Association of Assisted Reproductive Technology With Offspring Growth and Adiposity From Infancy to Early Adulthood

Key Points Question Is conception by assisted reproductive technology associated with growth and adiposity from infancy to early adulthood? Findings In this cohort study of up to 158 066 infants, children, adolescents and young adults from Europe, Asia-Pacific, and Canada, those conceived using assisted reproductive technology (in vitro fertilization or intracytoplasmic sperm injection, plus embryo transfers) were shorter, lighter, and thinner by most estimates from infancy up to early adolescence compared with their naturally conceived peers; however, the differences were small across all ages and reduced with older age, with 95% CIs sometimes including the null. Meaning These findings suggest that parents conceiving or hoping to conceive through assisted reproductive technology and their offspring should be reassured that differences in early life growth and adiposity are small and no longer apparent by late adolescence.

The study received approval from the ethics committee (CCPPRB) of Kremlin Bicêtre on 12 December 2002 and from CNIL (Commission Nationale Informatique et Liberté), the French data privacy institution. All subjects gave their informed consent for inclusion before they participated in the study. Consent for the child was obtained from both parents after the child's birth.

Etude Longitudinale Françcaise depuis l'Enfance (ELFE)
ELFE is a nationwide birth cohort, including 18 329 children born in 2011 in a random sample of 349 maternity units from mainland France 12 . Inclusion criteria were singleton or twins born after 33 weeks' gestation to mothers aged ≥18 years and not planning to move outside of metropolitan France in the next 3 years. Detailed information has been collected from parents using questionnaires ((including mode of conception and fertility treatment), data extraction from obstetrical file, and clinical assessment by general practitioner at age 2. Height and weight measures have been collected through parental report of measurements performed by health professionals retrieved from the child health booklet and from the 2-yr general practionner clinical exam. Data up to 39 months have been used for this analysis.
Up to 309 ART-conceived offspring and 9,632 NC offspring were included in this study (including multiple births). ELFE contributed results to the main analysis (ART vs. NC) and to additional analysis stratified by sex, ICSI/IVF, and sub-fertility, for height, weight, and BMI. Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
Ethical approvals for data collection in maternity units and for each data collection wave during follow-up were obtained from the national advisory committee on information processing in health research (CCTIRS: Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé), the national data protection authority (CNIL: Comission Nationale Informatique et Liberté) and, in case of invasive data collection such as biological sampling, the committee for protection of persons engaged in research (CPP: Comité de Protection des Personnes). The ELFE study was also approved by the national committee for statistical information (CNIS: Conseil National de l'Information Statistique). Informed consent was signed by the parents or the mother alone, with the father being informed of his right to deny consent for participation

EU Childhood Obesity Project (CHOP)
The European Childhood Obesity Project (CHOP) was a one-year multicentre double-blind randomized controlled intervention trial including 1678 children (registered at ClinicalTrials.gov: NCT00338689). Healthy singleton term infants born between 1 st October 2002 and 31 st July 2004 were recruited in five European countries (Belgium, Germany, Italy, Poland, Spain) during their first 8 weeks of life. They were randomized to cow-milk based formula with either higher or lower protein-content. Additionally, a reference group of breastfed children was included. The aim was to test whether feeding infant formula, which differ in their level of milk proteins, can influence infant growth and the risk of later childhood obesity ('early protein hypothesis'). After the intervention, children were prospectively followed up until the age of 11 years. More detailed information on the study design and results can be found elsewhere [13][14][15][16] . Data on the exposure (ART) and the

Growing Up in New Zealand (GUiNZ)
GUiNZ is a prospective birth cohort study that recruited 6,853 children via their pregnant mothers if they had an expected delivery date between 25 April 2009 and 25 March 2010 and were residing within a geographically defined region of New Zealand which was chosen because it could provide a cohort of births that would be representative of all current births in NZ, especially with respect to ethnic and socioeconomic diversity 22 . Birth parameters were retrieved via linkage to routine perinatal records (with maternal consent) and repeated child height and weight measurements were collected as part of field interviews when the children were 2 years and 4 years of age. Anthropometric measurements were undertaken by trained interviewers using a standardised approach used by the NZ Ministry of Health.
Up to 173 ART-conceived offspring and 4,274 NC offspring were included in this study. GUiNZ contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, for height, weight, BMI, and waist circumference. Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
Ethical approval for GUiNZ was received from the Ministry of Health Northern Y Regional Ethics Committee (NTY/08/06/055). Written informed consent was obtained from all participating mothers at recruitment and confirmed at each subsequent interview.

Growing up in Singapore Towards healthy Outcomes (GUSTO)
GUSTO recruited pregnant women aged 18 years and above, attending their first trimester antenatal dating ultrasound scan clinic at Singapore's two major public maternity units 23 . Women were eligible if 18 years and older, Singaporean citizens or permanent residents, with self-reported homogenous ethnic ancestry (Chinese, Indian, Malay), intended to deliver at the either of the recruitment hospitals and reside in Singapore for the next 5 years. Women greater than 14 weeks of gestation, receiving chemotherapy, psychotropic medications, or having an existing type I diabetes mellitus diagnosis at the time of recruitment were excluded. Women who ultimately did not agree to donate birth tissues (cord, placenta, cord blood) were also excluded. Women were asked to self-report whether the current pregnancy was conceived via IVF and use of assisted reproductive technologies, along with relevant treatment modalities, were confirmed via medical record review by a senior Weight was measured to the nearest 10 g using a digital scale (Seca Alpha, model 770; Seca, Hamburg, Germany). Children were weighed without shoes in the minimum clothing possible. Height was measured to the nearest 0·1 cm using a commercial stadiometer (Leicester Height Measure; Invicta Plastics, Oadby, UK) with the child standing barefoot, keeping shoulders in a relaxed position, arms hanging freely and head in the Frankfurt horizontal plane. Waist circumference was measured to the nearest 0·1 cm with the use of a nonelastic tape (Hoechstmass, Sulzbach, Germany) with the child standing, at the end of a gentle expiration, after placing the measuring tape on a horizontal plane around the trunk, at the level of the umbilicus, midway between the lower rib margin and the iliac crest. Bioelectrical impedance analysis (BIA) was used for the assessment of percentage body fat (Akkern BIA 101; Akkern Srl, Florence, Italy). Data on the socio-economic background of the families having at least one child participating in the study were collected from the parents (most preferably from the mother) during scheduled face-to-face interviews at school.
Up to 63 ART-conceived offspring and 2,182 NC offspring were included in this study. HGS contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, and IVF/ICSI, for all study outcomes (i.e., height, weight, BMI, waist circumference, body fat % and fat mass index). Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
Approval to conduct the study was granted by the Greek Ministry of National Education and the Ethics Committee of Harokopio University of Athens, and the study was conducted in accordance with the ethical standards specified in the 1964 Declaration of Helsinki. Parents who agreed to the participation of their children in the study had to sign the consent form and provide their contact details.

Italian Twins Register (ITR)
ITR 25 is a population-based registry of voluntary twins. Since its inception, 29,000 twins have been enrolled and about 20% of them are minors. The ITR collects information on a large spectrum of phenotypes by either selfreported questionnaires or clinical examinations. In the case of underage twins, the information is reported by the parents who sign an informed consent form.
The ITR offspring were aged between 6 months and 13 years at time of outcome assessments. Due to the wide age range at outcome assessment, ITR was analysed in 7 separate age groups that each included between 32 and 54 ART-conceived offspring (and between 140 and 819 NC offspring (multiple births only). ITR contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, and IVF/ICSI (for IVF only), for height, weight, and BMI. Data were available for all study confounders (maternal age, BMI, smoking, education, parity and offspring sex and age at outcome assessment) except for ethnicity.
The study was approved by the ethics committee of Istituto Superiore di Sanità (prot. Number CE-ISS 05-113). All included twins gave their consent to participate in the studies proposed by the ITR research group.

Millenium Cohort Study (MCS)
MCS is a nationally representative birth cohort study that followed 19,244 children born in the UK in 2000-2002 26 . Baseline interviews were conducted when the children were approximately nine months old, and followup interviews were conducted when the children were around 3, 5, 7, 11, 14 and 17 years old. MCS includes detailed information about the demographic, health and socio-economic characteristics of the respondents and their families. At the baseline interview, the cohort member's mother was asked whether they had used any fertility treatment to conceive.
Up to 30 ART-conceived offspring and 2,153 NC offspring were included in this study (including multiple births). MCS contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, subfertility, and IVF/ICSI, for all study outcomes (i.e., height, weight, BMI, waist circumference, body fat % and fat mass index). Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
Ethical approval for the Millennium Cohort Study was granted from the multi-centre research ethics committee. Following ethical approval for the study from an NHS Research Ethnics Committee (MREC), informed consent was obtained from parents, as well as from the children themselves as they grew up.

MUltiple BIrth Cohort Study (MUBICOS)
MUBICOS 25 has been established within the Italian Twin registry since 2010 but these cohorts do not overlap. About 360 families were enrolled and DNA was taken from parents and twins. Follow-up questionnaires have been administered at 6, 12, 18 and 36 months of age. All height and weight measures are self-reported by parents.
Up to 54 ART-conceived offspring and 101 NC offspring were included in this study (multiple births only). MUBICOS contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, and IVF/ICSI (for IVF only), for height, weight, BMI. Data were available for most study confounders (maternal age, BMI, smoking, education, parity and offspring sex and age at outcome assessment), except ethnicity.
The study was approved by the ethics committee of Istituto Superiore di Sanità (prot. Number CE-ISS 09-281). All included twins gave their consent to participate in the studies proposed by the ITR research group.

Nascita e INFanzia: gli Effetti dell'Ambiente (NINFEA)
NINFEA study is an Internet-based birth cohort established in 2005 in Italy (http://www.progettoninfea.it) 27 . A baseline questionnaire on general health and exposures before and during pregnancy is completed by mothers at enrolment, which may occur at any time during pregnancy. During the period 2005-2016 around 7,500 mothers were recruited. Further follow-up information was obtained with repeated questionnaires completed 6 and 18 months after delivery and when children turn 4, 7, 10 and 13 years. At each follow-up mothers reported their child current weight and height measurements, and if able to recall or retrieve from baby books or child health records retrospective measurements at pre-defined ages (3 months at 6-month, 12 months at 18-month, and 5 and 6 years at 7-year follow-up). Additional information on whether the measurements were recalled or taken from baby books is available.
Information on exposures was retrieved from the baseline questionnaire completed during pregnancy where mothers reported whether the pregnancy was planned or not, and in the case of affirmative response the following information was collected: i) number of months since she begun trying and became pregnant, ii) if she used any ART treatment, and iii) the type of ART as a checkbox (ovulation induction, intrauterine insemination, gamete intrafallopian transfer, in vitro fertilization, intra-cytoplasmic sperm injection, other technique). Information on the following confounding variables was collected in the baseline and 6-month follow-up questionnaires: maternal age (continuous), maternal BMI (continuous, derived from maternal pre-pregnancy weight and height reported at enrolment); smoking during pregnancy (yes vs. no, defined as any maternal smoking during pregnancy, independently whether sustained or not), educational level (lowprimary school or less, medium-secondary school, and high-university degree), maternal country of birth as a proxy for ethnicity (born in Italy vs. born outside Italy, with more than 95% of mothers born in Italy), and parity (nulliparous vs. multiparous, based on the number of previous livebirths and stillbirths).
Up to 253 ART-conceived offspring and 4,990 NC offspring were included in this study. NINFEA contributed results to the main analysis (ART vs. NC), and to additional analyses stratified by sex, sub-fertility, and IVF/ICSI, for height, weight, and BMI. Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
The Ethical Committee of the San Giovanni Battista Hospital and CTO/CRF/Maria Adelaide Hospital of Turin approved the NINFEA study (approval N. 0048362, and subsequent amendments). Informed consent was obtained from all the participants at enrolment and at each follow-up.

Norwegian Mother, Father and Child Cohort Study (MoBa)
MoBa is a nationwide, pregnancy cohort comprising family triads (mother-father-offspring) who are followed longitudinally. All pregnant women in Norway who were able to read Norwegian were eligible to participate. The first child was born in 1999 and the last in 2009 28,29 . Extensive longitudinal data were collected using nine questionnaires: three during pregnancy, and then follow-up questionnaires when the children were 6 months, 18 months, 36 months, 5 years, 7 years and 8 years of age. Data collected include general background and health information, including diet and lifestyle, a semi-quantitative food frequency questionnaire, information on birth and pregnancy outcomes, and on several aspects of child nutrition and development, as well as the physical and mental health of both mother and child. MoBa is linked to the Medical Birth Registry of Norway, which provides standardized information about the health of the mother during pregnancy, other essential medical information related to the pregnancy and birth, and standard post-natal measures of the child. The Medical Birth Registry (MBRN) is a national health registry containing information about all births in Norway.
Up to 2,097 ART-conceived offspring and 77,210 NC offspring were included in this study (multiple birth included). MoBa contributed results to the main analysis (ART vs. NC) to all additional analysis, for height, weight, BMI. Data were available for most study confounders (maternal age, BMI, smoking, education, parity, and offspring sex and age at outcome assessment), but not ethnicity.
The establishment and data collection in MoBa was previously based on a license from the Norwegian Data protection agency and approval from The Regional Committee for Medical Research Ethics, and it is now based on regulations related to the Norwegian Health Registry Act. MoBa is conducted according to the guidelines laid down in the declaration of Helsinki, and written informed consent was obtained from all participants. A detailed protocol of the study including the consent can be found elsewhere (http://www.fhi.no/morogbarn).

Piccolipiù
Piccolipiù is a prospective birth cohort study of 3,358 children born in selected maternal units located in five Italian cities (Florence, Rome, Trieste, Turin, and Viareggio) between 2011-2015. Piccolipiù study recruited singleton pregnant women aged at least 18 years old and giving birth in one of the selected maternity units. Mothers were recontacted when the child was 6, 12, 24, 48 months and 6 years old for follow-up questionnaires 30 .
Up to 86 ART-conceived offspring and 2,479 NC offspring were included in this study. Piccolipiù contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, and sub-fertility, for height, weight, BMI, and waist circumference. Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
The protocol of the study has been approved by the Ethics committees of the Local Health Unit Roma E (management centre), of the Istituto Superiore di Sanità (National Institute of Public Health) and of each local centre. Standard procedures for the protection of confidential individual information were applied according to the Italian law. Consent forms for participation was signed by the mother and also by the father, when both legally responsible for the newborn.

Southampton Women's Survey (SWS)
SWS is a population-based prospective birth cohort study of 12 583, initially non-pregnant, women aged 20-34 years, living in the city of Southampton, UK 31 . Assessments of lifestyle, diet and anthropometry were done at study entry in 1998-2002. Women who subsequently became pregnant with singleton pregnancies were followed up during pregnancy; and their offspring have been studied in infancy and childhood. Research nurses collected all anthropometric measurements on offspring and DXA scans were performed at various ages to determine body fat % and fat mass index Information on ART was obtained at the time of the first scan by questioning the mother.
Up to 36 ART-conceived offspring and 2,554 NC offspring were included in this study (singleton births only). SWS contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, for all study outcomes (i.e., height, weight, BMI, waist circumference, body fat % and fat mass index). Data were available for all study confounders (maternal age, BMI, smoking, education, ethnicity, parity and offspring sex and age at outcome assessment).
SWS study was conducted according to the guidelines laid down in the Declaration of Helsinki and was approved by the Southampton and South West Hampshire Local Research Ethics Committee (06/Q1702/104). Written informed consent was obtained from all participating women and by a parent or guardian with parental responsibility on behalf of their children.

The Trøndelag Health Study (HUNT)
The Trøndelag Health Study (HUNT) is a population-based study where all adult residents of the Nord-Trøndelag region, Norway have been invited to repeated surveys since the 1980s. Since the 1990s, all adolescents (aged 13-19 years) in the region have also been invited (the Young-HUNT Study) 32,33 . The participants have consented to data linkage to health registries, such as the Medical Birth Registry of Norway (MBRN), which includes information on virtually all births in Norway since 1967. In this study, we included participants from the Young-HUNT1 (1995-97), Young-HUNT2 (1999-2000 and Young-HUNT3 (2006-08) surveys, which included clinical measurements of height, weight and waist and hip circumferences. Information on mode of conception was obtained through linkage to information from the MBRN.
Up to 121 ART-conceived offspring and 9,711 NC offspring were included in this study (including multiple births). HUNT contributed results to the main analysis (ART vs. NC) to additional analysis stratified by sex, IVF/ICSI, and ET/FET, for height, weight, BMI, and waist circumference. Data were available for some study confounders (maternal age, parity and offspring sex and age at outcome assessment), but not for maternal BMI, smoking, education, or ethnicity.
The study is approved by the Regional Committee for Medical and Health Research Ethics and by the Norwegian Data Protection Authority, and all study participants gave consent to take part in the study.  Estimates represent the cohort-specifc confounder-adjusted mean differences in SD units [and 95% confidence intervals] in waist circumference at each age group between ART-conceived and NC offspring (ART minus NC). Estimates were adjusted (as fully as possible) for maternal age, parity, BMI, smoking, education, ethnicity (or country of birth), plus offspring sex and age at outcome assessment. Cohorts are arranged by the offspring's mean age at outcome assessment. Blue diamonds represent the pooled mean differences from random-effects meta-analyses. NC is the number of NC ofspring; ART is the number of ART-conceived offspring; I² represents the percentage of total variability due to between cohort heterogeneity. eFigure 6. Cohort-Specific Mean Differences in Body Fat Percentage Between Offspring Conceived via ART and Those Who Were NC Estimates represent the cohort-specifc confounder-adjusted mean differences in SD units [and 95% confidence intervals] in body fat % at each age group between ART-conceived and NC offspring (ART minus NC). Estimates were adjusted (as fully as possible) for maternal age, parity, BMI, smoking, education, ethnicity (or country of birth), plus offspring sex and age at outcome assessment. Cohorts are arranged by the offspring's mean age at outcome assessment. Blue diamonds represent the pooled mean differences from random-effects meta-analyses. NC is the number of NC ofspring; ART is the number of ART-conceived offspring; I² represents the percentage of total variability due to between cohort heterogeneity.

eFigure 7. Cohort-Specific Mean Differences in Fat Mass Index Between Offspring Conceived via ART and Those Who Were NC
Estimates represent the cohort-specifc confounder-adjusted mean differences in SD units [and 95% confidence intervals] in fat mass index at each age group between ART-conceived and NC offspring (ART minus NC). Estimates were adjusted (as fully as possible) for maternal age, parity, BMI, smoking, education, ethnicity (or country of birth), plus offspring sex and age at outcome assessment. Cohorts are arranged by the offspring's mean age at outcome assessment. Blue diamonds represent the pooled mean differences from random-effects meta-analyses. NC is the number of NC ofspring; ART is the number of ART-conceived offspring; I² represents the percentage of total variability due to between cohort heterogeneity. Estimates represent the confounder-adjusted pooled mean differences in SD units [and 95% confidence intervals] in length/height, weight, and body mass index at ages <3 months and 3-5 months between ART-conceived and NC offspring (ART minus NC), across all studies (bottom rows) and after refitting the meta-analysis models with each cohort study omitted in turn. Estimates were adjusted (as fully as possible) for maternal age, parity, BMI, smoking, education, ethnicity (or country of birth), plus offspring sex and age at outcome assessment. Cohorts arranged by offspring's mean age at outcome assessment. Estimates represent the confounder-adjusted pooled mean differences in SD units and 95% confidence intervals in length/height, weight, and body mass index at each age group between ART-conceived and NC offspring (ART minus NC), separately for fresh embryo transfer and frozen-thawed embryo transfer, before and after further adjustment for the potential mediators birthweight and gestational age. The confounder-adjusted estimates were adjusted (as fully as possible) for maternal age, parity, BMI, smoking, education, ethnicity (or country of birth), plus offspring sex and age at outcome assessment.