Assessment of Cardiovascular Health of Children Ages 6 to 10 Years Conceived by Assisted Reproductive Technology

This cohort study examines the association of conception by assisted reproductive technology with cardiovascular health outcomes among children in China ages 6-10 years.


Introduction
Assisted reproductive technology (ART) is widely used in the treatment of infertility. It is estimated that more than 8 million infants have been born after the use of ART worldwide since Louise Brown's birth, accounting for approximately 2% to 6% of births in high-income countries. 1,2 ART requires the in vitro manipulation of gametes and embryos in a synthetic culture environment, and these nonphysiological exposures may be associated with adverse outcomes in embryonic development and offspring health. 3 Some studies 4,5 found that ART may be associated with increased risk of unfavorable obstetric outcomes, including preterm birth, low birth weight, and infants born small for their gestational age. These are also risk factors associated with cardiovascular disease. 6,7 Evidence from the developmental origins of health and disease theory also suggests that adverse events (eg, restricted fetal growth and preterm birth) in early life are associated with increased the risk of cardiovascular disease later in life. 8 Therefore, the cardiovascular health of offspring conceived by ART has gained much attention. Some scholars have found an increased incidence of congenital heart defects among children conceived by ART, 9 and cardiac remodeling already presents in fetal life 10 and persists in postnatal life and at ages 2 to 6 years among offspring conceived by ART. [10][11][12] However, other studies did not find an increased risk of cardiovascular disease among children conceived by ART. 13,14 These studies include limited sample sizes, and the results were inconsistent. 11,12,15 Additionally, measurement of the heart among children aged 2 to 6 years may be inaccurate because of their low compliance to examination and relatively small heart size. Therefore, to investigate the association of ART with heart health outcomes, we used cardiology ultrasonography measurements to assess left ventricular cardiac structure and function among children aged 6 to 10 years conceived by ART compared with children in a matched control group conceived naturally.

Methods
This cohort study's protocol was approved by the institutional review board of the Center for Reproductive Medicine of Shandong University. Written informed consent was obtained from parents or guardians of all participants. This report followed the Strengthening the Reporting of

Physical and Lifestyle Measurements
Standardized protocols and instruments were used for all measurements among children conceived by ART and those in the matched control group. Weight and height were measured with children in light clothes and without shoes using a standardized scale. Weight was measured twice to the nearest 0.1 kg, and height was measured twice to the nearest 0.1 cm. The means of the 2 measures were calculated for data analysis. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Overweight and obesity were defined using age-specific and sex-specific BMI percentile cutoffs for Chinese children and adolescents. 16 Blood pressure (BP) was measured by trained staff (Z.Z., J.H., J.M., and M.F.) with an electronic device (Omron HEM-7012; Omron), which has been clinically validated. 17 After the child had at least 10 minutes of rest, BP was measured on the right arm using the appropriate-sized cuff, with the child seated. We obtained 3 consecutive BP measurements, and the mean value of the final 2 readings was used for data analysis. Elevated BP and high BP were defined as systolic BP (SBP) or diastolic BP (DBP) at or above the age-specific and sex-specific 90th and 95th percentile values, respectively, of the Chinese pediatric BP references. 18 Lifestyle factors, including frequency of fruit and vegetable intake, soft drink intake, physical activity (ie, low to moderate or vigorous exercise), and duration of screen time (ie, watching TV or playing computer games) and night sleep, were collected using the same questionnaire. was calculated as LVM (in grams) divided by height (in meters) 2.7 to correct for body size. 20 Left ventricular hypertrophy (LVH) was defined as LVMI at or above the age-specific and sex-specific 95th percentile based on 2273 US children who were healthy and did not have obesity. 21 Relative wall thickness (RWT) was calculated as (LVPWT + IVST) / LVDD. We then found adjusted RWT (aRWT) for age using the formula aRWT = RWT − 0.005 × (age − 10) for children aged 1 to 17 years. 22 High RWT was defined as an aRWT of 0.375 or more, which was the age-specific 95th percentile cutoff for aRWT. 22 Based on LVMI and aRWT, children were then divided into 4 groups: reference range geometry (ie, reference range LVMI and aRWT), concentric remodeling (CR; reference range LVMI and high aRWT), eccentric hypertrophy (EH; LVH and reference range aRWT), and concentric hypertrophy (CH; LVH and high aRWT).

Cardiology Ultrasonography Measurements and Definitions
The LV shortening fraction (LVSF) was calculated using internal ventricular diameters with the following equation: (LVDD − LVSD) / LVDD. Mitral inflow velocities, including peak velocities during early diastole (E) and late diastole (A), were measured, and E to A ratio was calculated.

Statistical Analysis
All data analyses were performed using SAS statistical software version 9.3 (SAS Institute). Study outcomes included BP and cardiac structure and function. The independent variable of interest was conception type (ART vs natural), and the covariates were gestational age at birth, birth weight, duration of breastfeeding, current (ie, at age 6-10 years) weight status, BP status, and lifestyle factors, including fruit and vegetable intake, soft drink intake, physical activity, and duration of screen time and sleep. Data were expressed as mean (SD) for continuous variables (all variables were in approximately normal distribution) and No. (%) for categorical variables. Early life factors, current weight, and lifestyle factors were compared between ART and control populations using t test or χ 2 test, as appropriate. The parameters of BP indices, cardiac structure, and function were compared between groups using t test. To rule out the association of covariates with outcomes, linear regression analysis was used to assess the associations of conception type (ie, ART vs natural) with BP indices and cardiac structure and function after adjustment for gestational age at birth, birth weight, duration of breastfeeding, current weight status, BP status (for outcomes of cardiac structure and function), and lifestyle factors. Additionally, given that it is hypothesized that fertilization type (ie, ICSI vs IVF) and embryo type (ie, fresh vs frozen) may have different associations with LV structure and function, 23,24 we performed subgroup analyses by fertilization type and embryo type.

JAMA Network Open | Cardiology
The prevalence of LVH, elevated BP, high RWT, and LV remodeling patterns were compared between groups using χ 2 test or Fisher exact test. A 2-sided P < .05 was considered statistically significant.
Data were analyzed from March 2019 through December 2019.

Characteristics of Study Groups
Among 764 children aged 6 to 10 years enrolled in the study, 382 children were conceived by ART  However, the distribution of gestational age at birth (ie, proportion of children born preterm, at full term, or postterm), current frequency of fruit and vegetable intake, physical activity, and mean sleep duration were similar between groups.

Blood Pressure and LV Structure and Function
Children conceived by ART, compared with children who were naturally conceived, had statistically

Discussion
To our knowledge, this cohort study is the largest study (with 382 children conceived by ART and 382 children matched in the control group) to examine the associations of ART with alterations in LV structure and function among Chinese children aged 6 to 10 years. Our study found that abnormal cardiac structure and dysfunction, including an increased risk of LVH and LV geometric remodeling, were more frequent among children conceived by ART compared with children in the control group.
These findings were independent of early life factors, current lifestyle factors, and fertilization type (ie, ICSI or IVF). However, frozen embryo transfer had some favorable cardiac outcomes compared with fresh-embryo transfer, which may be associated with decreased risks of several unfavorable birth outcomes (eg, being born small for gestational age, low birth weight, and preterm delivery) that are associated with pregnancies conceived from frozen embryo transfer vs fresh embryo transfer. 25 Several studies have consistently found that children conceived by ART had increased carotid intima-media thickness, increased pulse wave velocity, and decreased flow-mediated dilation. 10,[26][27][28] However, 3 studies 11,12,15  Evidence from the Framingham Offspring Study 29 suggests that LVM can persist from childhood to adulthood and may be a factor associated with cardiovascular disease in adulthood. There are 3 forms of LV geometric remodeling patterns (CR, EH, and CH) that are associated with risk of cardiovascular disease, while CH is considered to carry the greatest risk for cardiovascular disease. 30,31 To our knowledge, our study is the first to assess the associations of ART with LVH and LV geometric remodeling. We found that children conceived by ART had increased prevalence of LVH, high RWT, and LV geometric remodeling patterns (ie, CR, EH, and CH). We also found that CR was the most frequent pattern of LV geometric remodeling among children conceived by ART.

JAMA Network Open | Cardiology
There are several potential mechanisms that may explain the association of ART with abnormal LV structure and dysfunction. First, epigenetic perturbations induced by ART during the periconceptional period may play an important role in the fetal programming of future subclinical cardiovascular alterations. [32][33][34] Several studies found that ART may be associated with altered methylation levels among differential methylation regions of imprinted genes in the embryo and placenta. [35][36][37][38] The incidence of imprinting disorders among offspring conceived by ART is increased compared with offspring conceived spontaneously. 39,40 These epigenetic alterations may be associated with the infertility backgrounds of parents, superovulation, or factors associated with the ART procedure. 41,42 The alterations may persist into childhood and be associated with worsened cardiovascular health among offspring. 33,34 Second, increased oxidative stress (OS) may also be associated with altered cardiac structure and function among offspring conceived by ART. Several publications suggested that OS was crucial in the fetal programming of adulthood cardiovascular diseases. [43][44][45][46] The infertility backgrounds of parents, pregnancy complications, and the ART procedure could be associated with excessive OS, which may be associated with the cardiac health of offspring. 47 Third, it has been shown that ART may be associated with increased risk of impaired lipid and glucose metabolism, which is the pathophysiological basis of various cardiovascular diseases. 48-55 Therefore, ART may be associated with adverse LV structure and function among offspring through the disorder of lipid and glucose metabolism.

Limitations
Although we conducted a strict matched cohort study with a large sample size and adjusted for many confounders, several limitations of this study should still be noted. First, we cannot make conclusions concerning whether the observed association was associated with the ART procedure itself or other unknown confounders that were not measured in the present study. However, a previous study's findings 33 suggested that the ART procedure itself, rather than parent-associated factors, may be the main factor associated with this outcome. Second, it should be noted that because there are no unified criteria to define LVH, high RWT, or LV geometric remodeling patterns, we used sex-specific and age-specific 95th percentile cutoffs based on definitions established among Western children, 21,22 and the cutoffs may be too high to be used among Chinese children. Third, all children conceived by ART in this study were from a single medical center in Shandong, China; therefore, our findings should be generalized with caution. Additionally, although the differences between groups were significant, the absolute values of the changed parameters in the ART group were still within the reference range, which may limit the clinical relevance of our findings. However, considering the association between the cardiovascular parameter changes in childhood and adult cardiovascular disorders, a continuous monitoring was suggested in this ART-conceived population.

Conclusions
This study found increased levels of unfavorable changes in LV structure and function, including LV geometric remodeling, among children conceived by ART compared with children who were naturally conceived. These findings may have significance for clinical and public health. Because childhood is a critical window for early detection, potential intervention, and improvement of cardiac health among children conceived by ART, it may be possible to reverse the unfavorable alterations in their cardiac structure and function. Further studies are necessary to confirm the association