STUDY QUESTION Do twins conceived through assisted reproductive treatments (ART) grow differently from naturally conceived (NC) twins in early life? SUMMARY ANSWER Assessments at 6–8 weeks old and at school entry show that ART twins conceived from frozen embryo transfer (FET) grow faster than both NC twins and ART twins conceived from fresh embryo transfer (ET). WHAT IS KNOWN ALREADY Singletons born from fresh ET grow more slowly in utero and in the first few weeks of life but then show postnatal catch-up growth by school age, compared to NC and FET babies. Evidence on early child growth of ART twins relative to NC twins is inconsistent; most studies are small and do not distinguish FET from fresh ET cycles. STUDY DESIGN, SIZE, DURATION This cohort study included 13 528 live-born twin babies conceived by ART (fresh ET: 2792, FET: 556) and NC (10 180) between 1991 and 2009 in Scotland. The data were obtained by linking Human Fertilisation and Embryology Authority ART register data to the Scottish Morbidity Record (SMR02) and Scottish child health programme datasets. Outcome data were collected at birth, 6–8 weeks (first assessment), and school entry (4–7 years old) assessments. The primary outcome was growth, measured by weight at the three assessment points. Secondary outcomes were length (at birth and 6–8 weeks) or height (at school entry), BMI, occipital circumference, gestational age at birth, newborn intensive care unit admission, and growth rates (between birth and 6–8 weeks and between 6–8 weeks and school entry). PARTICIPANTS/MATERIALS, SETTING, METHODS All twins in the linked dataset (born between 1991 and 2009) with growth data were included in the analysis. To determine outcome differences between fresh ET, FET, and NC twins, linear mixed models (or analogous logistic regression models) were used to explore the outcomes of interest. All models were adjusted for available confounders: gestational age/child age, gender, maternal age and smoking, Scottish Index of Multiple Deprivation, year of treatment, parity, ICSI, and ET stage. MAIN RESULTS AND THE ROLE OF CHANCE In the primary birth weight models, the average birth weight of fresh ET twins was lower [–35 g; 95% CI: (−53, −16)g] than NC controls, while FET twins were heavier [71 g; 95% CI (33, 110) g] than NC controls and heavier [106 g; 95% CI (65, 146) g] than fresh ET twins. However, the difference between FET and NC twins was not significant when considering only full-term twins (≥37 weeks gestation) [26 g; 95% CI (–30, 82) g], while it was significantly higher in preterm twins [126 g; 95% CI (73, 179) g]. Growth rates did not differ significantly for the three groups from birth to 6–8 weeks. However, FET twins grew significantly faster from 6 to 8 weeks than NC (by 2.2 g/week) and fresh ET twins (by 2.1 g/week). By school entry, FET twins were 614 g [95% CI (158, 1070) g] and 581 g [95% CI (100, 1063) g] heavier than NC and fresh ET twins, respectively. Length/height and occipital frontal circumference did not differ significantly at any time point. LIMITATIONS, REASONS FOR CAUTION Although the differences between ART and NC reflect the true ART effects, these effects are likely to be mediated partly through the different prevalence of mono/dizygotic twins in the two groups. We could not explore the mediating effect of zygosity due to the unavailability of data. The confounding variables included in the study were limited to those available in the datasets. WIDER IMPLICATIONS OF THE FINDINGS Live-born twins from FET cycles are heavier at birth, grow faster than their fresh ET and NC counterparts, and are still heavier at school entry. This differs from that observed in singletons from the same cohort, where babies in the three conception groups had similar weights by school entry age. The results are reassuring on known differences in FET versus fresh ET and NC twin outcomes. However, FET twins grow faster and are consistently larger, and more ART twins depict catch-up growth. These may lead to an increased risk profile for non-communicable diseases in later life. As such, these twin outcomes require careful evaluation using more recent and comprehensive cohorts. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the EU H2020 Marie Sklodowska-Curie Innovative Training Networks (ITN) grant Dohartnet (H2020-MSCA-ITN-2018-812660). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A.

Study question Does embryo biopsy for preimplantation genetic testing (PGT) followed by fresh or vitrified-warmed embryo transfer affect children’s health up to 2 years of age? Summary answer Cleavage- or blastocyst-stage embryo biopsy followed by fresh or vitrified-warmed-embryo transfer had neither impact on anthropometry at birth, infancy or childhood nor on health outcomes. What is known already Literature data regarding neonatal outcomes after embryo biopsy for PGT show mixed results due to small sample sizes and/or the heterogeneity in terms of embryo biopsy (cleavage- or blastocyst-stage) and type of embryo transfer (fresh or frozen-thawed). Even fewer data exist on the impact of embryo biopsy on children’s health beyond infancy, including growth. Study design, size, duration This single-center cohort-study compared outcomes in singletons conceived after cleavage- or blastocyst-stage embryo biopsy either in a fresh or vitrified-warmed transfer cycle with results after non-biopsied embryo transfer between 2014 and 2018. Pregnancies after IVM, oocyte vitrification or oocyte/embryo donation were excluded. Eligible singletons living in Belgium were invited for examination at 3-6 months and 2 years. Anthropometric measurements at birth, infancy and childhood and health outcomes including surgeries, medication use and hospitalisations are reported. Participants/materials, setting, methods Birth characteristics were available for 630 and 222 children after cleavage-stage and blastocyst-stage embryo biopsy and for 1532 children after transfer of a non-biopsied embryo. Follow-up data were available for 426, 131 and 662 children, respectively. The impact on children’s health following embryo biopsy in vitrification and fresh cycles was the primary outcome. Other outcomes were the impact of timing of biopsy and of vitrification. Subgroup analysis according to infertility background was additionally performed. Main results and the role of chance Regarding the impact of embryo biopsy, either at the cleavage or blastocyst stage, no differences in anthropometrics were found at birth, infancy or childhood in vitrified-warmed transfer cycles compared to outcomes in non-biopsied vitrified-warmed transfer cycles, even after adjustment for neonatal, treatment and maternal characteristics. Likewise, no impact of embryo biopsy (cleavage stage) was found in fresh transfer cycles. The timing (day 3 or day 5/6) of embryo biopsy did not affect anthropometrics at birth, infancy or childhood. However, children born after cleavage-stage embryo biopsy followed by a vitrified-warmed transfer cycle had larger birth sizes than children born after cleavage-stage biopsy followed by a fresh transfer. Weight and height gain from birth to infancy and from infancy to early childhood were comparable in all biopsied and non-biopsied groups. Reassuringly, comparable rates of major congenital malformations, (severe) developmental problems, hospital admissions, surgical interventions and of chronic medication use up to the age of 2 years were observed in biopsied and non-biopsied groups. Subgroup analysis in children born to parents with an infertility diagnosis showed that birth parameters were not different in children born after embryo biopsy. Limitations, reasons for caution As the majority of the PGT cycles in our center are performed for a genetic indication only, the number of children born after embryo biopsy in infertile parents is rather limited and the results should therefore be interpreted cautiously. Wider implications of the findings Our findings add reassurance that embryo biopsy does not adversely affect the health of offspring up to 2 years of age. This is of particular importance given the widespread shift to blastocyst-stage embryo biopsy in PGT. Trial registration number Not applicable