There are few data from Latin America. We have examined PM2.5 and pregnancy outcomes in Lima. The study included 123,034 births from 2012 to 2016, at three public hospitals. We used estimated daily PM2.5 from a newly created model developed using ground measurements, satellite data, and a chemical transport model. Exposure was assigned based on district of residence (n = 39). Linear and logistic regression analyzes were used to estimate the associations between air pollution exposure and pregnancy outcomes. Increased exposure to PM2.5 during the entire pregnancy and in the first trimester was inversely associated with birth weight. We found a decrease of 8.13 g  overall and 18.6 g  in the first trimester, for an interquartile range (IQR) increase (9.2 µg/m3) in PM2.5. PM2.5 exposure was positively associated with low birth weight at term (TLBW) during entire pregnancy , and at the first , second , and third trimester  per IQR (9.2 µg/m3) increase. Higher exposure to PM2.5 was also associated with increased risk of small for gestational age (SGA). There were no statistically significant associations between PM2.5 exposure and preterm births (PTB). Exposure to higher concentrations of PM2.5 in Lima may decrease birth weight and increase the frequency of TLBW and SGA. Our study was inconsistent with the literature in finding no associations with preterm birth.The literature shows associations between maternal exposures to PM Air pollution is a major environmental concern worldwide. There is a large literature regarding particulate matter (PM) in air and its relationship with human health –3.2.5 ranging from 35 µg/m3 (Ate station) to 16 µg/m3 (Campo de Marte station). In 2015, it reached a mean value of 26 µg/m3 for 2015 [3).The metropolitan area of Lima, a megacity located in Peru, has been considered among the most contaminated cities in Latin America  with ann2.5 is a heterogeneous mixture of fine PM that can induce systemic inflammation, oxidative stress, and hemodynamic changes. In pregnant women, the placenta can be affected by PM reducing the blood flow, and delivering less oxygen and nutrients to the fetus [PMhe fetus . Studieshe fetus . This inhe fetus .Low birth weight at term (TLBW) (<2500 g) has been considered an important risk factor for noncommunicable diseases later in life such as coronary heart disease, stroke, type II diabetes, and hypertension . Preterm2.5 and TLBW, with odds ratios (ORs) ranging from 1.02 to 1.13 per 10 µg/m3 or interquartile range (IQR) increase, and with PTB, with ORs ranging from 1.02 to 1.14 [3 increase in PM2.5.Previous meta-analyses have shown an association between high PM to 1.14 –14. Thes2.5 come from developed countries, with very few from Latin America. Lima has heterogeneous levels of PM2.5, offering large exposure contrasts for pregnant women living in different parts of Lima. To our knowledge there are no studies related to the effect of outdoor PM air pollution on pregnancy outcomes in Lima.Most studies about PM2.5 has an association with lower birth weight, and would increase the risk of PTB, TLBW, and births that are small for gestational age (SGA). Therefore, the aim of the present study was to investigate the association between maternal exposure to PM2.5 overall during pregnancy, and at each trimester, in relation to adverse pregnancy outcomes in Lima, Peru, from 2012 to 2016.Our hypothesis was that higher PMLima is located in the central coast of Peru at 150 m above sea level and has an approximate population of 9,320,000 inhabitants [n = 39) of pregnant women who delivered in three large public hospitals: National Maternal Perinatal Institute, (INMP in Spanish) and San Bartolome National Hospital , and Santa Rosa hospital, all located in downtown Lima  represented only 4% of the total population of Lima, and hence their exclusion could have little bearing on the results.The metropolitan area of Lima is made up of 43 districts and divided into 4 zones: North Lima, Central Lima, East Lima, and South Lima. For the present study, we have assigned exposure based on the residential districts , mothers residing outside the metropolitan area of Lima (n = 466), malformations (n = 855), stillbirths (n = 1469), and outlier data where birth weight that was greater or less than three standard deviations from the mean corresponding to gestational age (n = 340), missing residence district (n = 150), duplicate records (n = 1941), missing mother’s age (n = 69), and days without PM2.5 estimates (n = 3080), due to missing satellite date on some days. The remaining data for live newborns from singleton births coming from women aged 15–49 years old and living in the Metropolitan area of Lima, numbered a total of 123,034 births.Birth records for the hospital were obtained from the three public hospitals for the years 2012–2016. These hospitals attend pregnant women from all over Lima. Two of them were exclusive maternal care facilities, and, together they attend 26% of the deliveries in Lima . From 13Z-score adjusted for gestational age, determined from INTERGROWTH tables (https://intergrowth21.tghn.org/).Birth weight (g) was defined as the first weight of the newborn measured in a calibrated scale by a trained hospital staff. PTB was defined by a delivery below 37 weeks of gestation. TLBW was defined as birth weight less than 2500 g at 37–42 weeks of gestation. SGA was defined as the 10th percentile of weight by gestational age. Cutoff weights to determined SGA were developed using the birth weight-2.5 data in Lima were available from March 2010 through December 2016, from monitors in ten stations located in the four zones of Lima and belonging to the Peruvian National Service of Meteorology and Hydrology , and monitors in six stations in southern Lima operated from 2011 to 2012 by Johns Hopkins University [2.5 exposure data from a model developed by Vu et al. [2.5 in Lima. The resulting model predicted daily PM2.5 in Lima at a 1 km2 spatial resolution and fits the observed data well, with an R-square of 0.70 (mean difference between ground and predicted measurements 0.09 µg/m3). The full methodology employed in this study can be finding in [Some ground-monitoring PMu et al. , Brieflynding in  as well.2.5 across her district for each day she was in the study. We then calculated several exposure variables for each birth: (1) average exposure to PM2.5 during the entire pregnancy defined as the average of pollutant concentration from week 1 to birth, and (2) average exposure by trimester of pregnancy, defined as the average of PM2.5 in the first (1–12 weeks), second (13–26 weeks), and the third (week 27 to birth) trimester of pregnancy.Our birth records provided data on district of residence but not exact address. Hence we assigned to each mother the population-weighted average PMGestational age at delivery was determined by the last menstrual period. For this variable, 105 values were missing in the database and were replaced by the gestational age determined by physical examination according to the Capurro methodology .2), and parity . BMI was missing from half of the database and we generally did not include it in our models, but when we did, the results for PM2.5 were generally similar to those omitted. Variables for prenatal care and the presence of preeclampsia during pregnancy were included in the analysis as dichotomous variables (yes/no).The following characteristics from mothers were also obtained from the database: mother’s age at delivery , body mass index (BMI) according to WHO  and proteinuria (≥300 mg/24 h) after 20 weeks of gestation in otherwise normotensive women.Percentage of poverty was defined as the percentage of households within a district with a self-reported family income below what is needed for sustenance, food, clothing, or shelter by the Peruvian Census . We dichSmoking was not considered as the rate among pregnant women in Peru is very low (<1%) and data were not consistently available . All theWe did not include hospital or district as either fixed or random effects because our exposure was assigned based on district of residence, and spatial contrast across districts was our primary exposure contrast, such that inclusion of either hospitals (which served primarily certain districts), or district itself, limited our ability to detect exposure contracts. We also had a number of individual-level potential confounders, limiting likely confounding by district.3, mean in districts above the midpoint, 22.8 μg/m3). Hence as a confounder GDM would be expected to bias our results to the null, making our estimates conservative.A study in the National Maternal Perinatal Institute in Lima showed that ~16% of pregnant women at that hospital were diagnosed with gestational diabetes mellitus (GDM); in that study GDM was diagnosed using an Oral Glucose Tolerance Test performed between 24 and 28 gestational weeks . However2.5 exposure and birth weight as a continuous variable. Logistic regression analysis was used to determine the relationship between PM2.5 exposure during pregnancy with TLBW, SGA, and PTB. Covariates which by themselves attained a p value < 0.1 were included one by one in the models, and those that retained statistical significance at p < 0.05 were included in the final model. Maternal age, parity, prenatal care, gestational age, poverty, and preeclampsia were included in all final models, with the exception of poverty, which was not included in the model for TLBW.Data were collected in an Excel spreadsheet and analyzed using SAS version 9.4 . Generalized linear models were used to explore the relationship between PM2.5, exposure was also categorized into quartiles (based on the data over the entire study period): Q1st: 12.74–16.83 µg/m3; Q2nd: 16.84–18.42 µg/m3: Q3rd: 18.43–26.04 µg/m3, and Q4th: 26.05–41.6 µg/m3, Exposure variables were also generated for each trimester of pregnancy. Regression models were run for exposure during the entire pregnancy and also by trimester of pregnancy. By way of reference, WHO recommends PM2.5 concentration below 10 µg/m3 as an annual mean, while the Peruvian Ministry of the Environmental in Peru recommends 25 µg/m3.In addition to continuous PMThe protocol was approved by Ethics Review Committee of Cayetano Heredia University (SIDISI code 101546).The computer code to conduct our analyses is available on request to the authors.From the overall population studied , among those with full-term births, 2074 (1.8%) were TLBW, and from the overall population, 8897 (7.2%) were PTB  and median 21.8 µg/m3, and an IQR of 9.2 µg/m3. The highest concentrations of PM2.5 were observed in the eastern zone of Lima, and the lowest concentrations in the center zone of Lima  exposure during the entire pregnancy, and during the first trimester, but not during the second and third trimester. An IQR (9.2 µg/m3) increase in PM2.5 concentrations during the entire pregnancy was associated with 8.13 g decrease in birth weight . We also found a decrease of 18.6 g  per IQR (9.2 µg/m3) increase in PM2.5 during the first trimester of pregnancy. In this trimester, the highest quartile of PM2.5 exposure (>25.3 µg/m3) was associated with lowest birth weight . We found the similar results when we restricted the data to full-term births. There was a decrease in birth weight largely restricted to PM2.5 in the first trimester.Table 2.5 and TLBW are shown in Table 3) increase in PM2.5 concentration observed during the entire pregnancy  and all trimesters  after controlling for age, parity, gestational age, preeclampsia, and prenatal care. In quartile analyses the highest risk was found in the fourth quartile of PM2.5.The results of the logistic regression analysis to assess the relationship between PM2.5 and SGA are shown in Table 3) increase in PM2.5 concentrations over the entire pregnancy , during the first  and third trimester , after controlling for age, parity, preeclampsia, and prenatal care. Categorical analyses found the highest risk concentrated in the highest quartile of PM2.5 exposure overall and in the first and third trimester.The results of the logistic regression analysis to assess the relation between PM2.5 and preterm birth (Table No significant associations were observed between exposure to PMth Table .Table 6A3) increase of PM2.5 in the first trimester while boys had a 12.6 g (95% CI 4.7–20.5) decrease. In girls, the OR of TLBW associated with an IQR increase in PM2.5 across all trimesters was 1.13 (95% CI 1.03–1.24), while boys had an OR of 1.09 (95% CI 1.00–1.19). For SGA, for an IQR increase in PM2.5 across all trimesters, girls had an OR of 1.05 (95% CI 1.00–1.10) while boys had an OR of 1.04 (95% CI 0.99–1.09). For PTB, there was virtually no effect of PM2.5 when analyses were conducted for each sex separately.We also explored possible differences between male and female births. We found some differences for birth weight, but little difference for TLBW or SGA. For girls, birth weight decreased 23.8 g (95% CI 15.9–31.8) per IQR . Eastern areas in Lima had higher values, while Central Lima had the lowest. The variability observed between districts is due to the morphological and atmospheric characteristics that determine the air quality in Lima. Coastal winds heading south to north, the Andes Mountain on the east, and thermal inversions, cause the pollution loads to be deposited in the eastern and northeastern areas of Lima [2.5 as our exposure measure.The present study aimed to determine the relationship between the exposures to outdoor PM2.5 was associated with birth weight, SGA, and TLBW. We observed a decrease in birth weight with higher PM2.5 over the entire pregnancy and in the first trimester, while risks for TLBW and SGA were elevated in each of the three trimesters. This is an important finding since the mean value of PM2.5 concentration for our mothers observed in Lima (22.3 µg/m3) is considered by the Peruvian authorities as normal (recommended level for PM2.5 is <25 µg/m3), but resulted in higher risk of adverse pregnancy outcomes.Our results showed that exposure to PM2.5 was associated with higher risk of LBW or TLBW. Our results for TLBW are generally consistent with these reviews.Three prior meta-analyses , 13, 24 2.5, during the entire pregnancy, and in particular in the third trimester [Regarding birth weight as a continuous variable, two meta-analysis , 13 foun2.5 exposure during the first trimester than during the second and third trimesters. Another study by Li et al. [2.5 found lower birth weight after exposure in both early and late gestational periods.However, not all studies have found the strongest effects on birth weight due to third trimester exposure. In one study by Kumar et al. , birth wi et al.  had simii et al.  exposing2.5 exposure during the first trimester could interfere with the normal development of the placenta, affecting placental mitochondria and, therefore, the ability of the placenta to support the growing fetus [Oxidative stress produced by PMng fetus . The fas2.5 exposure during the first trimester may also show a stronger association with body weight than the exposure during the second and third trimester, because the fetus size is relatively small during early gestation period. Thus, the same PM2.5 exposure during the early stage of gestation period may translate to a high dose of PM2.5, given the smaller fetus size [2.5 for exposure in both early and late in pregnancy.The PMtus size . On the 2.5, while we did not. We have no explanation for this discrepancy. In our data, PM2.5 seemed to decrease fetal growth but not lead to prematurity. It may be that the specific nature of the PM2.5 in Lima, which derives primarily from vehicular emissions rather than industrial sources, is associated with fetal growth but not prematurity.With respect to PTB, three prior meta-analyses , 14, 29 2.5 concentrations for each district of Lima. This model allowed the construction of consistent long-term historical measurements in replacement of the sparse data available from ground monitors, and the assignment of PM2.5 to local residential areas. In addition, we used the perinatal records of three large specialized public hospital located in the center of Lima, which received pregnant women from all the districts of the city.One strength of our study is the use of a predictive model that estimated daily PM2, as reported in other articles [One limitation of our study is the lack of data on co-pollutants such as ozone, or NOarticles , 31. Anoarticles , 33.3 for PM2.5, the Peruvian Ministry of Environment recommends a much higher limit of 25 µg/m3.Identification of an association of air pollution with adverse pregnancy outcomes has substantial public health implications in Lima. We note that while WHO recommends an annual limit of 10 µg/m2.5 with pregnancy outcomes in Lima. The results supported previous findings that environmental PM2.5 exposure can be hazardous to pregnancy, regarding birth weight, low birth weight, and births SGA, but not for PTB, where we found no effect for PM2.5. We found our most significant association of PM2.5 with birth weight during the first trimester. Our study provides new evidence regarding the effects of PM2.5 on pregnancy outcomes, suggesting that the existing Peruvian standards are too high.This is the first study that correlates the exposure to PM


This was especially the case for women whose AUD diagnosis was in the same year as their delivery . AUDs were associated with increased risk of adverse birth outcomes, especially when prevalent in the same year as a woman’s delivery. Our study confirms that the monitoring of expecting women with a diagnosis of alcohol-related problems may be useful in preventing adverse birth complications.For women who suffer from Alcohol Use Disorders (AUDs), the use of alcohol before and/or during pregnancy may result in various birth complications, including miscarriage, stillbirth, or preterm delivery. Thus, this study aimed to explore whether Alcohol Use Disorders (AUDs) are associated with increased risk of adverse birth complications and outcomes. A total of 76,799 deliveries between 2003 and 2013 in the Korean National Health Insurance Service National Sample Cohort (NHIS-NSC) were analyzed. Women with an AUD diagnosis preceding delivery were identified as individuals with alcohol dependence. A multivariate Cox proportional hazards model was used to estimate the hazard ratio of adverse birth complications and outcomes associated with alcohol dependence. Diagnosis of an AUD was associated with increased risk of adverse birth complications (Hazard Ratio [HR]: 1.15, 95% CI: 1.01–1.31,  During the COVID-19 pandemic, alcohol consumption has been increasing as individuals experience increased feelings of social estrangement, self-isolation, and a sense of loneliness .AUDs are a maladaptive pattern of heavy and harmful alcohol use that leads to various behavioral, cognitive, and physiological phenomena, including a strong desire to drink, increased tolerance, and physical withdrawal . GloballFor women who suffer from AUDs, the use of alcohol before and/or during pregnancy can result in various pregnancy and birth complications, including miscarriage, stillbirth, preterm delivery, and sudden infant death syndrome . In one Because alcohol may cause an angiogenic imbalance that affects lipid levels, inflammation, and oxidative stress , drinkinWhile many published reports have focused on an association between alcohol consumption during pregnancy and such outcomes, few studies have focused on the association between a diagnosis of alcohol addiction preceding pregnancy and subsequent consequences. Despite the fact that the risk for alcohol use during pregnancy increases if the mother used and/or abused alcohol before her pregnancy , few stuThus, this study aimed to examine the association between Alcohol Use Disorders (AUDs) and the risk of adverse birth complications and outcomes using nationwide cohort data.The data used in this study were obtained from the National Health Insurance Service National Sample Cohort (NHIS-NSC) in Korea for the years 2002 to 2013. These data were constructed with the sole purpose of providing public health researchers and policy-makers with representative, useful information regarding Korean citizens’ utilization of health insurance and health examinations . To consFor the identification of pregnancy, we employed the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis codes Z32.1, Z33, Z34.00, Z34.80, Z34.90, Z35, and O30, as seen in a previous study using the same dataset . IncidenFor the identification of AUDs, we employed the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis codes E24.4 , F10 , G31.2 , G62.1 , G72.1 , I42.6 , K29.2 , K70 , K85.2 , K86.0 , as seen in a previous study of alcohol use disorders .We controlled for numerous patient-related covariates when analyzing the association between alcoholism and adverse birth outcomes. Such variables included age of delivery, income, insurance coverage, employment status, region, and year of diagnosis.Age was divided into six 5 year groups  to reflect any differences in the association between alcoholism and adverse outcomes by maternal age at delivery. Insurance coverage was categorized into three groups: insurance , insurance (corporate), and Medical Aid. Based on the criteria of South Korea’s National Health Insurance (NHI) system, individuals who are self-employed are covered by regional insurance, while individuals employed by a company are covered by employer-based corporate insurance. Medical aid beneficiaries consist of all those who have an income below the government-defined poverty threshold or a disability which enables them to receive free inpatient and outpatient care through the government.Region was divided into three categories according to population density: metropolitan , city , and other .2 tests to examine the distribution for adverse birth complications and outcomes according to each variable. Subsequently, we performed survival analyses using a Cox proportional hazards model to examine the factors significantly associated with adverse pregnancy outcomes. Cases with an event of birth complication or abortion were coded as 1, and all other cases were coded as 0. The follow-up period in the study was from 1 January 2003 to 31 December 2013 with the length of survival recorded in days. The HR and 95% confidence interval (CI) were computed from the Cox proportional hazard model, and statistical significance was determined by a two-tailed test with a p-value of 0.05 as the threshold. All statistical analyses were performed using SAS software, version 9.4 .We first examined the frequencies and percentages of each categorical variable at each patient’s baseline and performed χAll methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Institutional Review Board of Yonsei University’s Health System (Y-2019-0174). The need for informed consent was waived since the NHIS-NSC provides anonymous cohort data to researchers for scholarly use.This p = 0.0302) of adverse birth outcomes, especially if her AUD diagnosis was made in the same year as her delivery . Age at delivery was also significantly associated with increased adverse birth outcomes: compared to the 20–24 years’ age group, women who gave birth in the 25–29 age group , 30–34 age group , 35–39 age group , and ≥40 age group  had increased odds of adverse birth outcomes.p = 0.0129) compared to women with regional insurance coverage, as did women who had already given birth 2 times , three times , and four or more times . Women who gave twin births  were at increased odds of adverse birth outcomes compared to women who gave single births also.Working with corporate insurance coverage also had increased risk of adverse birth outcomes  and other regions  had decreased risk of adverse birth outcomes.On the contrary, compared to women living in metropolitan regions, women living in the city  and Intrauterine Growth Restriction .Our findings show that AUD diagnosis is significantly associated with increased risk of adverse birth outcomes, especially when the diagnosis is made in close proximity to delivery (same year as delivery). To our knowledge, few studies to date have employed an ICD-10 diagnosis of AUDs as a marker for predicting adverse birth outcomes, despite the fact that self-reported measures of alcohol consumption during pregnancy are presumed to be highly unreliable.Although instruments like the T-ACE/T-ACER-3, TWEAK, or Substance Use Risk Profile-Pregnancy (SURP-P) have been empirically validated for use among pregnant women , it is uUntil more research on biomarkers like meconium ethyl glucuronide (EtG) are developed to detect alcohol consumption during pregnancy , employiWhile evidence has been conflicting regarding the association between alcohol diagnosis prior to and during pregnancy and risk of adverse birth outcomes, one prospective cohort of 1303 pregnant women in the United Kingdom found that intakes of more than 2 units of alcohol/week before pregnancy, and in trimesters 1 and 2, were significantly associated with increased risk of babies with lower birth weight, lower birth centile, and preterm birth compared to non-drinkers . LikewisLike previous studies, our study also found an association between high number of births and the birth of twins with increased adverse birth outcomes. A high number of previous births has been associated with poor pregnancy outcomes in a number of studies ,28, and Surprisingly, AUD diagnosis was not associated with increased risk of many individual birth complications: AUD diagnosis was only associated with increased risk of intrauterine growth retardation in our subgroup analysis. Many studies have explored the association between prenatal alcohol exposure and intrauterine growth retardation ,30,31, iThe limitations of our study derive from the limited accuracy and reliability of the ICD-10 classification of alcoholism within our study population. Although it must be assumed that the classifications were made by healthcare professionals fully aware and abiding of specific criteria for maximum diagnostic reliability, chronic alcoholism is a difficult condition to diagnose. Furthermore, diagnosing alcoholism among individuals with disability can be extremely challenging for clinicians; heavy drinking associated with alcoholism can contribute to, or result from, several different psychiatric syndromes ranging from depression to sociopathy and dementia, that make diagnosis confusing and inaccurate .Second, our study centered on the time until adverse birth outcome following year of diagnosis (2003), which was around ten years. Therefore, during this time period, various confounding variables will have influenced the patient’s risk of adverse birth outcomes, relative to medical advancements, seasonal biological changes, and social, economic, and political factors. As of yet, there are not enough previous studies with regard to a nationally representative population of Koreans when it comes to measuring the impact of alcoholism and adverse birth outcomes. It is difficult to see whether the figures we calculated are similar to that of the statistics found in previous studies for Koreans.Also, while our study specifically focused on adverse birth outcomes that occur during birth and mostly affect the mother, further studies should also attempt to examine the long-term and more severe consequences of prenatal alcohol exposure on child development, i.e., fetal alcohol spectrum disorders (FASDs). Prenatal alcohol exposure has been associated with a range of mental and/or physical disabilities among individuals whose mothers consumed alcohol during pregnancy . PreviouLastly, while we assumed that alcohol consumption during pregnancy would be prevalent if an AUD diagnosis was made in the same year as delivery, it is impossible to know whether or not this was so. This was a limitation of our secondary data, but it is highly recommended that future studies employ instruments like the T-ACE/T-ACER-3, TWEAK, or Substance Use Risk Profile-Pregnancy (SURP-P) in simultaneity with AUD diagnoses, for a more accurate prediction of alcohol consumption during pregnancy .Despite these limitations, our study has a number of strengths. It is one of the first reports to address the association between AUD diagnosis and adverse birth complications, while controlling for the proximity between diagnosis time and birth. To our knowledge, no study in South Korea has attempted to evaluate the association between alcohol consumption during pregnancy and adverse birth outcomes, mostly because data on alcohol consumption during pregnancy are very rare, if not non-existent, in our country as of now. Furthermore, our data consisted of a nationally representative sample of women who were examined by medical professionals for the NHIS database. Most importantly, instead of using retrospective self-reports of alcohol consumption during pregnancy, we measured the possibility for alcohol-related problems during pregnancy through an ICD-10 diagnosis of AUDs, which allowed for an accurate identification of expecting women with past/present alcohol-related problems.Overall, such findings suggest that AUDs are significantly associated with increased risk of adverse birth outcomes, especially when prevalent in the same year as a woman’s delivery. It is highly recommended that healthcare professionals routinely ask all pregnant women about their alcohol consumption, and pay special attention to women with a previous diagnosis of AUDs.Validated behavioral interventions for AUDs in pregnancy include motivational enhancement therapy and cognitive behavior therapies such as brief psychodynamic psychotherapy, interpersonal therapy, educational interventions, and supportive counseling . Pharmacbefore they become pregnant [As AUDs are associated with neurobiological adaptations that reduce an individual’s cognitive control, clinicians must be non-judgmental, non-directive, and private ,38. Empapregnant .


P < 3.53 × 10–127) and enrichment of functional annotations (P < 1.62 × 10–39) between birth weight and childhood body mass index (BMI)/obesity. The pleiotropic loci were enriched for regulatory features in skeletal muscle, adipose and brain tissues and in cell lines derived from blood lymphocytes. At 5% false discovery rate, 6 loci were associated with birth weight and childhood BMI and 13 loci were associated with birth weight and childhood obesity. Out of these 19 loci, one locus (EBF1) was novel to childhood obesity and one locus (LMBR1L) was novel to both birth weight and childhood BMI/obesity. These findings give evidence of substantial shared genetic effects in the regulation of both fetal growth and childhood obesity.Childhood obesity is a global public health problem. Understanding the molecular mechanisms that underlie early origins of childhood obesity can facilitate interventions. Consistent phenotypic and genetic correlations have been found between childhood obesity traits and birth weight (a proxy for in-utero growth), suggesting shared genetic influences (pleiotropy). We aimed to (1) investigate whether there is significant shared genetic influence between birth weight and childhood obesity traits, and (2) to identify genetic loci with shared effects. Using a statistical approach that integrates summary statistics and functional annotations for paired traits, we found strong evidence of pleiotropy ( Complex interactions of genetic susceptibility6, environmental exposures8 and in-utero programming12 contribute to childhood obesity. Fetal growth and development during the intrauterine period shape physiological and structural processes that impact risk for childhood obesity (COB)9. Birth weight, a marker of fetal growth and prenatal environment, has been consistently associated with COB20. Notably, a significant positive genetic correlation has been found between birth weight and COB/childhood body mass index (CBMI)21, suggesting the possibility for genetic links these phenotypes may share. Insight into the shared genetic underpinnings of these early life phenotypes facilitates knowledge on the potential common molecular pathways and intervention targets of abnormal birth weight and COB/CBMI. However, pleiotropic evidence on these traits remains scarce.Childhood overweight or obesity is a global public health problem26. In an analysis of the GWAS catalog27 it was found that 16.9% of the reported genes were associated with multiple traits28. With the advent of large scale independent GWASs on multiple early life traits and functional annotation database31, innovative statistical approaches can be applied to identify and test for novel pleiotropic loci.Recent genome wide association studies (GWAS) have reported that shared genetic effects (pleiotropy) might explain a substantial proportion of correlations between complex human traits32. Both birth weight and COB/CBMI are under strong genetic control35, with relatively high heritability estimates of 25–40% for birth weight35 and 30–70% for COB/CBMI34. However, the heritability of the traits explained by genetic variants identified using published GWAS for birth weight36, COB37 and CBMI40 was less than 10%. Therefore, detecting novel genetic loci via pleiotropic analysis can help close the gap in missing heritability.In addition, accounting for shared genetic effects improves statistical power for detecting genetic variants that are associated with complex traitsEBF1) was novel to childhood obesity and one locus (LMBR1L) was novel to both birth weight and childhood BMI/obesity.To date, pleiotropic associations of birth weight and COB/CBMI have not been exclusively investigated. In the current study, we used an innovative statistical approach that integrates pleiotropy and functional annotation data with the following aims: (1) to investigate whether there is significant shared genetic influence between birth weight and COB/CBMI (pleiotropy enrichment test), (2) to examine whether the shared risk variants are functionally more enriched in birth weight and COB/CBMI as compared to only considering single traits in models that incorporate a variety of genetic annotations in different tissues and cell types (annotation enrichment test), and (3) to identify genetic loci with shared genetic effects on birth weight and COB/CBMI. Our analysis found abundant evidence of pleiotropy and significant enrichment of functional annotations for shared risk variants associated with birth weight and COB/CBMI. We identified genetic loci with overlapping influence on both birth weight and COB/CBMI. Most of those loci have been associated with childhood BMI/obesity in previous GWAS but not with birth weight, whereas one locus  consortium. Two sets of GWAS summary statistics were available for birth weight, one involving European ancestry individuals (BWEU) and a second from trans-ancestry meta-analysis (BWTR). The GWAS summary statistics for CBMI and COB were from European ancestry individuals. Therefore, tests of pleiotropy were conducted on four trait pairs: BWEU-CBMI, BWEU-COB, BWTR-CBMI, and BWTR-COB.Data on summary statistics including 41 which implements a statistical approach to explore the genetic architecture of complex traits by integrating pleiotropy and functional annotation information, including prioritizing risk genetic variants. GPA performs hypothesis test for evaluating enrichment of pleiotropy and functional annotations using a likelihood ratio test approach. For each trait pair, we performed tests of pleiotropy and enrichment of functional annotations based on 4 functional categories: combined annotation dependent depletion (CADD) (http://www.cadd.gs.washington.edu)42, expression quantitative trait loci (eQTL) 43, transcription factor binding sites (TFBS) (ftp://ccg.epfl.ch/snp2tfbs/mapped_files/annotated)44, and DNase I hypersensitivity sites (DHS) (https://github.com/joepickrell/1000-genomes)45.Pleiotropy analysis was performed via the R package GPA–277 < P < 3.51 × 10–127) under CADD annotation, 2.6 to 5.2 (1.18 × 10–277 < P < 3.51 × 10–127) under eQTL annotation, 2.6 to 5.1 (2.52 × 10–276 < P < 6.52 × 10–127) under DHS annotation, and 2.6 to 5.1 (1.18 × 10–277 < P < 6.52 × 10–127) under TFBS annotation  tested was done using CADDed Table . Enrichm42. Out of the 16 tests, SNPs associated with pairs of birth weight and childhood obesity traits were more likely to be functionally deleterious than SNPs associated with neither trait in 15 tests ; however, 1 test under TFBS category for BWEU-COB pair was not significant (P = 0.21)  were observed in tissues from skeletal muscle, adipose, brain, heart, esophagus, thyroid, adrenal, colon, small intestine and whole blood. The most significant DHS enrichments were observed in cell lines derived from blood lymphocytes (e.g. CD4+), skin (e.g. iPS), cancer (e.g. HeLa), embryo (e.g. embryonic stem cells) and in tissues from fetal brain. The most significant transcription factor enrichments were Interferon Regulatory Factor 1, AT-Rich Interaction Domain 3A and Testicular receptor 4  were jointly associated with birth weight and childhood obesity traits , one locus (rs6887211 in EBF1) was novel to childhood obesity traits but not birth weight, and one locus (rs7958572 4 Kb upstream to LMBR1L) was novel to both birth weight and childhood obesity traits  and the LMBR1L locus was only suggestively associated with birth weight (P = 2.4 × 10–3) and childhood obesity traits (P = 7.3 × 10–6) in previous GWAS39.At 5% false discovery rate, 19 loci , nearly all alleles associated with decreased birth weight were associated with higher CBMI/COB risk . The top enriched IPA Canonical Pathways included RAR Activation and Estrogen Receptor Signaling  tool  or vertical pleiotropy  we performed colocalization analysis using the coloc R packaget Tables  and S10.49. Investigating such effects in early life traits can facilitate our understanding of their complex genetic architecture and in developing early life interventions to promote long-term health51. The present study revealed substantial genetic pleiotropic effects between birth weight and childhood obesity traits. We also found that biologically functional SNPs are more likely to be associated with both birth weight and COB traits compared to SNPs that are not functional, consistent with evidence of high evolutionary conservation of pleiotropic genes and their consequences52. Lastly, we identified 19 genetic loci with pleiotropic effects, including loci in EBF1 and LMRBL1 that have not been associated with birth weight and/or COB phenotypes in previous GWAS. With the majority (68.4%) of the 19 loci reaching the significance threshold in analyses involving European-only or trans-ethnic birthweight GWAS and the remaining loci exhibiting high posterior probability of association (0.85–0.93), the results suggest that the effects of many pleiotropic loci are likely shared across ancestries. In all, these findings facilitate our understanding of the genetic mechanism that may underlie associations of early life growth with COB traits.There is growing evidence that several genetic variants can influence two or more traitsEBF1) was found to be associated with increased risk of COB traits. The association of EBF1 with birth weight and gestational duration is well-recognized55. EBF1 is highly expressed in adipocytes  and pericardial adipose tissue volume56. Previous studies found substantial overlaps between childhood and adult obesity GWAS loci62. While there is relatively weak LD between the lead SNP showing pleiotropic effect in our study (rs6887211) and rs1650505 (r2 = 0.06 in CEU), we acknowledge that the novelty of the locus in childhood BMI may be in part due to the small study power of the source GWAS used for childhood BMI. Furthermore, our novel SNP and its LD proxies (r2 ≥ 0.8) overlapped with DNase I hypersensitivity sites of blood lymphocytes and fetal muscle further indicating that many components of the metabolic and inflammatory pathways are positively and directly regulated by EBF1 as emphasized in a prior study63. These findings collectively indicate EBF1’s role in fetal growth and general adiposity which highlights that it should be considered as a key player in understanding the association of early life growth with the development of COB.The birth weight-decreasing allele of the novel pleiotropic SNP rs6887211  was found to be associated with decreased risk of COB. LMBR1L has regulatory effects on the canonical Wnt signaling pathway. It stabilizes the beta-catenin destruction complex64 that is required for regulating CTNNB1 expression, and is responsible in making the beta-catenin protein which plays a key role in the canonical Wnt signaling pathway68. The Wnt signaling pathway is conserved in various organisms and plays important roles in development, cellular proliferation, and differentiation69. Previous studies have demonstrated that the Wnt signaling pathway regulates adipogenesis and maintains the undifferentiated state of pre-adipocytes by inhibiting adipogenic gene expression 71. In addition, the Wnt signaling cascade plays fundamental roles in placental development by regulating trophoblast differentiation and invasion, and aberrant Wnt signaling activation can have downstream consequences on fetal growth 74. Our finding here for LMBR1L, together with the regulatory impact of LMBR1L on Wnt signaling and the functional relevance of the Wnt pathway in placental function, suggests a novel hypothetical mechanism by which LMBR1L contributes to the link between fetal growth and development of COB. Future studies such as eQTLs in tissues relevant to early life traits can facilitate better inference from the pleiotropic SNP to a potential functional gene.The birth weight-decreasing allele of the novel pleiotropic SNP in 20, we observed that birth weight-decreasing alleles of pleiotropic SNPs may be associated with either increased or decreased risk of COB. We found that the birth weight-reducing alleles of most of the pleiotropic loci we found  have previously been associated with decreased risk of COB, suggesting that concurrent genetic mechanisms may play an important role in the well-recognized positive correlation between low birth weight and low risk of COB. We also found that in 3 out of our 19 pleiotropic loci  the birth weight-decreasing alleles were associated with increased risk of COB. Genetic loci near EBF1and SEC16B genes have been associated with birth weight, SGA and adiposity traits 76; however, the association of NCOA1 with birth weight or COB traits remains elusive. Interestingly, co-activators like NCOA1 are fundamental in uterine growth by regulating placental morphogenesis, embryo survival and interacting with estrogen receptors in the human placenta to enhance estrogen signaling which has downstream consequences on birth weight78. NCOA1 is also highly expressed in the hypothalamus region of the brain  or low birth weight is associated with both lower and higher risk of COBain Fig.  which is81. Previous studies83 have demonstrated that the cell‑mediated immune response is impaired in obese children along with an over‑representation of the immune and inflammatory response in adipose tissues of children. We also found the pleiotropic SNPs to be significantly enriched in Retinoic Acid (RA) signaling pathway and have cis-regulatory effects on gene expression in adipose tissue and muscle skeletal. Multiple studies have documented that RA regulates adipogenesis86. RA acts as a high affinity ligand for the nuclear receptor peroxisome proliferation-activated receptor β/δ which is a master regulator of lipid metabolism and glucose homeostasis. Deactivation of these receptors decreases lipid catabolism in adipose tissue and skeletal muscle, increasing the risk of obesity. Lastly, we found the pleiotropic SNPs to be significantly enriched in intracerebral signaling pathway by having regulatory effects on several cerebral tissues. Brain-derived neurotrophic factor (BDNF), a key neurotrophin with multipotent impact on brain signaling87 plays an important role in regulating energy homeostasis and metabolism90. BDNF acts on hypothalamic PVN and VMH neurons to suppress appetite and mediate the anorexigenic effects of MSH acting on the MCH-4 receptor89. Deficit in BDNF levels or signaling is attributed to the development of obesity92, indicating that pathways of intracerebral signaling may be related to COB. Our results suggest that these same causal biologic pathways likely influence early life growth, but further research is needed to understand how these pathways that influence COB also influence birth weight.Functional enrichment tests and pathway analysis revealed three key biological processes that may jointly contribute to the association of early life growth and development of COB. Our study found that SNPs associated with both birth weight and COB traits mapped to genes that were significantly enriched in immune system pathways and regulated the signaling of immune cells and estrogen. Estrogen plays an essential role in regulating immune response, through its interactions with the receptors on the immune cells and their functioningWe recognize that our study has limitations. First, despite the large sample sizes of the consortia-based meta-analysis GWAS studies included in our study, there were differences in sample sizes and number of SNPs among the various GWAS studies. These contrasts may contribute to power differences in identifying pleiotropic loci. Additionally, some of the observed associations might be due to independent associations of the locus on birth weight and COB traits, due to the correlation of the traits in a causal pathway or some unmeasured characteristics. Second, the inference on joint associations of the variants in our study is based on estimates from the GPA model which performs optimally under low to moderate genetic correlation between the traits; however, under complex and weaker correlations the estimates of the GPA model can be biased. Third, some cohorts contributed to both childhood BMI and birthweight GWAS. Since GPA does not account for sample overlaps, we do not know whether the estimates are biased. Fourth, our analysis did not find functional enrichment of the pleiotropic loci between birth weight and COB under the TFBS annotation set, which requires further investigation in larger samples. An important strength of our study is the integrated modelling of functional annotations and GWAS summary statistics data from pairs of traits. This multi-trait approach has been most conducive in testing for functional enrichment and identifying novel genetic loci with their shared impacts on multiple traits, expanding our understanding of the genetic links between fetal growth and COB traits.In conclusion, this study found that pleiotropic genetic influences and enrichment of functional annotations are substantially pervasive in the genetic architectures of birth weight and COB traits. The novel loci found in the analysis and the pathways through which the associated genes act have the potential to unravel the genetic basis that underlines associations between early growth and development of COB.P values and direction of effects in GWAS meta-analysis of birth weight36, CBMI39 and COB37 was obtained from the publicly available data of EGG  and transethnic  meta-analysis . Individuals who were part of multiple births, who reported their birth weight in multiple visits with the mean difference in their birth weight being > 1 kg and individuals whose birth weight was < 2.5 kg or > 4.5 kg were excluded36. The results from the GWAS of own birth weight (as opposed to offspring birth weight), and without adjustment for maternal genotype, were used in this analysis.Summary statistic data including GG Table . The GWA42, eQTL43, TFBS44 and DHSs45 to functionally annotate the SNPs. Under CADD framework, implemented in CADD v1.2 (http://www.cadd.gs.washington.edu)42, a deleteriousness score  is generated using the integrated functional and evolutionary importance of each variant from 63 annotation sources. Phred-like scores (ranged 1–99) are further generated based on the rank of each variant relative to 8.6 billion substitutions in human reference genome . Each variant in our analysis receiving a score ≥ 15 was assigned an annotation of 1 (deleterious) while scores < 15 were assigned an annotation of 0 (non-deleterious)42. In addition to CADD, which comprises a composite score, we used annotations from a variety of tissues and cell lines to elucidate the regulatory mechanisms of the risk variants. The eQTL annotations was obtained from dbGaP accession number phs000424.vN.pN on 02/11/20 43 which consisted of cis-eQTL files on 49 different tissues. We took the intersection of these eQTL with the common variants of BW and COB/CBMI and the variants that overlapped were annotated as 1 and others as 0. The TFBS annotation (ftp://ccg.epfl.ch/snp2tfbs/mapped_files/annotated/)44 had data on 195 different transcription factors. Similar to eQTL, we took the intersection of the TFBS to annotate the common variants of birth weight and COB/CBMI. The DHSs annotation data, which has also been used in other studies93, was downloaded from (https://github.com/joepickrell/1000-genomes)45 and comprised of 402 binary annotations that included maps of DNase-I hypersensitive sites from different primary tissues, cell lines and cell types95.We used 4 annotation databases CADD41. For convenience, we briefly introduce the GPA model and its notations below.We used a unified statistical approach that integrates summary statistics with functional annotations for paired traits using probabilistic models implemented in genetic analysis incorporating pleiotropy and annotation (GPA)P values from two GWAS have been collected in an M × 2 matrix, p = [pjk], where pjk denotes the P value of the jth SNP in the kth GWAS, k = 1,2 (in our case) and M is the number of SNPs. In the GPA model, these P values are assumed to come from a mixture of null (un-associated) and non-null (associated), with probability π0 and π1 = 1 − π0, respectively. GPA uses the Uniform distribution on  and the Beta distribution with parameters  to model the P values from the null and non-null groups, respectively.Suppose the j1 and pj2 is the P value of the jth SNP in GWAS 1 and 2. GPA further incorporates functional annotation as follows. Let an M-dimensional vector A collect functional information from an annotation source, where Aj ∈ {0,1} indicates whether the jth SNP is a functional unit according to the annotation source. For example, given an eQTL data, if the jth SNP is an eQTL, then Aj = 1, otherwise Aj = 0. The relationship between Zj and Aj is described as:00 is the probability of a null SNP being annotated, q10 is the probability of the first trait-associated SNP being annotated, q01 is the probability of the second trait-associated SNP being annotated, and q11 is the probability of jointly associated SNP being annotated. GPA then implements an efficient EM-algorithm to obtain the estimates of the model parameters: {π00, π10, π01, π11, q00, q10, q01, q11, α}.Let Zj ∈ {00,10,01,11} indicate the association between the jth SNP and the two traits: Zj = 00 means the jth SNP is associated with neither of them, Zj = 10 means it is only associated with the first trait, Zj = 01 means it is only associated with the second trait, and Zj = 11 means it is associated with both trait. Thus, the four-group model is represented as:0: π11 = (π10 + π11)( π01 + π11), versus H1: not H0. Similarly, to assess the significance of enrichment for annotation it uses LRT with H0: q00 = q11, versus H1: q00 ≠ q11. GPA also calculates the standard errors for the model parameters along with their covariance matrix based on an empirically observed information matrix.To assess the significance of enrichment for pleiotropy between two traits it uses the likelihood ratio test (LRT) with H00, π10, π01, π11}) and controls for false discovery based on the local false discovery rate (FDR). The local FDR is defined as the probability that the jth SNP is either not associated with any trait (Fdr0) or is associated with the first trait (Fdr1), second trait (Fdr2) or both trait  given its P value and annotation information.After estimating the parameters, GPA assigns each SNP four posterior probabilities (PP) . IPA is a web-based software application that allows analysis of data obtained from several sequencing platforms. IPA enables for targeted search of information on genes, proteins, chemicals, diseases, and drugs. IPA’s data analysis provides clarity in understanding the significance of data or targets of interest in relation to larger biological systems. Statistical significance of overrepresented canonical pathways was determined using Fisher’s exact test after adjustment for multiple testing using the Benjamini–Hochberg method. Statistical significance was based on P < 0.05 in pathways with at least two molecules.Pathway analysis elucidates the underlying biological processes in which genes are related by common functionality. To detect such relationships of the identified pleiotropic genetic loci in our study, we used QIAGEN’s IPA tool  in a region, we performed colocalization analysis for each of the 19 pleiotropic loci using the European ancestry GWAS summary statistics. For each genetic locus, the lead SNP with the highest posterior probability of joint association with birthweight and childhood obesity traits as well as SNPs within 500 kb window on either side of the lead SNP defined a colocalization region. Analysis was performed using the coloc R packageThe NIH Office of Human Subjects Research Program granted the study an exemption from IRB review (OHSRP ID Number: 18-NICHD-00412) per 45 CFR 46 and NIH policy for the use of specimens/data.Supplementary Information.


At birth, 22 lambs were assessed through Apgar score at 5 minutes and after 1 hour. Measurement of body weight was also carried out immediately at birth and weekly during 30 days after birth. Thoracic diameter showed a significant increase between 91-105 days and 121-135 days. Conversely, abdominal diameter had a progressive growth until 106-120 days, and then, a steady development was observed. Biparietal diameter showed progressive growth only towards days 91 and 105. For the humerus length, we verified a significant increase between 106-120 days and 121-135 days, remaining unaltered onwards; while femur length continued to grow until lambing. The linear regression analysis between birth weight and biparietal diameter at 60-75 days was high . In conclusion, ultrasonographic analysis of fetal biparietal diameter at mid-pregnancy can be used as a predictor of lamb weight at birth. Moreover, assessment of femur length at final pregnancy can be employed for fetal and neonatal development estimation.Ultrasonographic examination of pregnant ewes can enable the identification of perinatal abnormalities and establish prenatal assistance responsible for minimizing morbidity and perinatal mortality. Therefore, we aimed to evaluate the feasibility of a fetal biometric analysis by ultrasonography to predict neonatal vitality and lamb growth during the first month of life. A longitudinal study was conducted with 13 healthy ewes, subjected to ultrasonographic examination every 15 days from 60 Real-time ultrasound is widely employed for pregnancy diagnosis in sheep. Among several advantageous, it is possible to identify twin pregnancies, especially in early stages, estimate gestational age and fetal growth, in addition to evaluating placental development . UltrasoThe evaluation of fetal growth and development can be accomplished primarily through fetal biometry, which consists mainly of ultrasonographic measurements of fetal structures, as well as of the placental unit. Fetal biometry also allows estimating gestational age and time of lambing . In sheeThe main measures taken in sheep are the cerebrospinal coccygeal length, biparietal diameter, circumference and area of the skull, diameter of the thorax and abdomen area and length of long bones such as femur, humerus, tibia and metacarpus . GreenwoVitality evaluation of the neonatal lamb at birth can be performed by taking into account the Apgar score , variatiTherefore, we aimed to evaluate the feasibility of a fetal biometric analysis by ultrasonography to predict neonatal vitality and lamb growth during the first month of life.The present study complied with the ethical requirements for the use of animals in experiments, and was approved by the Bioethics Committee of the Faculty of Veterinary Medicine and Animal Science, University of São Paulo (protocol number 2039/2010).A longitudinal study was conducted with the use of 13 healthy Santa Inês ewes. In order to assure the appropriate sample size, an analysis was conducted with the SAS Power and Sample Size 12 . For an acceptable statistical power (at least 0.8), 13 pregnant ewes were sufficient to demonstrate significant differences in the data. A retrospective analysis showed a power of 0.99.ad libitum. Ewes were naturally bred with the same ram. The gestational period was considered 24 hours after the first day of mating , ewes were diagnosed as pregnant, as well as having single or multiple pregnancies by identifying one or two fetal independent beating hearts, respectively. Out of 13 ewes, 9 females carried twins and 4 ewes had single pregnancies.Throughout gestation, all ewes were subjected to abdominal trichotomy and were maintained at standing position for transabdominal ultrasonography. We used a linear 5 MHz dynamic B mode real-time transducer . Ultrasonographic examination was performed every 15 days from the 60Fetal biometry was evaluated by means of the thoracic diameter measurement, abdominal diameter, biparietal diameter, humerus, femur and placentome length. Fetal abdomen was analyzed transversely at the level of the umbilical cord insertion and abdominal diameter was measured  Figure A. BipariAt the beginning of the vaginal labor, females were continuously monitored and one fetus in posterior presentation dystocia was excluded from this experiment. Immediately at birth, newborns were dried and vigorously rubbed at chest area. Then, oral and nasal cavities were gently aspirated with a urethral probe coupled to a secretion aspirator.At birth, 5 minutes and after 1 hour of birth, 22 lambs were assessed through the Apgar score (0-10), according to All data were evaluated using the SAS System for Windows . The effect of time of evaluation was estimated by the repeated measures analysis of variance (Mixed Procedure of SAS). Differences between time were analyzed using parametric and non-parametric tests, according to the residual normality (Gaussian distribution) and variance homogeneity. Whenever one of these assumptions was not respected data were transformed. If transformations were not successful, non-parametric tests were employed. To compare the moments of observation the Least Significant Difference (LSD) test was used.Response variables were also submitted to a Pearson Correlation Analysis. Correlation coefficients were used to measure the degree of relationship between continuous variables. Additionally, linear regression model was applied to evaluate the association between birth weight and ultrasonographic fetal biometry variables. Linear regression analyses were also used to generate equations for the estimation of lamb birth weight.Results were described as untransformed means ± SE. Statistical differences between classificatory variables for a certain response variable were considered to occur if P < 0.05.The thoracic diameter showed a significant increase between 91-105 days and 121-135 days, during which the thoracic area developed mostly . Conversth day of pregnancy onwards  was significantly lower when compared to those of 5 min (8.8 ± 0.9) and 60 min (9.9 ± 0.3). Neonatal lambs have evolved to satisfactory Apgar score (>7) already at 5 minutes after birth. At 60 minutes, the Apgar score was statistically superior to previous moments of evaluation.The birth weight of lambs born from twin pregnancies was 3.17 Kg ± 0.64 being significantly smaller (P<0.01) in relation to lambs of single pregnancies (4.4 Kg ± 0.8). No difference in birth weight was evidenced between male (3.3 Kg ± 0.9) and female (3.1 Kg ± 0.6) lambs. In addition, after one month of life, lambs from twin pregnancy weighted 9.24 Kg ± 1.44, significantly inferior (P<0.01) to lambs from single pregnancy (12.97 Kg ± 0.21).2=0.96; P<0.0001; coefficient of variability of 3.3%) gendered the following linear regression equation: Birth weight = -1.8933 + 1.7249 x biparietal diameter 60-75 days.There was a positive correlation between biparietal diameter at 60-75 days of pregnancy and birth weight , as well as between abdominal diameter at 60-75 days and birth weight . The femur length at days 136 until lambing correlated positively  with birth weight. The regression analysis between birth weight and biparietal diameter at 60-75 days  towards lambing, suggesting an initial placental maturation that maintained unchanged throughout pregnancy. Conversely, th to the 133th day of pregnancy , from which it remained unchanged. In fact, biparietal diameter reaches 49% of its total growth at mid-pregnancy in sheep, showing a deceleration of development from the 75regnancy . Cephaliregnancy . Interes the dam . Ideally the dam . TherefoOur study shows a progressive increase in abdominal diameter throughout pregnancy, more pronounced until 91-105 days. Similarly, th day of pregnancy. However, we did not find a correlation with birth weight, thus, not being a feasible predictor of fetal development at term pregnancy, although thoracic measurement is reported to be highly correlated with fetal age and weight  is an accurate predictor of lamb weight at birth and the assessment of femur length at final pregnancy can be employed as guide for the diagnosis of fetal restriction growth. Both analysis allow for the evaluation of fetal development and the estimative of neonatal development, thus predicting the need of a differential assistance towards lambing and to underdeveloped neonatal lambs. However, further studies on uterine and fetal vascularization, such as uterine artery and fetus cerebral artery hemodynamics may provide a better understanding on fetal development and well-being.


We document increased old-age mortality rates among Swedish mothers of twins compared with mothers of singletons, using administrative data on mortality for 1990–2010. We argue that twins are an unplanned shock to fertility in the cohorts of older women considered. Deaths due to lung cancer, chronic obstructive pulmonary disease, and heart attacks—all of which are associated with stress during the life course—are significantly increased. Stratifying the sample by education and pension income shows the highest increase in mortality rates among highly educated mothers and those with above-median pension income. These results are consistent with the existence of a double burden on mothers’ health resulting from simultaneously child-rearing and working.The online version of this article (10.1007/s13524-020-00868-6) contains supplementary material, which is available to authorized users. In times of demographic change and aging populations, policy-makers in many countries are concerned about the development of the work force. In order to buffer the systematic aging and potential shrinking of the work force, policies that encourage an increase in female labor supply are often on the agenda. At the same time, family policies aiming at an increase in fertility rates are discussed Jaumotte . Howevertwin mothers) with those of mothers of singletons at first birth (nontwin mothers). The Swedish data allow us to follow mothers over a long period, and to our knowledge, we are the first to study the effects of twins on mothers’ old-age mortality. We find that twin mothers have a 3.8 percentage point (13%) higher all-cause mortality risk compared with mothers of singletons. This finding provides some initial evidence on the relationship among family, work, and health. However, although twinning is an exogenous shock,Our analysis is based on administrative birth and death registries from Sweden. To study the effect of childbearing and labor force activity on maternal health, an ideal set-up would provide exogenous variation in both labor market activity and childbearing. In our study, we use twin births as an unplanned fertility shock. Initially, we compare health outcomes of mothers who had twins at first birth  for women with high education and/or high pension income because these women more often combine working and child-rearing.3To understand potential mechanisms and to study a double-burden effect of family and work, we pursue two strategies.The second strategy to understand the mechanism behind the increased mortality of twin mothers is to analyze specific stress-related diseases. The underlying hypothesis is that the double burden of working and caring for children at the same time creates stress, which may particularly affect stress-related mortality. Here, one challenge is the measurement of lifetime stress. We analyze two specific groups of medical diagnoses related to stress during life: cardiovascular diseases (heart attacks and strokes) and smoking-related diseases (lung cancer and chronic obstructive pulmonary disease (COPD)  treatments to obtain exogenous variation at the extensive margin. Interestingly, they found that the effects of fertility on earnings are larger at the extensive margin than at the intensive margin. Given this evidence, it seems reasonable to extrapolate that there also are important double-burden effects at the extensive margin.Given that we compare twin and nontwin mothers, all women in our sample have at least one child. Thus, we are able to study the effect of fertility only at the intensive margin—that is, comparing having a singleton versus twins at first birth. A comparison at the extensive margin  would be at least as interesting as the intensive margin comparison: by definition, women without children do not experience the double burden of children and work. However, it is hard to find exogenous variation on the extensive margin effect. In fact, in a lone exception, Lundborg et al.  used theOur study is linked to the literature on the interactions among fertility, working life, and maternal health. Kahn et al.  were amoA growing literature has examined the relationship between family life and female careers. Several studies found that childbearing has negative effects on female labor force participation, earnings, and wages , even if they do not live in the same household or have died. The register contains parental information for persons born in 1932 or later, including information on year and month of birth  had twins at first birth.7Among all mothers born between 1925 and 1935, 8.6% were not observed in 1990 because they died (75%) or moved abroad (25%) before 1990. In Table APrimary schooling includes elementary schooling (Folkskola), which comprised seven years for most of the cohorts considered here, and junior secondary schooling . The designation of secondary schooling means that mothers had some education beyond the primary level but no tertiary education. This level also includes vocational training at the upper secondary level. Tertiary schooling indicates that mothers experienced some higher education: that is, the attainment of some college or of a university education or a PhD.We have a set of socioeconomic characteristics from population registers. Table On average, the mothers were 60 years old in 1990, had their first child at age 24.5, and have 2.4 children. The majority of the mothers completed primary education (59%), about 30% completed secondary education, and roughly 11% earned a tertiary degree. The age at first birth is, on average, three years higher in the highly educated group compared with the lower-educated group, and the average number of children is about the same.Overall, about 66% of the women aged 55–65 are still active on the labor market (positive labor income) in 1990. However, the fraction of working women varies considerably by education. Whereas 89% of the women with a tertiary degree receive labor income, only 57% of mothers with primary schooling receive income from work at those ages. About 29% of the women in our sample died between 1991 and 2010, with large variation by education. About one-third of the lower-educated mothers died in the 20-year time window we consider, compared with only 26% (19%) among mothers with medium (high) levels of education. We see similar patterns for the different causes of death.,10From the population registers, we also obtain information on pension income at age 72. Pension income is the best available proxy for lifetime labor force participation in our data set.8yi is the outcome variable, and  is an indicator equal to 1 if mother i gave birth to twins at first birth. Depending on the specification, the outcome variables are indicators equal to 1 if the mother died from any cause between 1991 and 2010, from a heart attack or stroke, or from lung cancer or COPD between 1991 and 2010. The control variables, xi, include dummy variables for seven education levels, dummy variables for mothers’ birth cohorts, and a quadratic polynomial in age at first birth.11,12To investigate differences between twin and nontwin mothers, we specify the following linear regression model:One worry when comparing twin mothers with nontwin mothers is that twinning may not be entirely random. For instance, Bhalotra and Clarke  documentWe have several strategies for addressing these issues. First, as mentioned earlier, we control for age at first birth in all our analyses. Second, to investigate a possible selection bias stemming from dizygotic twins, we compare estimates between mothers of all twins and only same-sex twins . Because monozygotic twins necessarily have the same sex, their share must be higher among same-sex twins


Supplemental Digital Content is available in the text. Air pollution has been associated with hypertension and preterm birth. We examined if prenatal exposure to air pollutants was associated with gestational hypertension and if its association with preterm birth was modified by maternal hypertension.2), particulate matter <10µm (PM10) and <2.5µm (PM2.5), and carbon monoxide (CO) for different averaging periods over pregnancy. We estimated odds of preterm birth and multiplicative interaction between each pollutant and hypertensive disorder.Data were from birth certificates and hospital discharge records of 252,205 women in San Joaquin Valley of California from 2000 to 2006. Air quality data were assigned from 24-hour averages of nitrogen dioxide (NO2 and CO during the first trimester with higher odds among hypertensive women, and PM2.5 and CO during the last 6 weeks with higher odds among normotensive women. For 28–31 week births, there was effect modification by hypertensive status for PM10 exposure for the entire pregnancy, first, and second trimester with hypertensive women consistently having lower odds of preterm birth than normotensive.Among normotensive women, odds of preterm birth were slightly higher for higher exposure to all pollutants over the entire pregnancy. Patterns were similar among women with a hypertensive disorder. Among 32–36 week births, there was effect modification for exposure to NO10 and early preterm birth, and CO and PM2.5 at the end of pregnancy, but overall, hypertension did not modify the relationship between pollution and preterm birth.There was some evidence of effect modification in the direction counter to our hypothesis for exposure to PM How air pollution influences risk of preterm birth has not been fully elucidated. We explored one of the primary indications of preterm birth, hypertension in women. To fill a knowledge gap regarding potential differences in the effect of air pollution on preterm birth in pregnant women with a hypertensive disorder compared with those without, we estimated potential effect modification using data from a highly polluted region of California.2 A meta-analysis of exposure to ambient air pollution and pregnancy-induced hypertension also reported increased risks for particulate matter and nitrogen dioxide (NO2).3 While there has been some heterogeneity in the results of these studies, especially between different pollutants and timing of exposure, air pollution is believed to cause endothelial dysfunction and increase blood pressure through autonomic nervous system imbalance, oxidative stress, and systemic inflammatory response,6 thus making this relationship mechanistically feasible.Exposure to ambient air pollution, specifically particulate matter, has been associated with increases in blood pressure and hypertension in previous studies.7 and a previous analysis of our cohort observed associations between increased exposure to particulate matter [particulate matter <10µm (PM10)] and [particulate matter <2.5µm (PM2.5)] and preterm birth, with the strongest associations observed for exposure during the second trimester.8 Hypertensive disorders have also been associated with preterm birth. Preeclampsia, a hypertensive condition affecting pregnancy, contributes substantially to preterm birth,9 and chronic hypertension has been associated with both spontaneous10 and medically-indicated preterm birth.12Ambient air pollution exposure during pregnancy has also been associated with preterm birth2, PM10, PM2.5, and traffic density] and preterm birth may be modified by the presence of a hypertensive disorder. We examined possible effect modification by hypertensive status, separately by timing of preterm birth and each pollutant. We also assessed the potential for pregnancy-induced hypertension to mediate any relationship between air pollution and preterm birth.Thus, using data from a highly polluted area of California, we sought to determine if the relationship between exposure to air pollutants  was the main outcome. Timing of pollutant exposure did not include end of pregnancy in an attempt to account for temporality due to general diagnosis of pregnancy-induced hypertension earlier in pregnancy. An ad hoc indirect effects mediation analysis was then performed for a particular pollutant if an association was observed. For the second analysis, chronic hypertension  was also included. For timing of exposure in the latter analysis, we additionally included the last 6 weeks of pregnancy to account for differences in gestational age and thus differing third trimester lengths and averaging periods between term and preterm births. Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (95% CI) and we considered a 20% higher odds to be “meaningful.” We stratified by the presence of a hypertensive disorder to test for effect modification of the association between each pollutant and preterm birth. We also tested for multiplicative interaction using a Wald test.All analyses were adjusted for covariates believed to be confounders . We also performed additional sensitivity analyses, one restricting to spontaneous preterm birth (preterm labor or premature rupture of membranes), one additionally adjusting for season of conception, and one excluding women with diabetes. All analyses were performed using SAS version 9.4 .Early preterm births  were more likely among women who were under age 20, Black non-Hispanic, and high school educated. Preterm births were more likely among women with medical care funded by Medi-Cal, who did not receive prenatal care in the first trimester and who were of low neighborhood socioeconomic status compared with term births. Women with pregnancy-induced hypertensive disorders were more likely to deliver preterm and those with preexisting hypertension were slightly more likely to deliver preterm compared with normotensive women . Correlations between the pollutants and traffic density were low. The associations between the individual air pollutants and pregnancy-induced hypertension are shown in Table 10 and pregnancy-induced hypertension comparing the highest quartile of exposure to the lower three quartiles. There was no association when restricted to exposure in the first or second trimesters. There was a suggestive association between exposure to PM2.5 and pregnancy-induced hypertension for exposure averaged over the entire pregnancy and for exposure in the highest quartile during the second trimester. Associations for the other air pollutants and exposure periods were null. The indirect effects analysis resulted in a β ~ 0 (results not shown).The pollutants were highly correlated with each other. The Pearson correlations were 0.75, 0.57, and 0.76 between CO and NO2.5. Patterns were similar for women with a hypertensive disorder.Associations between each air pollutant and preterm birth, stratified by presence of a maternal hypertensive disorder and gestational age, are presented in Table 2 among women with a hypertensive disorder, for which there was significant effect modification (P = 0.04), and PM2.5, for which the significance test for effect modification approached significance (P = 0.06).For preterm births (32–33 weeks), results for first trimester exposures were slightly different among women with a hypertensive disorder. There were higher odds for exposure to NO2.5 during the last 6 weeks of pregnancy and CO during the first trimester. However, patterns were inconsistent by exposure timing. Odds of preterm birth for exposure to CO were higher among hypertensive women than normotensive women for exposure during the first trimester and lower among hypertensive women for exposure during the last 6 weeks.In the later preterm births, we also observed effect modification for exposure to CO and PMhttp://links.lww.com/EE/A56). Patterns were not the same among women with a hypertensive disorder except for exposure to PM2.5. There was also effect modification by hypertensive status for exposure to PM10 across the entire pregnancy, during the first trimester and during the second trimester with hypertensive women consistently having lower odds of preterm birth than normotensive women.For preterm births between 28 and 31 weeks gestation, among women without a hypertensive disorder, there were also slightly higher odds for higher exposure over the entire pregnancy to all pollutants , among women without a hypertensive disorder, there were slightly higher odds for higher exposure to CO and PMThere were no substantial differences after exclusion of women with diabetes or after adjustment for season of conception. When the sample was restricted to spontaneous preterm birth, many of the previous estimates were no longer statistically significant, probably due to the smaller sample size (results not shown).This study aimed to disentangle the complex relationship among exposure to air pollution, maternal hypertensive disorders, and preterm birth. After stratification by maternal hypertensive disorders, associations between exposure to air pollution and preterm birth were variable. In general, there were moderately higher odds of preterm birth after exposure to air pollution among women without hypertension. Overall, the patterns were similar for women with a hypertensive disorder.10 among births between 28 and 31 weeks, the estimates suggest an increased risk among women without a hypertensive disorder and no association or lower odds among those with a hypertensive disorder. There were a few exceptions like the first trimester exposures for late preterm births, but overall, the results were counter to our hypothesis. This analysis also included many comparisons and thus it is possible that some of the observed associations were due to chance.There were a few instances of statistically significant effect modification. However, most estimates were not meaningfully different between women with and without a hypertensive disorder. For those that were different such as the estimates for PM2.513 and another observed lower fecundability among couples exposed to higher levels of PM2.5.14 It is possible that women with high levels of exposure to air pollutants experienced fetal loss before a preterm birth could be observed or that especially sensitive women were unable to become pregnant. This may also be more likely among women with a hypertensive disorder. Some studies on pollution and fertility or miscarriage have observed similar results and some have not15 but these studies did not explore the potential differences by comorbid status.There were also a few inverse associations and they tended to be for exposure at the end of pregnancy and stronger among women with a hypertensive disorder with a few exceptions. One could posit that these results could be attributed to unobserved fetal loss earlier in gestation. One prospective study observed faster time to fetal loss with higher exposure to PM3 and further investigation into mediation is warranted.Mechanistically, we hypothesized that the effect of exposure to air pollution might be modified by presence of a hypertensive disorder. Thus, we also explored the potential for hypertension to act as a mediator between air pollution and preterm birth. However, we did not observe a meaningfully higher odds of pregnancy-induced hypertension with higher exposure to air pollutants in this sample and thus, were not able to perform a full mediation analysis. This association has been previously observed16 However, underreporting of hypertension or preeclampsia is unlikely to be related to air pollution exposure and thus misclassification should be non-differential, resulting in attenuation of the estimates. Data on other potential confounders such as smoking and obesity were also not available or adequately collected and thus could not be adjusted for in this analysis.Our large, population-based sample came from a diverse area of California with high levels of pollution. Residences of our maternal participants were geocoded which allowed for very detailed air pollution exposure classification at specific time periods in gestation. The study was not designed to assess maternal comorbidities and characterization of hypertensive disorders relied on administrative data. OSHPD is a valuable source of information but sensitivity of administrative data and recording errors are a potential concern. A validation study of maternal conditions and obstetric conditions in California discharge data found diagnoses of preeclampsia to have a sensitivity and positive predictive value of about 80% but found diagnoses of hypertension to have a sensitivity of only about 60% and positive predictive value of 75%.10 for early preterm birth, but overall, hypertension did not modify the relationship between pollution and preterm birth. The inverse associations observed, especially among women with a hypertensive disorder, warrant further research into the potential effects of air pollution on early fetal loss to fully elucidate the mechanism by which air pollution influences preterm birth.This study adds some additional evidence to the hypothesis that exposure to higher levels of air pollution, especially particulate matter, is associated with preterm birth. There was some evidence of effect modification by maternal hypertensive status for exposure to PMThe authors declare that they have no conflicts of interest with regard to the content of this report.Supported by F32HD096754 to investigator K.A.W from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. This work was also supported by R21 ESO14891, P20 ES018173, P01ES022849, and R00ES021470 from the National Institute of Environmental Health Science, and the March of Dimes Prematurity Research Center at Stanford University. This publication was made possible by US Environmental Protection Agency STAR Grant RD83459601 and RD83543501. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the US EPA. Further, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication.The data are publicly available from the Office of Statewide Health Planning and Development (OSHPD). The data are not available for replication because specific approvals from OSHPD and the California Committee for the Protection of Human Subjects must be obtained in order to access them.We thank Bryan Penfold of Sonoma Technology, Inc. for traffic data processing and traffic density estimation.