchange is determined by the size of the fetuses, blood gas concentrations in the mother, and the latter’s capacity for transfer and transport. Thus, modifications to any of these parameters can generate a state of hypoxia and subsequent disruptions of the acid–base balance, such as metabolic acidosis . It is bThe results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth, though there is a wide range in birth weights due to the ample morphological variability characteristic of this species. We observed that the puppies born from bitches in categories three and four had the highest birth weights , with a difference greater than 100 g from the pups in C1 and C2. The LP puppies in category 1—the lightest ones (189.85 g)—had fewer physio-metabolic alterations during the first minute of life; while the litters that weighed 16.1–35.8 kg showed more physio-metabolic alterations, which impacted their adaption to extrauterine life and caused respiratory and metabolic acidosis and hypocalcemia, all of which contributed to the total mortality of 17% as a consequence of intrapartum hypoxia.Furthermore, all the Type II SB were heavier  than the LP born in the same group. The above suggests that puppies with higher weights experience greater difficulty during expulsion through the birth channel, which increases the risk of suffering an acute process of intrauterine asphyxia that affects their chances of survival. The highest mortality rates for the heavier categories (C3 and C4) were 20.58% and 24.48%, respectively. Therefore, this study established the mother′s weight (16.1–35.8 kg) as a risk factor for fetal asphyxia in eutocic births and, consequently, a high percentage of intrapartum mortality. New studies will allow the integrated evaluation of other variables as risk factors related to in utero asphyxia in eutocic births and their influence on postpartum survival.


Most published newborn growth references are based on conventional monitoring data that usually included both low- and high-risk pregnancies. We sought to develop a set of neonatal growth standards constructed from only a large sample of low-risk pregnancies. A total of 24,375 naturally conceived singleton live births with gestational ages of 24–42 weeks were collected in 69 hospitals in thirteen Chinese cities between 2015 and 2018. Unhealthy infants or those with high-risk mother were excluded. Smoothed percentile curves of six anthropometric indicators were established using the Generalized Additive Model for Location, Scale and Shape. The 3rd, 10th, 25th, 50th, 75th, 90th, and 97th percentile references for birth weight, length, head circumference, weight/length, body mass index, and ponderal index were calculated for neonates with gestational ages of 24–42 weeks. This set of neonatal growth standards with six anthropometric indicators can provide more tools for growth and nutrition assessment and body proportionality in neonatal clinical practice. These standards might also help to show the differences between growth curves based on low-risk and mixed low- and high-risk pregnancies. Whether sample population are drawn from low-risk pregnancies or mixed low- and high-risk pregnancies is the key to distinguish growth standards and growth references8. The sample populations for growth standards are selected based on relatively healthy and adequately nourished pregnant women and represent relative healthy patterns of growth of neonates that can answer how infants ought to grow rather than how they do grow9. Considering the difference of the reference samples, a standard may have more clinical utility than a population reference8.Intrauterine growth is associated with infant survival, future growth and development, and health conditions. An appropriate growth standard or reference is an essential tool for neonatal growth assessment at birth. Therefore, some countries have established newborn growth standards based on low-risk pregnancies with a normal outcome10 is ill-suited for assessing the growth and development of today’s Chinese newborns due to several limitations. Recent monitoring data has shown that the growth level of newborns is substantially higher compared to the 1988 reference13. Further, the 1988 reference only covers newborns with a gestational age (GA) of greater than 28 weeks, which does not meet the current need of assessing infants with a GA of less than 28 weeks as more extremely preterm babies are being born. To overcome these limitations, we aimed to develop a set of growth standards for neonates with GA of 24–42 weeks based on a large sample of low-risk pregnancies. Three commonly used indicators—birth weight, length, and head circumference—that allowed better definition of small for GA (SGA) and large for GA (LGA) were employed. In addition, three anthropometric ratios—weight/length, body mass index (BMI), and ponderal index (PI)—that allowed for better assessment of symmetric vs asymmetric abnormalities in growth were utilized. We also examined the differences among our standards, the INTERGROWTH-21st standards15 and the new US curves16.The growth reference for Chinese newborns constructed in 198817. Considering the low numbers of extremely and early preterm babies, four cities in the surrounding regions of the nine cities including Tianjin, Shenyang, Changsha, and Shenzhen were added to supplement the sample sizes of preterm babies with GA of 32 weeks and below.From June 2015 to November 2018, a cross-sectional survey of newborn babies with a GA of 24–42 weeks in 13 cities in China was carried out. Of these 13 cities, nine, including Beijing, Harbin, Xi’an, Shanghai, Nanjing, Wuhan, Guangzhou, Fuzhou, and Kunming, were selected to investigate newborn babies with GA of 24–42 weeks. These nine cities were also the sample cities on the National Survey on the Physical Growth and Development of Children in China, which was a representative national survey of the countrySingle naturally conceived live births with a GA of 24–42 weeks were included. Infants who were not healthy or whose mothers were at high health risk were excluded according to the following exclusion criteria: ① unclear GA; ② severe congenital malformation at birth or known chromosomal abnormality; ③ edema or hematoma during physical measurement; ④ parents of non-Chinese origin; ⑤ mothers were not permanent residents in surveyed cities and lived in surveyed cities for < 2 years; ⑥ maternal height < 145 cm; ⑦maternal age < 18 years or > 40 years; ⑧ mothers who were smoking, alcoholic or drug dependent over the three months before or during pregnancy; ⑨ mothers who had continuously taken adrenal cortex hormones or other immunosuppressive agents for > 1 month during pregnancy; ⑩ mothers of full-term babies with any of the following conditions during pregnancy: severe anemia (Hb ≦ 60 g/L), gestational diabetes, preeclampsia, eclampsia, hyperthyroidism or hypothyroidism, heart and kidneys dysfunction, chronic hypertension; ⑪ mothers of preterm babies with any of the following conditions during pregnancy: severe anemia (Hb ≦ 60 g/L), gestational diabetes that cannot be effectively controlled by diet and exercise intervention, severe preeclampsia, eclampsia, hyperthyroidism or hypothyroidism that cannot be effectively controlled by drug therapy, severe heart and kidneys dysfunction.+0 to 24+6 weeks for the 24 week GA group.GA was jointly determined based on the mother’s last menstrual period (LMP) and the results of ultrasound examination in the first three months of pregnancy. GA based on LMP was used when the GA difference between the two methods was ≦ 1 week, and GA based on ultrasound examination was used when the GA difference was > 1 week. GA groups were divided by week, such as 24Sample size was estimated according to both statistical accuracy requirements for establishing percentile curves and observed numbers of newborns at each GA. For full-term babies with GA of 37–41 weeks, the sample size for each GA group was about 100 per sex and city; for preterm babies with GA of 29–36 weeks, the sample size for each GA group was about 50. Full-term babies with GA of 37–41 weeks were sampled by cluster sampling. Samples were evenly distributed by season, and babies randomly selected from each season. All eligible full-term babies with GA ≧ 42 weeks or preterm babies with GA ≦ 28 weeks within selected hospitals were included in this study. The study was reviewed and approved by the Ethics Committees of the Capital Institute of Pediatrics (No.SHERLL-2015009). Written informed consent at the top of questionnaires was obtained from all respondents . All methods were performed in accordance with the relevant guidelines and regulations.18. Each measurement was collected independently by two trained doctors or nurses. If the difference between the two measurements exceeded the maximum allowable difference , a third measurement was taken, and then those two measurements not exceeding the allowable difference were recorded.Birth weight was measured within 12 h of birth with an electronic scale . Birth recumbent length was measured within 24 h with infantometer  for term babies and preterm babies of large GA and new patent infantometer (Patent No. zl201520996396.X)  for preterm babies of small GA. Birth head circumference was measured within 24 h with a flexible non-stretchable plastic tape . Birth weight, length, and head circumference were measured twice and recorded twice in a standardized measurement procedureUniform measuring tools were equipped for all sites, including infantometer and new patent infantometer for length measurement, non-stretchable plastic tape for head circumference measurement, standard weights  for calibration of electronic scale, and standard steel tape (accuracy 1 mm) for calibration of infantometer and plastic tape. The electronic scales in each site were used for investigation after evaluation and calibration of standard weights with a maximum allowable difference of 10 g. Calibration was taken every week with maximum allowable differences of weight 10 g, length 0.5 cm, and head circumference 0.5 cm. Questionnaires were completed by pairs of trained doctors or nurses, with one recording the answers and the other reviewing. The completed questionnaires in each city were sent to the Beijing Steering Committee for final check and data entry. EpiData 3.0 software was used for double entry and logic check of the questionnaires.2], and [weight (kg)/length (m)3], respectively. During data cleaning, we excluded 2 missing weight values, 8 missing length values, and 16 missing head circumference values. Few measures not within ± 5 standard deviation (SD) of the mean of overall sex- and GA-specific values was also excluded . The final sample sizes contributing to the establishment of the growth curves for each indicator are listed below: weight , length , head circumference , weight/length , BMI , and PI . During the establishment of the growth curves, normality test, and skewness and kurtosis analysis were assessed for each indicator. Data analyses used SAS v9.4 (SAS Institute Inc).The mean of two measurements for birth weight, length and head circumference was used for data analysis and calculation of anthropometric ratio. Weight/length, BMI, and PI were calculated according to the following formula: [weight (kg)/length (m)], [weight (kg)/length (m)19, was employed to establish smoothed percentile growth curves of male and female newborn babies with GA of 24–42 weeks. Curve fitting was performed using the GAMLSS 4.3–1 library running under R 3.1.2. Goodness of fit of the GAMLSS models was assessed by the Schwarz Bayesian Information Criterion that is justified as a general criterion for model selection and by Q-Q plots that assesses the age-conditional normality of the transformed data21. After comparative testing of alternative methods  used to generate the growth curves, birth weight, weight/length, BMI, and PI percentile curves were established using the GAMLSS with Box-Cox t (BCT) distribution with cubic splines, and birth length and head circumference using the GAMLSS with Box-Cox power exponential (BCPE) distribution with cubic spline. All these GAMLSS models did not need to be weighted because the difference of each indicator between non-weighting and equal proportional weighting were negligibly small16. The differences between fitted percentiles and empirical values at each week were examined for all the six anthropometric indicators.The Generalized Additive Model for Location, Scale and Shape (GAMLSS) which is a general framework for fitting regression models where the distribution of the response variable allows for highly skewed and kurtotic continuous distribution2, and weight gain during pregnancy was 13.8 (± 5.0) kg.A total of 24,375 singleton live births with GA of 24–42 weeks were collected in 69 hospitals in 13 Chinese cities, including 12,264 preterm babies  and 12,111 full-term babies . Table Tables Figure Overall, the percentile curves of birth weight, length, head circumference, and weight/length in China presented similar growth trajectories with the INTERGROWTH-21st standards, but also expressed distinct differences for length at 37–42 weeks and weight/length at 24–32 weeks ; and there is a large disparity for birth BMI from 24 to 41 weeks . For example, the distance in our weight curves from low-risk individuals was shorter compared to other weight curves from Chinese routine monitoring data that included both low- and high-risk individuals23. Assessment of growth and nutrition of the neonates is essential for a positive outcome in later life. However, no single anthropometric measure fully reflects growth, development and health of newborn babies, so we established percentile curves of multiple anthropometric measures, with each measure revealing distinct relationships with specific health risks or diseases. Birth weight is typically used to define the classification of newborn size as small, appropriate, or large for a specified GA at birth24. Birth length is helpful in evaluating whether postnatal catch-up growth is appropriate26. Birth head circumference reflects intrauterine brain development and predicts the prognosis of nervous system development27. A consensus was reached on the definition of growth restriction as birth weight < 3rd percentile or at least 3 out of 5 of the following: birth weight < 10th percentile, length < 10th percentile, head circumference < 10th percentile, prenatal diagnosis of fetal growth restriction, and maternal pregnancy complications28. Traditional classification based on birth weight centiles for GA does not reflect body fat in both term and preterm newborns29, while weight/length greatly aids in predicting newborn fat mass and fat-free mass as well as body proportionality30. BMI is useful for measuring body proportionality for newborn infants32. PI is a customary measure to evaluate whether abnormalities in growth in preterm infants are symmetric or asymmetric35. In sum, our established reference values of six indicators can provide more tools for growth and nutrition assessment  and nutrition assessment/body proportionality  in neonatal clinical practice.The health of the mother and the newborn is the foundation of sustainable development for individuals, families, and societies as it is closely linked with health throughout lifeOur study has several strengths. First, our standards were based on low-risk pregnancies with a normal outcome. Second, besides the commonly used weight, length, and head circumference, we also established percentile curves of weight/length, BMI, and PI that help assess whether abnormal babies in growth are symmetric or asymmetric. Under strict exclusion criteria, we included a relatively large sample size of preterm infants that guaranteed more reliable percentile curves; however, the screening efficacy of this set of new standards for SGA or LGA and body proportionality still needs to be further validated and evaluated.Based on a contemporary, large-scaled, population-based cross-sectional nationally representative sample from low-risk pregnancies with a normal outcome that represents optimal intrauterine growth, we developed a set of neonatal growth standards for 24–42 weeks of gestation, including reference values of six anthropometric indicators that can provide more tools for growth and nutrition assessment and body proportionality in neonatal clinical practice. In addition, our study aids in better understanding the differences in the shapes of growth curves between based on data from low-risk pregnancies only or from mixed low- and high-risk pregnancies.


Preterm birth is the leading cause of infant morbidity and mortality globally . DetermiRecently, novel methods for establishing an infant’s GA have emerged that may be able to overcome some of the existing limitations. One such approach involves using an established public health procedure, a heel prick blood spot typically used for newborn screening . NewbornLevels of the analytes measured by newborn screening vary by the GA of an infant at birth . While tFurther validation of this approach is necessary to determine to what extent local factors may affect metabolic profiles, which in turn could impact the accuracy and generalisability of metabolic-derived estimates of GA. We are currently in the process of implementing and further evaluating this approach to GA estimation in multiple low-resource settings in sub-Saharan Africa and South Asia, including Kenya, Zambia, Zimbabwe, and Bangladesh . Heel prLogistical challenges with obtaining samples within an appropriate timeframe  and then shipping them to an external laboratory under conditions that preserve the quality of the blood spots are important to address. Ultimately, the goal will be to develop in-country capacity and point-of-care diagnostics that will permit these analyses at the time of birth to inform care for individual infants. There are also cultural concerns to be understood and addressed that may affect willingness of families to provide either heel prick or cord blood samples. Another challenge is the adaptation of algorithms developed using blood spot data to estimate population rates of SGA. Existing algorithms can be enhanced using machine learning approaches to improve model performance, and could be further reiterated for SGA-specific estimations .Is this approach worth the cost? According to our estimates, the incremental cost per preterm infant correctly classified by the metabolic algorithm is US$11 542, and per SGA infant is US$688 . DetermiTo optimise benefit for costs, additional potential value to the collection of these samples should be evaluated. An immediate obvious benefit is the potential to guide the care of appropriately recognised preterm infants, provided that metabolic data can be analysed and processed promptly. Moreover, the analysis of the samples can provide newborn screening value by identifying infants at risk of treatable conditions such as congenital hypothyroidism and hemoglobinopathies.Looking to the future, work is under way to determine whether there is additional value to the analysis of blood spots, including their predictive value for a host of conditions including early neonatal mortality and neonatal sepsis ,14. BlooWe envision a future where a heel prick at birth could inform caregivers of the GA of a child, distinguish SGA from preterm infants, identify treatable conditions and determine risk for specific health conditions. This data could also provide population-level estimates of preterm birth to guide health system planning and resource allocation for improved cost-effectiveness and impact of maternal and child health policies and programmes.


The male child had negligible DMR associations. The DMR associated genes for each group involve previously identified preterm birth associated genes. Observations identify a potential paternal germline contribution for preterm birth and identify the potential epigenetic inheritance of preterm birth susceptibility for the female child later in life. Although expanded clinical trials and preconception trials are required to optimize the potential epigenetic biomarkers, such epigenetic biomarkers may allow preventative medicine strategies to reduce the incidence of preterm birth.Preterm birth is the major cause of newborn and infant mortality affecting nearly one in every ten live births. The current study was designed to develop an epigenetic biomarker for susceptibility of preterm birth using buccal cells from the mother, father, and child (triads). An epigenome-wide association study (EWAS) was used to identify differential DNA methylation regions (DMRs) using a comparison of control term birth versus preterm birth triads. Epigenetic DMR associations with preterm birth were identified for both the mother and father that were distinct and suggest potential epigenetic contributions from both parents. The mother (165 DMRs) and female child (136 DMRs) at  Complications of being born preterm were the leading cause of mortality in children less than five years of age in 20152. Children who survive preterm birth are at increased risk of developing future adverse health outcomes, including cognitive disabilities, seizures, visual and hearing impairment, and cardiovascular problems11. Although there are many risk factors associated with preterm birth including genetic variants, exposure to environmental toxicants, presence of multiple fetuses, preeclampsia and ethnicity, more than half of premature birth cases have an unknown etiology13. Reliable biomarkers for preterm birth could greatly help in predicting which pregnancies are at risk and would improve clinical management and health outcomes for the children.Preterm birth (PTB) is childbirth that occurs at less than 37 weeks of gestation. Worldwide, preterm birth rates are estimated at 11%, accounting for about 14.8 million of the live births of 20148. Although many associations between mid-trimester ms-hCG and/or ms-AFP levels and adverse pregnancy outcomes are statistically significant, the sensitivity and positive predictive value are too low for them to be clinically useful as screening tests for preterm birth14. Other proposed biomarkers of PTB risk include selected inflammatory cytokines18, metabolic lipid products19, specific gene mRNA transcripts21, cervicovaginal proteome22, and micro-RNA transcripts24. Urinary oxidative stress metabolites have also been proposed as biomarkers of preterm birth26. These biomarkers are not extensively used and are not considered efficient or ideal27. Either the assays for proteins and metabolites are technically challenging and expensive, or the specificity and sensitivity of the assays in predicting preterm birth need to be improved27.A number of potential biomarkers for preterm birth have been identified. Maternal serum levels of alpha-fetoprotein (ms-AFP) and human chorionic gonadotropin (ms-hCG) have been used clinically31. Epigenetics is defined as “molecular factors and processes around DNA that regulate genome activity, independent of DNA sequence, and that are mitotically stable”32. Epigenetic factors and processes include DNA methylation, histone modifications, non-coding RNA, and chromatin structure changes33. Assays for DNA methylation have the advantage of using smaller sample size due to high sensitivity of the assays, as well as being less expensive and technically demanding than assays for proteins and metabolic products. DNA methylation changes can also be detected in easily obtained surrogate samples , such as cheek buccal epithelial cells34. This is due to the fact that epigenetic differences can be heritable, so all somatic cells derived from the embryo of an individual have cell-specific epigenetic changes derived from the germline33. Altered DNA methylation sites caused by fetal toxicant exposure, abnormal nutrition, or stress have been found in previous studies to be associated with increased risk of disease in exposed offspring and their descendants 37.Previous studies have proposed that epigenetic alterations should be considered for use as biomarkers to predict preterm birth38 and tissues of children born preterm. Studies that compared DNA methylation in umbilical cord blood between preterm and full-term children found from 31 to 296 differentially methylated sites40. One study found DNA methylation differences in umbilical cord tissue between preterm and full-term children39. These results indicate that DNA methylation changes may occur with preterm birth and suggest that DNA methylation changes are worth investigating as a viable biomarker for predicting preterm birth. Although all cell types have the same DNA sequence present, a limitation of examining DNA methylation changes in a mixed cell population, such as blood with over 20 different cell types, is that each cell type has a unique epigenome and DNA methylation profile driving the cell type specificity32. Thus, small changes in the relative numbers of different cell types in a mixed population can suggest an epigenetic difference, but are in fact due to the changes in cell population numbers33. Therefore, purified individual cell types are preferred to effectively assess epigenetic differences and potential disease biomarkers42.There is evidence that epigenetic differences are associated with preterm birth in the placenta. infertility from no known cause, and not related to low sperm count or motility) were found to have 217 differential DNA methylation regions (DMRs) at a p value of p < 1e−05 compared to sperm samples from fertile men43. In addition, 56 DMRs were found between initially infertile men who responded to follicle stimulating hormone (FSH) therapy versus those who did not, suggesting that DNA methylation may be used as a biomarker of responsiveness to this therapy43. Recently it was reported that a set of 805 DMRs in sperm was potentially associated with men having an increased risk of having a child with autism44. Previously, it has been shown that DNA methylation at the SLC9B1 gene in blood samples from pregnant women between 24 and 32 weeks gestation can predict whether the fetus is at risk for fetal intolerance of labor, which can cause fetal hypoxia, and is an indication for performing a Caesarean section45. In a recent study, we have used buccal cells as an easily obtained purified cell population to identify epigenetic  biomarkers for female rheumatoid arthritis46. Although sperm epigenetic biomarkers reflect epigenetic inheritance of disease in offspring and subsequent generations, a surrogate cell such as buccal cells can reflect early embryo impacts on all somatic cells to be used for disease assessment47. Together, these studies indicate that epigenetic biomarkers of preterm birth susceptibility or pathology potentially exist and are worthy of further development. Identification of maternal biomarkers associated with preterm birth could help in the prediction and clinical management of at-risk pregnancies and allow for better preventative care for preterm birth children. Clinical management protocols that could be used to reduce the incidence of preterm birth and infant morbidity include: enhanced surveillance of at-risk pregnancies, timely use of prenatal steroids and tocolytics, application of protective uterine monitoring, hospitalization and operative delivery. Epigenetics may also point the way to specific gene targets for future pharmaceutical agents where epigenetically identified “at risk” women could be given gene-specific therapeutics.Changes in DNA methylation at particular genomic loci have been reported as biomarkers associated with human diseases. Sperm samples from men with idiopathic infertility  and premature (< 37 week gestation) populations and compared. The goal was to find in maternal and paternal buccal cells DMRs which could distinguish preterm from term birth. Clearly the infant epigenetic biomarker is not used to predict potential preterm birth, but can potentially be used to assess later life disease susceptibility in the individual. These epigenetic biomarkers identified can now be prospectively tested for their positive and negative predictive power in subsequent investigations. The generational study presented suggests potential epigenetic inheritance aspects for preterm birth.48. Any contaminating bacterial molecular data can be removed during the analysis. Buccal cells were obtained from participants with a home collection swab kit and sent directly to the lab for storage and analysis. The participants were recruited prior to collection or analysis from Indiana University (IU) Health Hospitals  and Franciscan Health, Indianapolis, Indiana. Approvals to conduct the study were obtained from Indiana University Institutional Review Board (IRB) #1901985132 and the Franciscan Institutional Review Board (IRB), #1489434-5. Informed consent and HIPAA authorization was obtained from all participants and from a parent and/or guardian for participants that were minors prior to the clinical sample collection. The buccal cells were collected from the mother, father, and newborn child (triads) to assess epigenetic biomarkers in each group separately. The triad samples were collected, approximately nine days following delivery. This period was used to allow the PTB case child to mature and allow an effective buccal cell collection. The full term (FT) birth controls had 21 triad participants and the pre-term birth (PTB) cases had 19 triad participants. Although the majority were of non-Hispanic white Caucasian backgrounds, a number of triads in each population were of African American descent, Supplemental Table p ≤ 0.001), Supplemental Table The objective of the study was to develop an epigenetic  biomarker for preterm birth (PTB). One of the least invasive and easiest purified cell types to collect is a buccal swab from the cheek, which is greater than 90% pure squamous epithelial cells49, as described in the Methods. This procedure can provide a genome-wide assessment of greater than 90% of the genome, compared to approximately 50–70% for bisulfite sequencing or less than 1% for array analysis50. Differential DNA methylation regions (DMRs) were identified by comparing the control and PTB case samples for each mother, father, or child triad. DMRs identified were obtained for each group and presented in Fig. p < 1e−04 was used for all subsequent data analysis, which was selected as it also provided a reasonable false discovery rate (FDR). The number of adjacent DMR 1 kb windows are shown at a significance level of p < 1e−04 and the majority of DMR for each group had a single 1 kb window with some higher numbers of significant adjacent windows, Fig. p < 1e−04, except for the mother and female child, which shared 31 DMRs in common, Fig. p < 1e−04 DMRs with the other groups at a p < 0.05 threshold. This extended overlap demonstrated much higher levels of overlaps with maternal DMRs having a 49% overlap with the paternal, 58% with the female child, and 30% with the male child. Paternal DMRs had a 75% overlap with the mother, 64% with the female child, and 47% with the male child. The female child overlaps were higher and ranged from 34 to 58%, while the male child overlap ranged from 18 to 28%, Fig. DNA was isolated from the buccal cell collections and analyzed with a methylated DNA immunoprecipitation (MeDIP) procedure to obtain methylated DNA for subsequent sequencing (Seq) for an MeDIP-Seq protocolp value and FDR), log-fold methylation change (increase positive or decrease negative) for each DMR, gene associations (within 10 kb of gene) and functional categories for the associated genes. The chromosomal locations of the DMRs (red arrowheads) for each group are presented in Fig. 51, which represents the majority (> 90%) of the genome, but some DMRs are observed at higher 8–10 CpG/100 bp density associated with CpG islands50.The lists of DMRs and genomic information are presented in Supplemental Table A principal component analysis (PCA) of the DMRs for the control and case comparison for each group are presented in Supplemental Figure 44. The accuracy for the test set mother was 50%, father was 40%, and female child 60%. However, after the analyses of the unblinded samples, a very heterogenous equal mixture of moderate, very, and extreme PTB were present. In addition, some batch effects within the assay were detected. Due to the low sample size (n = 5) of the test set and heterogeneity of the samples, this blinded test set analysis was potentially compromised and marginally successful, so not utilized for further analysis. As now discussed in the Discussion section, expanded clinical trials with larger sample size and larger test sample size are required to optimize and validate the epigenetic biomarkers (DMRs) identified.A blinded test set of samples were collected to help validate the predictive ability of the PTB samples identified. Five triads for control and five triads for PTB case were collected for analysis. These samples were blinded to the WSU investigators during the analysis and prediction. This test set was analyzed and the data used in dendrogram, machine learning and PCA analysis, as previously describedThe final analysis investigated the DMR associated genes with each mother, father, and child DMR sets. The DMRs within 10 kb of a gene were considered to include proximal and distal promoter regions, as well as the gene. The DMR associated genes listed in Supplemental Tables A presentation of the mother, father, and child DMR associated genes with network links, as determined by Pathway Studio , are presented in Fig. 1. Even when preterm children survive, they are at higher risk of developing chronic disease conditions5. These include hypertension, diabetes, metabolic and lipid disorders, heart disease, kidney disease, sleep apnea, and all cause mortality5. This is in part due to the stresses placed on the late-stage fetus, impacting their normal development. These impacts are studied in light of the Developmental Origins of Health and Disease (DOHAD) hypothesis. Previous studies have correlated many adult-onset diseases with fetal and early life developmental stresses54. The potential to predict preterm birth, and provide interventions to reduce its incidence, would have a significant impact on human health.Preterm birth is a major health concern worldwide, affecting more than one in 10 pregnancies55, most previous studies have found that paternal lifestyle factors do not predict gestation length56. The current study identified epigenetic changes in both the mothers and fathers of children born preterm, suggesting potential maternal and paternal epigenetic components. Future expanded epigenetic analysis applied to both the mother and father may better assess risk of preterm birth, compared to assays of the mother alone.In this study buccal swab samples were collected from mothers, fathers and newborn infants approximately nine days following birth in cases where preterm birth occurred, and similarly in control full-term births. The buccal epithelial cells were analyzed for sites of DNA methylation in genomic regions when differences in methylation (DMRs) were detected. Mothers, fathers, and children all showed DMR signatures related to preterm birth Figs. , 2. MaleThe statistical confidence and accuracy of the prediction needs to be improved with expanded clinical trials with larger numbers of samples and trials monitoring individuals prior to conception of the child. Although, the current study demonstrates that epigenetic biomarkers in maternal and paternal buccal cells may be useful, larger studies are needed for predicting preterm birth. In the current study, buccal samples were collected from mothers and fathers immediately after the birth of their child. In the future, prospective studies with sample collection during pregnancy and prior to birth will be needed in order to develop a more clinically relevant predictive assay. Although a prospective study is anticipated to have similar DMR biomarkers, this remains to be confirmed.57. Another example is the ovarian cancer Ca125 biomarker which has a low accuracy for screening with both false positives and false negatives being problematic. However, for both PSA and Ca125, monitoring an individual over time does improve the accuracy of the assay to over 70% for monitoring, but not screening59. In addition, use of additional biomarkers in concert with the PSA and Ca125 has been found to improve the accuracy of screening to approximately 50%60. Due to the general low accuracy of such disease biomarkers, there have been a number of qualification and verification parameters put in place to improve and allow greater discovery efforts to be made for disease biomarkers62. Clearly disease biomarkers are essential for future medicine, but the current major protein-based biomarkers developed have limited use for general screening due to low accuracy. The current study provides large numbers of unique epigenetic-based DMR sites throughout the genome, which appear to relate to preterm birth. This is a unique molecular approach that may improve biomarker development. The study’s observations are encouraging and support the concept that epigenetic biomarkers derived from surrogate marker cells may be used as a biomarker for preterm birth. However, like PSA and Ca125, further clinical trials are needed to refine and validate the use of epigenetic biomarkers to predict preterm birth.In considering the accuracy of the epigenetic biomarkers observed, it is important to optimize with expanded clinical trials that include subpopulations of various sources of PTB such as obesity or preeclampsia. Interestingly some major disease biomarkers work approximately within a 50% accuracy range with either false positives or false negatives to consider. For example, for the major male prostate disease biomarker of Prostate Specific Antigen (PSA) for prostate cancer, the standard PSA cut-off of 4 ng/mL has low sensitivity. With this cut-off only 20.5% of the prostate cancer cases test positive and nearly 80% of prostate cancer cases are missed. The specificity at this cut-off is high (93.6%), meaning only 6.2% of men who do not have prostate cancer falsely test positive63 collected peripheral blood leukocyte samples from African American women at the start of labor that delivered either preterm  or at term . DNA methylation was assessed using the HumanMethylation450 BeadChip by Illumina. No DNA methylation biomarkers for preterm birth were identified, but these researchers did report that there were many DNA methylation changes that were shared between mothers that delivered preterm and their infants63. In a larger study of African American women, Hong et al.64 collected peripheral blood leukocyte samples at the time of labor from 150 women who delivered preterm, and 150 who delivered at term. DNA methylation was assessed using the Illumina HumanOmni2.5-4v1 array. Forty-five DMR were identified, of which two were found to be retained in a follow-up replication analysis64. Knijnenburg et al.65 performed a study that evaluated genomic variants, gene expression and DNA methylation simultaneously in whole blood samples taken in the day or two after birth. Two hundred seventy preterm and five hundred twenty-one full term maternal samples were evaluated. DNA methylation was assessed using the Illumina Methylation 450K array. No genomic variants were associated with preterm birth. However, 215 differentially expressed genes and two DMRs were found to be associated with preterm birth. There were greater numbers of molecular differences associated with very early preterm birth (< 28 weeks of gestation). Analysis of the 44 cases of these very early births showed that 217 genetic variants, 838 differentially expressed genes and 811 DMRs were associated65. A combined approach like this that uses multiple types of biomarkers shows promise for developing accurate clinical assays to predict preterm birth in the future. As previously mentioned, a limitation of all these studies is the use of mixed cell populations, which can suggest the presence of an epigenetic change, but which is in fact due to alterations in cell population numbers33. Purified individual cell types are more effective to both identify and assess epigenetic differences as disease biomarkers42.Previous studies have attempted to identify changes in DNA methylation in pregnant women that could be used as biomarkers of preterm birth. Parets et al.65. These array platforms are biased toward detection of DMR in high density CpG islands, which constitute less than 1% of the genome50. However, the majority of the genome has a low density (1–3%) 1–3 CpG/100 bp density 50. The MeDIP procedure used in the current study is biased toward detection of DNA methylation in regions of lower CpG density of < 5 CpG/100 bp, which corresponds to > 95% of the genome50. Using the genome-wide MeDIP procedure to identify DNA methylation alterations increases the feasibility of finding robust epigenetic biomarkers of preterm birth.A number of the previous studies have used the Illumina array platform to identify DMRs as biomarkers of preterm birth46.In the current study, only approximately half of the DMRs had nearby associated genes. Although the regulatory role of the DMRs to alter gene expression requires further investigation, the potential functional correlations of the DMR-associated genes for each group were evaluated. Genes involved in cytoskeleton, transcription and signaling were prominent in the gene sets Figs. , 5. AmonIn conclusion, genome-wide differential DNA methylation regions for preterm birth were detected in buccal cells of mothers, fathers, and female children. This provides a “proof of concept” that DNA methylation analysis of buccal swabs of parents may be used to potentially predict preterm birth. However, the accuracy and predictive ability of the biomarker needs to be improved with future clinical trials, as discussed. Such a preterm birth risk or susceptibility biomarker would allow for better obstetrical management to prevent preterm birth, mitigate morbidity in unprevented preterm births , and thus improve the health and long-term outcomes for many children. Unanticipated preterm births continue to catch providers by surprise, and often lead to major morbidities such as intraventricular hemorrhage, severe lung disease and other irreversible injuries. The presence of preterm birth associated DMRs in parental buccal cells suggests potential parental early life exposures and/or ancestral impacts are involved in the etiology of preterm birth. Rodent models have shown that environmental exposures in early pregnancy when epigenetic programming occurs in the fetus impact DMRs in every somatic cell type in the body across the life span of the exposed fetus and its descendants. Parents’ buccal cells, thus, may have the epigenetic changes resulting from ancestral exposure and can potentially be used as biomarkers for risk of preterm birth. This assay could also potentially be used in the future to identify environmental exposures and risk factors that promote preterm birth.St. Franciscan Hospital and Indiana University School of Medicine. IU Health Hospitals  and Franciscan Health, Indianapolis, Indiana, USA provided samples for the current study. Informed consent and HIPAA authorization was obtained from all participants prior to the clinical sample collection. The study protocol was approved by the Indiana University Institutional Review Board (IRB) #1901985132 and the Franciscan Institutional Review Board (IRB), #1489434-5. All research was performed in accordance with relevant guidelines/regulations. Informed consent and HIPAA authorization was obtained from all participants prior to sample collection. For sample collection involving human participants that are minors, informed consent from a parent and/or legal guardian for study participation was obtained prior to sample collection. Buccal samples were collected from the mother, father, and child in instances where pre-term birth occurred (case), or where term birth occurred (control), approximately nine days following birth. This period was used to allow the case PTB child to mature and allow and effective buccal cell collection. The demographic data for these subjects is presented in Supplemental Table Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μL of cell lysis solution and 3.5 µL of Proteinase K (20 mg/mL). This suspension was incubated at 55 ºC for 3 h, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µL microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µL of protein precipitation solution  was added to the lysis solution. The sample was incubated on ice for 15 min, then centrifuged at 4C for 30 min. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1000 µL ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at − 20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 min, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μL H2O. DNA concentration was measured using the Nanodrop .Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual DNA samples  were diluted to 130 μL with 1 × Tris–EDTA  and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 mL microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer  to 400 μL, heat-denatured for 10 min at 95 C, then immediately cooled on ice for 10 min. Then 100 μL of 5X IP buffer and 5 μg of antibody  were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 C. The following day magnetic beads  were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μL per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 mL 1XPBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1–2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1xIP buffer  as the initial volume of beads. 50 μL of beads were added to the 500 μL of DNA-antibody mixture from the overnight incubation, then incubated for 2 h on a rotator at 4 C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1–2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μL digestion buffer with 3.5 μL Proteinase K (20 mg/mL). The sample was incubated for 2–3 h on a rotator at 55 C, then 250 μL of buffered Phenol–Chloroform- Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 s and then centrifuged at 14,000 rpm for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μL chloroform were added to the supernatant from the previous step, vortexed for 30 s and centrifuged at 14,000 rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μL of glycoblue (20 mg/mL), 20 μL of 5 M NaCl and 500 μL ethanol were added and mixed well, then precipitated in -20 C freezer for 1 h to overnight. The precipitate was centrifuged at 14,000 rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μL cold 70% ethanol in − 20 C freezer for 15 min then centrifuged again at 14,000 rpm for 5 min at 4 C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μL H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212).The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina  starting at step 1.4 of the manufacturer’s protocol to generate double stranded DNA. After this step the manufacturer’s protocol was followed. Each sample received a separate index primer. NGS was performed at WSU Spokane Genomics Core using the Illumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 5–35 million reads per sample, and 6–7 sample libraries each were run in one lane.66. Reads were filtered and trimmed to remove low quality base pairs using Trimmomatic67. The reads for each sample were mapped to the GRCh38 human genome using Bowtie268 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools69. To identify DMR, the reference genome was broken into 1000 bp windows. The MEDIPS R package70 was used to calculate differential coverage between control and exposure sample groups. The edgeR p value71 was used to determine the relative difference between the two groups for each genomic window. Windows with an edgeR p-value less than 10–4 were considered DMRs. The DMR edges were extended until no genomic window with an edgeR p-value less than 0.1 remained within 1000 bp of the DMR. CpG density and other information was then calculated for the DMR based on the reference genome. DMR were annotated using the NCBI provided annotations. The genes that overlapped with DMR were then input into the KEGG pathway search73 to identify associated pathways. The DMR associated genes were then sorted into functional groups by reducing Panther74 protein classifications into more general categories. All MeDIP-Seq genomic data obtained in the current study have been deposited in the NCBI public GEO database (GEO #: GSE194227).Basic read quality was verified using information produced by the FastQC program44.Blinded test set analysis was performed to classify test samples into case or control groups. Samples from ten novel trios were collected to evaluate the efficacy of using the DMR sets identified as a biomarker for preterm birth. The test samples were processed identically to the samples used in the main analysis. PCA and cluster dendrogram analyses were used to search for test samples that clustered with the known samples when only DMR sites were considered. Additionally, linear discriminant analysis (LDA) and random forest (RF) classification was performed to identify which blinded samples were preterm birth, as previously describedApprovals to conduct the study were obtained from Indiana University Institutional Review Board (IRB) #1901985132 and the Franciscan Institutional Review Board (IRB), #1489434-5.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.


The purpose of this study was to evaluate the gross motor development of early-term infants using the Alberta Infant Motor Scale (AIMS). A total of 1087 healthy infants (559 early-term and 528 full-term infants born at 39+0 to 41+6 weeks of gestation) were studied. Mean AIMS scores were compared between the two groups at monthly intervals. The impact of gestational age on total AIMS scores was assessed by linear regression, after adjustment for chronological age, sex and SGA. Mean total AIMS scores, albeit within normal range, were significantly lower in early-term than full-term infants at the 2nd, 6th, 7th, 8th and 12th month of age; differences between groups were within three points. In multivariate regression analysis, a longer gestation by one week had a positive impact on total AIMS score during the first year of life . In conclusion, early-term infants exhibit worse gross motor performance during the first year of life in comparison with their full-term peers; however, the differences between the two groups are small.Early-term birth (37 It has outcomes ,11, commoutcomes , school outcomes ,15. Howeoutcomes . So far,outcomes , furtherThe Alberta Infant Motor Scale (AIMS) is a reliable, practical, observational and standardized tool for the assessment of gross motor development of infants from birth to the age of independent walking 0–19 months). Infant motor performance is assessed by AIMS in four postures: prone, supine, sitting and standing . The AIM months. +6 weeks of gestation.This study aimed to assess, using the AIMS, the gross motor development of early-term infants in comparison with more mature infants born at 39 to 41+0 to 41+6 weeks of gestation, participated in this cross-sectional study. They were recruited from obstetric and pediatric clinics, state and private day care centers, and private pediatric offices located in different areas of the Prefecture of Attica. Demographic data based on medical birth records or maternal self-reports were recorded. Infants born preterm (<37+0 weeks), or with perinatal problems, neurological disorders or any acute or chronic diseases were not enrolled. The study population was categorized into two distinct groups: (a) early-term infants (37+0 to 38+6 weeks of gestation) and (b) full-term infants (39+0 to 41+6 weeks of gestation). Gestational age definition was based on medical reports of the obstetricians and neonatologists at birth. Depending on birth weight, size at birth was characterized as appropriate for gestational age (AGA) if birth weight was between the 10th and 90th percentile, or small for gestational age (SGA) if birth weight was below the 10th percentile for the corresponding gestational age and gender using the INTERGROWTH-21st growth chart [Healthy Greek singleton infants, aged 0 to 19 months, who were born from 37th chart .In all participants, the gross motor development was evaluated once during the study period using the AIMS. Assessments were performed by the same examiner who was blinded on the infants’ gestational age at birth. The infants were assessed at their house, in obstetric and pediatric clinics or in private pediatric offices, but always with the presence of the caregiver so that the infants could feel safe and comfortable.The purpose and design of the research was approved by the ethics committee of our institution and informed parental consent prior to enrollment was obtained.The AIMS consists of fifty-eight (58) items evaluated at 4 different postures . The components assessed in each item are based on the following three elements of movement: weight bearing, posture and antigravity movements. For each item observed during the evaluation, one (1) point is credited for the infant, whereas zero (0) points are credited when the item is not observed. The sum of all items observed equals to the total raw AIMS score, which can range from zero to fifty-eight. The total raw AIMS score can be converted into a percentile rank. High percentile ranks point out maturity in gross motor development; on the contrary, infants scoring below the 5th percentile are at high risk of gross motor delay ,19. The t-test or the Wilcoxon rank-sum test, as appropriate. The percentile rank of total AIMS score was also assessed and the number of infants exhibiting total AIMS scores below the 5th percentile was compared between the two study groups. The chi square test was used for comparison of categorical variables. Multiple regression analysis was applied to evaluate the effect of early-term birth on AIMS scores after adjusting for chronological age, sex and size at birth (SGA vs. AGA). p-value ≤ 0.05 was considered statistically significant. The data were analyzed using the STATA 13 and IMP SPSS 25.0  statistical packages.Mean total raw AIMS score and sub-scores at prone, supine, sitting and standing posture were compared at monthly intervals between early-term and full-term infants in order to identify any differences in gross motor development between the two groups. The Shapiro–Wilk test was applied to assess normality. Comparisons were performed using the Student’s p < 0.001). There was no difference in gender distribution between the early-term  and full-term group  (p = 0.513). In addition, the number of infants being born SGA (10 in the early-term group vs. 9 in the full-term group) did not differ significantly between the two study groups (p = 0.445).A total number of 1087 infants participated in this study. Of them, 559 infants were early-term and 528 infants were full-term, born at 39 weeks of gestation or beyond. Mean ± SD birth weight was 3140 ± 352 g and 3387 ± 382 g in the early-term and full-term group, respectively .However, after the first year of life, no significant differences were found in total AIMS scores or sub-scores between the two study groups. Moreover, the number of infants exhibiting total AIMS scores below the 5th percentile for age did not differ significantly between the early-term and full-term group .Linear regression was applied following stratification by age (≤12 months vs. >12 months). After adjusting for age, gender and size at birth, early-term birth was found to be associated with a statistically significant reduction in total AIMS score but only in the group of ≤12 months of age . Chronological age was an independent predictor of total AIMS score for both groups: ≤12 months  and >12 months of age  . When gep < 0.001), sitting  and standing sub-scores  in the group of infants aged ≤12 months of age.Regression models were also run for the sub-components of AIMS at prone, supine, sitting and standing posture. After adjusting for age, gender and size at birth, early-term birth was recognized as an independent predictor for prone  that normally coincide with important milestones’ achievement, namely independent sitting and standing, respectively. At 6, 7 and 8 months of age, the sub-scores for prone posture were mostly affected, also influencing the total AIMS scores. At 12 months of age, differences in sub-scores at sitting and standing posture contributed to the lower total AIMS score in early-term infants. The observed differences in motor performance between groups may be due to differences in trunk control development between early-term and full-term infants. Recent research has linked the segmental trunk control levels with gross motor performance and demonstrated that preterm infants exhibit a delay in trunk control development as compared to full-term infants ,29. FurtSuboptimal gross motor performance in our early-term healthy infants, without any perinatal morbidity or complications after birth, may be explained by the interruption of the normal brain maturation process in utero. It has been shown previously that the weight of human fetal brain increases linearly with gestational age, even during the last few weeks of full-term gestation . Thus, dIn our study, although significant differences were found in motor development between the early-term and full-term group at specific months of age, the mean values of AIMS scores at those months in early-term infants were well above the cut-off of the 5th percentile, thus within the normal range. Moreover, the number of infants exhibiting total AIMS scores below the 5th percentile for age did not differ significantly between groups. Furthermore, the observed differences in mean total AIMS scores between the two groups of infants, although significant, were rather small (within three points). As we do not have, at present, the minimal clinically significant difference of the AIMS, the clinical interpretation of the three-point difference is unknown.The major strength of our study is the relatively large sample size that augments the power of the results. However, although more than a thousand infants were studied, the number of infants was rather small in some age categories . Moreover, the results of this study should be considered in light of some methodological limitations. Gestational age at birth was assessed retrospectively based on medical reports of the obstetricians and neonatologists at birth. However, it should be mentioned that obstetricians in Greece follow the guidelines issued by medical societies, thus we expect that the results are not biased. Furthermore, it was not always reported whether the delivery was spontaneous, induced or acute-prolonged. Although we only included healthy infants in the study, such information would place some of our participants at greater risk for unfavorable neurodevelopmental outcomes. Additionally, although we adjusted for infants’ gender and SGA, other potential confounders of neurodevelopment  were not recorded; adjustment for those characteristics and socioeconomic circumstances known to affect neurodevelopment should be performed in future research. Finally, as this is a cross-sectional study, participants were assessed only once. However, in an attempt to more precisely evaluate the differences in motor development between groups, individual time trajectories for a long time period would possibly be more appropriate.In this study, we were able to demonstrate that early-term infants exhibit worse gross motor performance during the first year of life in comparison with their full-term peers. The differences between the two groups were small, but in combination with the remarkable rate of early-term births, and the evidence for additional intellectual delays in these infants, they may bring about a significant risk for this population. Efforts should be made for the prevention of early-term deliveries, as far as possible, and for the early detection of any neurodevelopmental disorders in these infants, so that early and appropriate interventions are offered.