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Clinical Trial
. 2021 Sep 23;12(1):5613.
doi: 10.1038/s41467-021-25899-4.

Analyses of child cardiometabolic phenotype following assisted reproductive technologies using a pragmatic trial emulation approach

Jonathan Yinhao Huang  1 , Shirong Cai  2   3 , Zhongwei Huang  3   4 , Mya Thway Tint  2   3 , Wen Lun Yuan  2   5 , Izzuddin M Aris  6 , Keith M Godfrey  7 , Neerja Karnani  2 , Yung Seng Lee  2   8 , Jerry Kok Yen Chan  9   10 , Yap Seng Chong  2   3 , Johan Gunnar Eriksson  2   3   11   12 , Shiao-Yng Chan  2   3
Affiliations
Clinical Trial

Analyses of child cardiometabolic phenotype following assisted reproductive technologies using a pragmatic trial emulation approach

Jonathan Yinhao Huang et al. Nat Commun. .

Abstract

Assisted reproductive technologies (ART) are increasingly used, however little is known about the long-term health of ART-conceived offspring. Weak selection of comparison groups and poorly characterized mechanisms impede current understanding. In a prospective cohort (Growing Up in Singapore Towards healthy Outcomes; GUSTO; Clinical Trials ID: NCT01174875) including 83 ART-conceived and 1095 spontaneously-conceived singletons, we estimate effects of ART on anthropometry, blood pressure, serum metabolic biomarkers, and cord tissue DNA methylation by emulating a pragmatic trial supported by machine learning-based estimators. We find ART-conceived children to be shorter (-0.5 SD [95% CI: -0.7, -0.2]), lighter (-0.6 SD [-0.9, -0.3]) and have lower skinfold thicknesses (e.g. -14% [-24%, -3%] suprailiac), and blood pressure (-3 mmHg [-6, -0.5] systolic) at 6-6.5 years, with no strong differences in metabolic biomarkers. Differences are not explained by parental anthropometry or comorbidities, polygenic risk score, breastfeeding, or illnesses. Our simulations demonstrate ART is strongly associated with lower NECAB3 DNA methylation, with negative control analyses suggesting these estimates are unbiased. However, methylation changes do not appear to mediate observed differences in child phenotype.

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Conflict of interest statement

Y.S.C., S.Y.C., and K.M.G. have received reimbursement for speaking at conferences sponsored by companies selling nutritional products, and are part of an academic consortium that has received research funding from Abbott Nutrition, Nestec, and Danone. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Associations between ART status and anthropometry, adjusted for pre-pregnancy characteristics.
Point estimates and Wald-type 95% confidence intervals show the adjusted mean difference in anthropometric measures between ART-conceived and spontaneously conceived (SC) children at each study visit, with SC children as the referent. Differences were estimated by multivariable linear regressions adjusted for maternal age, education, ethnicity, household income, height, pre-pregnancy BMI, parity, and any tobacco exposure in the home; paternal age, height, weight, diabetes history, and high blood pressure history; child sex, and polygenic risk score for adult adiposity with no multiple testing adjustments. Multiple imputations by chained equations were used to estimate associations while simultaneously accounting for missing covariate values. Sample sizes at each visit (SC/ART): 1091/83; 967/69; 953/71; 916/68; 872/70; 892/68; 874/67; 807/55; 832/60; 864/64; 801/57; 833/64; 812/61; 800/63; 771/59; 762/59. Shaded regions connect confidence intervals to aid in visualizing trends and provide no additional information regarding estimates between visits. cm centimeters, kg kilograms, SDS   standard deviation scores.
Fig. 2
Fig. 2. Associations between ART status and skinfold thickness and blood pressure, adjusted for pre-pregnancy characteristics.
Point estimates and Wald-type 95% confidence intervals show the adjusted mean difference in skinfold thickness (a) and blood pressure (b) measures between ART-conceived and spontaneously conceived (SC) children at each study visit, with SC children as the referent. Differences were estimated by multivariable linear regressions adjusted for maternal age, education, ethnicity, household income, height, pre-pregnancy BMI, parity, and any tobacco exposure in the home; paternal age, height, weight, diabetes history, and high blood pressure history; child size, and polygenic risk score for adult adiposity with no multiple testing adjustments. Multiple imputations by chained equations were used to estimate associations while simultaneously accounting for missing covariate values. Skinfold sample sizes (a) for each visit (SC/ART): 1038/81; 762/48; 776/54; 827/62; 765/55; 810/63; 779/59; 777/62; 750/55; 743/57. Blood pressure sample sizes (b) for each visit (SC/ART): 716/53; 662/44; 701/48; 647/47. Note, not all measures were taken at each visit. Shaded regions connect confidence intervals to aid in visualizing trends and provide no additional information regarding estimates between visits. cm centimeters, m meters, kg kilograms, mmHg milimeters mercury, SDS   standard deviation score; colors used to distinguish outcome measures.
Fig. 3
Fig. 3. Difference in anthropometrics, skinfold thickness, and blood pressure comparing ART-conceived fetuses versus a putatively subfertile cohort, adjusted for pre-pregnancy characteristics.
Point estimates and Wald-type 95% confidence intervals show the adjusted mean difference in anthropometric (a), skinfold thickness (b), and blood pressure (c) measures between ART-conceived and spontaneously conceived (SC) children from a subfertile cohort (N = 93) selected on the basis of medical history that may prompt couples to seek fertility specialist care including the history of two or more past miscarriages; medications with potential fertility indications (e.g., aspirin, hormones, thyroid, weight loss); history of PCOS, endometriosis, ovarian cysts, fibroids, or thyroid disorders (hyper- or hypo-). Differences were estimated by multivariable linear regressions adjusted for maternal age, education, ethnicity, household income, height, pre-pregnancy BMI, parity, and any tobacco exposure in the home; paternal height and weight; child size, and polygenic risk score for adult adiposity with no multiple testing adjustments. Multiple imputation by chained equations were used to estimate associations while simultaneously accounting for missing covariate values. Sample sizes for each visit were (SC/ART): (a) 93/83; 83/69; 86/71; 84/68; 80/70; 79/68; 79/67; 72/55; 80/60; 84/64; 84/57; 86/64; 80/61; 80/63; 82/59; 77/59; (b) 91/81; 69/48; 73/54; 82/62; 81/55; 82/63; 75/59; 77/62; 80/55; 75/57; (c) 75/53; 72/44; 70/48; 66/47. Shaded regions connect confidence intervals to aid in visualizing trends and provide no additional information regarding estimates between visits. cm centimeters, m meters, kg kilograms, mmHg milimeters mercury, SDS   standard deviation score; colors used to distinguish outcome measures.
Fig. 4
Fig. 4. Associations between ART status and fetal cord tissue DNA methylation.
Associations between ART status and 281 candidate CpGs (a), three top NECAB3 CpGs (b), and three top NECAB3 CpGs stratified by ethnicity (c), were estimated by multivariable linear regression adjusted for maternal ethnicity, age, parity, and pre-pregnancy BMI, and child sex. Horizontal thresholds represent P = 0.05; P = 1.8 × 10-4 (Bonferroni correction); P = 3.6 × 10−8 (EWAS-threshold per Saffari et al. 2018). CpGs are grouped by chromosome with colors indicating P < 0.05. b, c show distributions of standardized methylation values with two-sided t test P values indicated. Sample sizes for all plots = 1094 spontaneous (598 Chinese, 205 Indian, 291 Malay); 83 ART (64 Chinese, 11 Indian, 8 Malay).
Fig. 5
Fig. 5. Monte Carlo-simulated effects of ART mediated through fetal cord tissue methylation, overall sample.
The solid lines show the estimates and Wald-type 95% confidence intervals for the hypothetical average direct treatment effect of assigning all subjects to ART (versus spontaneous conception) while holding methylation for (a) cg03904042 (NECAB3) or (b) cg27146050 (HIF3A) at the untreated (SC) level. Point estimates and standard errors were estimated by parametric g-computation in 100 bootstrapped datasets. The dashed and shaded regions show the specific indirect effect of fixing the methylation level to that caused by ART itself (compared to the level that would have been observed without treatment) among ART-conceived children. Sample sizes across visits are identical to Fig. 1. cm centimeters, SDS   standard deviation score; colors used to distinguish outcome measures. Shaded regions connect confidence intervals to aid in visualizing trends and provide no additional information regarding estimates between visits.
Fig. 6
Fig. 6. Monte Carlo-simulated effects of ART mediated through fetal cord tissue methylation, target trial.
The solid lines show the estimates and Wald-type 95% confidence intervals for the hypothetical average direct treatment effect of assigning all subjects to ART (versus spontaneous conception) while holding methylation for (a) cg03904042 (NECAB3) or (b) cg27146050 (HIF3A) at the untreated (SC) level, in an emulated pragmatic trial among subfertile couples. Point estimates and standard errors were estimated by parametric g-computation in 100 bootstrapped datasets. Analyses are similar to those in the overall cohort but restricted to pregnancies from ART (N = 83) and spontaneous conceiving couples with infertility indications (N = 93). Sample sizes across visits are identical to Fig. 3a. cm centimeters, SDS   standard deviation score; colors used to distinguish outcome measures. Shaded regions connect confidence intervals to aid in visualizing trends and provide no additional information regarding estimates between visits.
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