Artigos

INTRODUCTION

Adverse factors acting in the embryonic, fetal and early life period can result in adaptations in human development1,2. These adaptations occur due to a biological programming process that produces structural, physiological and metabolic changes, which influence the occurrence of diseases across the lifespan1,2.

Studies have suggested that prenatal exposure to antimicrobials might be associated with the development of overweight and obesity during childhood3,4,5, although it is still unclear whether the association of maternal antimicrobial use during pregnancy with overweight and obesity during childhood is causal. Pathophysiological mechanisms may explain this relationship. For example, the interference in the mother's microbiome caused by the use of antimicrobials during pregnancy, which plays an important role in the fetal placental circulation6,7, may alters colonization and maturation of the infant intestinal microbiome by increasing susceptibility to obesity8.

Observational studies about the association between the use of antimicrobials during pregnancy and overweight and obesity in children have shown conflicting findings. In the US, participants of a cohort with 436 children who were exposed to antimicrobials during the second or third trimester of pregnancy had 84% higher risk of obesity than those unexposed8. Furthermore, a study conducted in China identified that the use of antibiotics during pregnancy was positively associated with the risk of obesity at five years of age. Conversely, in a cohort of 43,435 Danish children, prenatal exposure to small-spectrum antimicrobials was not associated with overweight at ages 7 and 114. Also, in a cohort of 53,320 children, the use of antibiotics during pregnancy was not associated with BMI-z in childhood at five years of age9.

Although prospective observational studies provide important insights, the relationships between the use of antimicrobials during pregnancy and the development of excess weight in early childhood are still poorly understood. In addition, the relationships observed in population-based studies are susceptible to bias due to residual or unmeasured contextual confounding factors10. Moreover, to date, most studies that investigated the association between the use of antimicrobials during pregnancy and early childhood excess weight lack information about the time of use, type of spectrum, the class of the antimicrobial, and critical confounding variables as maternal obesity and gestational diabetes11.

Therefore, the aim of this study was to investigate the association between the use of antimicrobials during pregnancy and excess weight in Brazilian children at three, 12, 24 and 48 months of age.

METHODS
Population and sample
This study used data from the 2015 Pelotas (Brazil) Birth Cohort study. This population-based cohort enrolled 99% of children born in all maternity hospitals in Pelotas, a city located in southern Brazil with approximately 350,000 inhabitants, whose mothers lived in the urban area of the municipality or in Jardim América in 2015 (n=4219 mothers, 4275 children). An antenatal study was conducted with 73.8% of women whose children were enrolled in the cohort (n=3155). For this, all 123 health units and private clinics that provided antenatal care in the city were visited or contacted weekly between May 2014 and December 2015 to identify pregnant women who were expected to deliver their babies between January 1 and December 31, 2015.

Mothers and children were invited to further data collections when infants were three (97.2%), 12 (95.4%), 24 (95.4%) and 48 months old (95.4%). Domiciliary interviews were conducted at ages 3 and 12 months. In contrast, at 24 and 48 months, the follow-up assessments were conducted in a research clinic in the Center for Epidemiological Research of the Federal University of Pelotas. Of the 4,275 children born in 2015 and whose mothers provided information about the use of medicines during pregnancy, 4,056, 3,948, 3,718 and 3,757 had anthropometric data when they were aged three, 12, 24 and 48 months, respectively. Data collection and measurements were standardized and collected by trained research assistants through RedCap12. Further details about the study design and protocols have been published previously13.

Use of antimicrobials during pregnancy
Information about the use of antimicrobials during pregnancy was collected using standardized questionnaires during the antenatal and perinatal assessments using the following questions: "Did you use any medication during pregnancy?"; "What are the names of the medicines that you used during pregnancy?"14. Subsequently, the International Anatomical Therapeutical Chemical (ATC) classification of the World Health Organization15 was used to classify the drugs by therapeutic groups up to the fifth level. For this study, the use of medicines in the 'group J', which are anti-infectious for systemic use, were considered.
Additionally, antimicrobials were classified according to their spectrum into narrow-spectrum or broad-spectrum. Narrow-spectrum antimicrobials are considered those that act on a few types of bacteria, for example only gram +, or only gram -, and broad-spectrum those capable of inhibiting several types of bacteria. In this paper, the following antimicrobials were considered to be narrow-spectrum: J01MB02 nalidixic acid; J01CA04 amoxicillin; J01CA01 ampicillin; J01CE01 benzylpenicillin; J01DB first-generation cephalosporins; J01XX05 methenamine; J01XE01 nitrofurantoin; J01MA06 norfloxacin; J04AB02 rifampicin; J01CE02 phenoxymethylpenicillin; J01CF04 oxacillin; J01XA01 vancomycin, and the following were considered to be broad-spectrum: J01MB02 nalidixic acid; J01CA04 amoxicillin; J01CA01 ampicillin; J01CE01 benzylpenicillin; J01DB first-generation cephalosporins; J01XX05 methenamine; J01XE01 nitrofurantoin; J01MA06 norfloxacin; J04AB02 rifampicin; J01CE02 phenoxymethylpenicillin; J01CF04 oxacillin; J01XA01 vancomycin16.

The following binary (yes/no) variables regarding the use of antimicrobials during pregnancy were investigated: a) Use of at least one antimicrobial during pregnancy; b) use of antimicrobial during the 1st trimester of pregnancy; c) use of antimicrobial during the 2nd trimester of pregnancy; d) use of antimicrobial during the 3rd trimester of pregnancy. Additionally, the number of trimesters of pregnancy in which antimicrobials were used (none; one; two or three), use of at least one low-spectrum antimicrobial during pregnancy (yes/no), and the use of at least one broad-spectrum antimicrobial (yes/no) were analyzed.

Anthropometric assessment
At ages three, 12, 24 and 48 months, children had their weight (g) and length/height (cm) measured by trained research assistants using standardized protocols. Weight was measured using a portable electronic scale (capacity of up to 50 kg and accuracy of 10g) at three months. For measurement of weight at 12 and 24 months, children were weighted on their mothers' laps using SECA 803 scales (SECA, Germany) with 100 g precision; child's weight was obtained as the difference between total weight (mother and children) and the weight of the mother. In all assessments, children were naked, and mothers were wearing light clothes when they were weighted. At age 48 months, a portable digital scale of the Tanita® with a maximum capacity of 150 Kg and accuracy of 100g was used. Child length at three and 12 months was measured using a portable anthropometer of the SANNY® model ES2000 with an amplitude of 20 to 105 cm and precision of 0.5 cm. At 24 months, a fixed Harpenden® with a range of 30 to 110 cm and precision of 0.1 cm was used, whereas a fixed Harpenden® with maximum height of 2.06 m and accuracy of 1 mm was used when children were aged 48 months. At 24 and 48 months, 90% of measurements were performed in a research clinic in the Center for Epidemiological Research. In the remaining 10% of participants, when measurements had to be performed at home, validated portable anthropometers and stadiometers were used16.

In each follow-up assessment, body mass index-for-age in z-scores was calculated according to the World Health Organization (WHO) Child Growth Standards17. WHO ANTHRO macros for STATA were used according to the methodology recommended by the WHO18. For this study, excess weight was defined as BMI-for-age z-score ? 1 standard deviation17.

Covariates
The following confounding variables were measured using standardized questionnaires and included in the analyses: number of pregnancies (one/ two/three or more); number of prenatal consultations (<6/7 or more); maternal smoking during pregnancy (no/yes); maternal consumption of alcohol during pregnancy (no/yes), hypertension (no/yes) and self-reported diabetes mellitus (no/yes) during pregnancy, maternal skin color (white/black/brown); maternal age (13-19 / 20-24 / 25-29 / 30-34 / 35 or more); maternal education (0-4 / 5-8 / 9-11 / 12 years or more); maternal marital status (with partner / no partner) and pre-gestational maternal BMI (? 24.9 / 25 to < 30.0 / ? 30.0 kg/m2).

Statistical analysis
Descriptive statistics were used to describe and compare sociodemographic characteristics of the original cohort with the analytical sample of each follow-up assessment. Absolute and relative frequencies were used to describe the use of antimicrobials during pregnancy. Mean and standard deviation of BMI-for-age in z-score, and prevalence of excess weight in each age were described according to the use of antimicrobials during pregnancy (as previously described).

The associations of each variable used to describe the use of antimicrobials during pregnancy with BMI-for-age z-score and excess weight at each age were examined using, respectively, linear regression and Poisson regression models with adjustment for robust variance. Crude and adjusted analyses were conducted. The potential confounders were considered, but only those associated with the dependent variable (p-value of < 0.20) were included in the final model. Given the number of comparisons and the potential for type 1 error, interpretation of findings was based on the magnitude of associations (regression coefficients) and overlap of 95% confidence intervals (CI). All analyses were performed in the Stata®15.0.

Ethical aspects
This study was approved by the School of Physical Education Ethics Committee at the Federal University of Pelotas (CAAE registration number: 26746414.5.0000.5313). In all interviews written informed consent was obtained from children’s parents or legal guardians.

RESULTS
Sociodemographic and health characteristics of the participants in the original cohort and each follow-up are presented in Table 1. There were no marked differences between the original cohort and those who participated in the follow-up assessments. At the time of delivery, most women were aged 20-34 years, had white skin color, had a partner, had 9-11 years of formal education, and nearly 20% of women were obese before pregnancy. Most women reported seven or more prenatal consultations (76.1%), 44.5% had only one child, 16.5% reported smoking during pregnancy, and 7.4% consumed alcohol. One in four women reported hypertension during pregnancy, and 8.6% had gestational diabetes (Table 1).

The prevalence of total antimicrobial use during pregnancy was 43.4% (95%CI 41.9; 44.9). The proportion of women who used antimicrobials in each trimester was approximately 20% (18%, 21%, and 21% in the first, second and third trimester, respectively); 30.4% of women reported use in only one trimester, and 13.0% reported use of antimicrobials in two or three trimesters of pregnancy. One-third of the women used antimicrobials classified as a small spectrum, and only 2.9% used antimicrobials classified as broad-spectrum (Table 2). The most used antimicrobials were cephalexin (23.6%), amoxicillin (15.8%), nitrofurantoin (15.6%) and ampicillin (5.9%) (Supplementary Table).

The mean BMI-for-age z-score was 0.06 (SD= 0.02), 0.68 (SD= 0.02), 0.56 kg/m2 (SD= 0.02) and 1.08 (SD= 0.04), at three, 12, 24 and 48 months, respectively. The prevalence of excess weight was 17.6% (95%CI 16.5; 18.8) at three months, 39.4% (95%CI 37.9; 40.9) at 12, 32.4% (95%CI 30.9; 33.9) at 24 and 43.8% (95%CI 42.2; 45.4) at 48 months. Overall, not marked variations in the distribution of BMI-for-age and excess weigh at each age according to use of antimicrobials during pregnancy was observed (Table 2)

Crude and adjusted associations of use of antimicrobials during pregnancy with BMI-for-age and excess weight at three, 12, 24 and 48 months are presented in Tables 3 and 4, respectively. Overall, the use of antimicrobials during pregnancy was not associated with either BMI-for-age or excess weight, regardless of the age. Of the 56 associations investigated, statistically significant findings (based on 95%CI) were only observed on three occasions, although with a small magnitude of associations. Children from mothers who used antimicrobials during the second trimester had on average 0.12 lower z-score of BMI-for-age at age 3 months than children whose mother did not use antimicrobials in the second trimester. At age 12 months, the z-score of BMI-for-age was slightly higher (?: 0.11; 95%CI: 0.01; 0.20) in children whose mothers used one antimicrobial during pregnancy than in those children of mothers who did not use antimicrobials. Children aged 48 months, whose mothers used antimicrobials in the first trimester of pregnancy, had a higher risk of excess weight than those of mothers who did not use antimicrobials in the first trimester [RR= 1.13 (95%CI 1.04; 1.23)] (Table 4).

DISCUSSION
It is well known that the exposure of the fetus to certain substances can affect many aspects of child development19,20. In this unique representative population-based cohort study conducted in a large middle-income country, we used a range of different exposure variables, a comprehensive list of potential confounders and two indicators of children nutritional status in four time points to explore the extent to which maternal use of antimicrobials during pregnancy may impact children BMI-for-age and excess weight during the first 48 months postpartum. Overall, the findings of this study suggest that antimicrobial use during pregnancy does not influence the nutritional status of children up to age 48 months.

The proportion of women who used at least one antimicrobial during pregnancy in our study (43%) was higher than the prevalence of antimicrobial use by pregnant women in large population-based studies conducted in New Zealand (35.7%)21 and the United States (25.2%)22. Probably, this may be due to the higher prevalence of urinary infection (UTI) in the pregnant women in our study, which was 45% (data not shown in results), since UTI is the most prevalent infection in pregnant women23. Another likely explanation may be due to the population, as they come from a population with lower income than in the United States and New Zealand, a lower level of education, which leads to more urinary and genital infections. In a study carried out in the city of Rio de Janeiro, Brazil, with a final sample of 1091 pregnant women, 45.9% had UTI23, data very similar to ours.
Moreover, as previously reported24, the prevalence of antimicrobial use tends to be the lowest in the first trimester. This can be caused by the high prevalence of urinary and genital infections in the second and third trimester of pregnancy25. Furthermore, early in the pregnancy, women may be more afraid to use medications due to the risk of causing adverse effects on the fetus.

Findings from previous studies that investigated the association between the use of antimicrobials during pregnancy and childhood nutrition status have shown conflicting results. Cassidy-Bushrow et al. (2018)26 evaluated 24 months old infants and found no evidence of association between the use of antimicrobials during the prenatal period and the excess weight. Similar to our findings, they also found isolated positive association when they analyzed the use of antimicrobials per trimester of pregnancy, yet with a small magnitude of associations observed26. Conversely, Lin et al. (2020)27 have found that among 4,909 children aged one to four years, exposure to antimicrobials during pregnancy was associated with a higher risk of excess weight only in female children aged 12 months. In our study, sensitivity analyses were conducted and showed that associations observed did not vary by sex (data not shown).

Regarding the type of antimicrobial used, Cassidy-Bushrow et al. (2018)26 found a significant association between the use of macrolides during pregnancy with BMI-for-age (BMI z-score 0.37; SD:0.18; p= 0.039). They found no association with other types of antimicrobials26. Macrolides are broad-spectrum antibacterial. In the present study, most pregnant women used small-spectrum antimicrobials, and no association with overweight was found according to the type of antimicrobials used.

A recent systematic review of 23 studies which analyzed data from over 1.2 million participants found that prenatal antimicrobial exposure is not associated with an excess of weight or childhood obesity. However, subgroup analyses were performed in this review indicating an increased risk of excess weight when use was in the second trimester of pregnancy (RR = 1.13; IC95% CI: 1.06; 1.22; p = 0.001)28. Thus, the results of this review are in accordance with the isolated findings found in our and previous studies29.

A study with 10,000 school-age children in Denmark found an association between the use of antimicrobials with excess weight in school children whose birth weight was more than 3500g and whose mothers used antimicrobials during pregnancy (PR=1.30, 95%CI 1.05;1.61)3. In that study, antimicrobial exposure in the second trimester (PR: 1.39, 95% CI: 1.11;1.73) and in the third trimester of pregnancy (PR: 1.43, 95% CI: 1.17;1.76) were associated with increased excess weight in school-age children. Furthermore, the prevalence of excess weight increased according to the number of antimicrobials prescribed to the mother (p=0.001) but not with the type of antimicrobial3. Sensitivity analyzes in our study were conducted and did not show differential associations according to birth weight status of children (data not shown).

Among members of Kaiser Permanente Northern California cohort study30, with data collected from 2007 to 2015, there was no increased risk of obesity in children whose mothers had controlled infection, either by type of antimicrobials or in terms of dose-response ratio. However, untreated maternal infection (i.e., those mothers who required an antimicrobial but did not use it) was associated with a higher risk of obesity (OR= 1.09 CI: 1.03; 1.16). These results were confirmed utilizing a sensitivity analysis among discordant siblings, i.e., pairing with children of the same mother with pregnancy without infection or using antimicrobials, which allowed additional control of unmeasured confounding factors, including genetic, intrauterine maternal and family factors.

Limitations of our study should be acknowledged. Maternal diet during pregnancy was not assessed, which can directly interact with the children's weight. However, our analyses accounted for maternal pre-pregnancy BMI as a potential confounder. Self-reported antimicrobial use may lead to misclassification of antimicrobials, as mothers may confuse the names of medications or not remember if they used antimicrobials. However, this may be more important to symptomatic or less impactful treatments in the context of pregnancy, which is not the case for the treatment of infections. The time and duration of antimicrobial use were not assessed in our study, only the frequency of use per trimester of pregnancy. However, our analyses of the number of trimesters that antimicrobials were used showed similar results to those from trimester-specific use.

Conclusion
Despite the controversies regarding the association between the use of antimicrobials during pregnancy and excess weight in children, the findings of our study suggest that the use of antimicrobials during pregnancy may not impact children excess weight up to 48 months postpartum.

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