Methylation differences between assisted reproductive technology-conceived and naturally conceived children near BRCA1 and NBR2

Previous studies have shown cord-blood DNA methylation differences in newborns conceived using assisted reproductive technologies (ART) compared to those conceived naturally. However, whether these ART-related DNA methylation differences vary with children’s sex is unknown. We hypothesize that the DNA methylation differences in cord blood between ART-conceived and naturally conceived newborns also varies by the sex of the child, with distinct patterns of differential methylation present in males and females. We investigated sex differences in cord-blood DNA methylation variation according to conception by ART using the Illumina MethylationEPIC platform, comparing 456 ART-conceived versus 507 naturally-conceived girls, and 503 ART-conceived and 473 naturally-conceived boys. We identified 37 differentially methylated CpGs according to ART-conception among girls, and 70 differentially methylated CpGs according to ART-conception among boys, when we used a 1% false discovery rate to account for multiple testing. Ten CpGs were differentially methylated according to conception by ART in both sexes. Among the genes that were associated with these CpGs, we found the BRCA1; NBR2 gene (two CpGs) was hypermethylated in girls while the APC2 (two CpGs) and NECAB3;ACTL10, (four CpGs) related to cellular signaling were hypomethylated in boys. These findings confirm the presence of sex-specific epigenetic differences, illustrating the nuanced impact of ART on the fetal epigenome. There is a need for further explorations into the implications for sex-specific developmental trajectories and health outcomes in ART-conceived children.

STUDY QUESTIONIs cord blood DNA methylation associated with having been conceived by medically assisted reproduction?SUMMARY ANSWERThis study does not provide strong evidence of an association of conception by medically assisted reproduction with variation in infant blood cell DNA methylation.WHAT IS KNOWN ALREADYMedically assisted reproduction consists of procedures used to help infertile/subfertile couples conceive, including ART. Due to its importance in gene regulation during early development programming, DNA methylation and its perturbations associated with medically assisted reproduction could reveal new insights into the biological effects of assisted reproductive technologies and potential adverse offspring outcomes.STUDY DESIGN, SIZE, DURATIONWe investigated the association of DNA methylation and medically assisted reproduction using a case–control study design (N = 205 medically assisted reproduction cases and N = 2439 naturally conceived controls in discovery cohorts; N = 149 ART cases and N = 58 non-ART controls in replication cohort).PARTICIPANTS/MATERIALS, SETTINGS, METHODSWe assessed the association between medically assisted reproduction and DNA methylation at birth in cord blood (205 medically assisted conceptions and 2439 naturally conceived controls) at >450 000 CpG sites across the genome in two sub-samples of the UK Avon Longitudinal Study of Parents and Children (ALSPAC) and two sub-samples of the Norwegian Mother, Father and Child Cohort Study (MoBa) by meta-analysis. We explored replication of findings in the Australian Clinical review of the Health of adults conceived following Assisted Reproductive Technologies (CHART) study (N = 149 ART conceptions and N = 58 controls).MAIN RESULTS AND THE ROLE OF CHANCEThe ALSPAC and MoBa meta-analysis revealed evidence of association between conception by medically assisted reproduction and DNA methylation (false-discovery-rate-corrected P-value < 0.05) at five CpG sites which are annotated to two genes (percentage difference in methylation per CpG, cg24051276: Beta = 0.23 (95% CI 0.15,0.31); cg00012522: Beta = 0.47 (95% CI 0.31, 0.63); cg17855264: Beta = 0.31 (95% CI 0.20, 0.43); cg17132421: Beta = 0.30 (95% CI 0.18, 0.42); cg18529845: Beta = 0.41 (95% CI 0.25, 0.57)). Methylation at three of these sites has been previously linked to cancer, aging, HIV infection and neurological diseases. None of these associations replicated in the CHART cohort. There was evidence of a functional role of medically assisted reproduction-induced hypermethylation at CpG sites located within regulatory regions as shown by putative transcription factor binding and chromatin remodelling.LIMITATIONS, REASONS FOR CAUTIONSWhile insufficient power is likely, heterogeneity in types of medically assisted reproduction procedures and between populations may also contribute. Larger studies might identify replicable variation in DNA methylation at birth due to medically assisted reproduction.WIDER IMPLICATIONS OF THE FINDINGSNewborns conceived with medically assisted procedures present with divergent DNA methylation in cord blood white cells. If these associations are true and causal, they might have long-term consequences for offspring health.STUDY FUNDING/COMPETING INTERESTS(S)This study has been supported by the US National Institute of Health (R01 DK10324), the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement no. 669545, European Union’s Horizon 2020 research and innovation programme under Grant agreement no. 733206 (LifeCycle) and the NIHR Biomedical Centre at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The UK Medical Research Council and Wellcome (Grant ref: 102215/2/13/2) and the University of Bristol provide core support for ALSPAC. Methylation data in the ALSPAC cohort were generated as part of the UK BBSRC funded (BB/I025751/1 and BB/I025263/1) Accessible Resource for Integrated Epigenomic Studies (ARIES, http://www.ariesepigenomics.org.uk). D.C., J.J., C.L.R. D.A.L and H.R.E. work in a Unit that is supported by the University of Bristol and the UK Medical Research Council (Grant nos. MC_UU_00011/1, MC_UU_00011/5 and MC_UU_00011/6). B.N. is supported by an NHMRC (Australia) Investigator Grant (1173314). ALSPAC GWAS data were generated by Sample Logistics and Genotyping Facilities at Wellcome Sanger Institute and LabCorp (Laboratory Corporation of America) using support from 23andMe. The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (Contract no. N01-ES-75558), NIH/NINDS (Grant nos. (i) UO1 NS 047537-01 and (ii) UO1 NS 047537-06A1). For this work, MoBa 1 and 2 were supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES-49019) and the Norwegian Research Council/BIOBANK (Grant no. 221097). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, Project no. 262700.D.A.L. has received support from national and international government and charity funders, as well as from Roche Diagnostics and Medtronic for research unrelated to this study. The other authors declare no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

BackgroundTelomere length (TL) has been reported to be associated with conditions such as endometriosis and polycystic ovary syndrome, with some studies finding associations with shorter TL and others with longer TL. In men, studies mostly report associations between shorter TL and sperm quality. To our knowledge, no studies have thus far investigated associations between TL and fecundability or the use of assisted reproductive technologies (ART).MethodsThis study is based on the Norwegian Mother, Father, and Child Cohort (MoBa) Study and uses data from the Medical Birth Registry of Norway (MBRN). We included women (24,645 with genotype data and 1054 with TL measurements) and men (18,339 with genotype data and 965 with TL measurements) participating between 1998 and 2008. We investigated associations between leukocyte TL (LTL) and fecundability (defined as the probability to conceive within a given menstrual cycle), infertility (defined has having spent 12 months or more trying to conceive without success), and ART use. We also repeated the analyses using instrumental variables for LTL consisting of genetic risk scores for LTL and genetically predicted LTL.ResultsApproximately 11% of couples had experienced infertility and 4% had used ART. LTL was not associated with fecundability in women (fecundability ratio [FR], 0.98; 95% confidence interval [CI], 0.92–1.04) or men (FR, 0.99; CI, 0.93–1.06), nor with infertility in women (odds ratio [OR], 1.03; CI, 0.85–1.24) or men (OR, 1.05; CI, 0.87–1.28). We observed an increased likelihood of using ART with increasing LTL in men (OR, 1.22; CI, 1.03–1.46), but not in women (OR, 1.10; CI, 0.92–1.31). No significant associations were observed using the instrumental variables for LTL.ConclusionsWe found no indication that LTL is a suitable biomarker for assessing fecundability, infertility, or ART use. Additional studies are required to replicate the association observed between LTL and ART use in men.

BackgroundAssisted reproductive technologies (ART) may perturb DNA methylation (DNAm) in early embryonic development. Although a handful of epigenome-wide association studies of ART have been published, none have investigated CpGs on the X chromosome. To bridge this knowledge gap, we leveraged one of the largest collections of mother–father–newborn trios of ART and non-ART (natural) conceptions to date to investigate sex-specific DNAm differences on the X chromosome. The discovery cohort consisted of 982 ART and 963 non-ART trios from the Norwegian Mother, Father, and Child Cohort Study (MoBa). To verify our results from the MoBa cohort, we used an external cohort of 149 ART and 58 non-ART neonates from the Australian ‘Clinical review of the Health of adults conceived following Assisted Reproductive Technologies’ (CHART) study. The Illumina EPIC array was used to measure DNAm in both datasets. In the MoBa cohort, we performed a set of X-chromosome-wide association studies (‘XWASs’ hereafter) to search for sex-specific DNAm differences between ART and non-ART newborns. We tested several models to investigate the influence of various confounders, including parental DNAm. We also searched for differentially methylated regions (DMRs) and regions of co-methylation flanking the most significant CpGs. Additionally, we ran an analogous model to our main model on the external CHART dataset.ResultsIn the MoBa cohort, we found more differentially methylated CpGs and DMRs in girls than boys. Most of the associations persisted after controlling for parental DNAm and other confounders. Many of the significant CpGs and DMRs were in gene-promoter regions, and several of the genes linked to these CpGs are expressed in tissues relevant for both ART and sex (testis, placenta, and fallopian tube). We found no support for parental DNAm-dependent features as an explanation for the observed associations in the newborns. The most significant CpG in the boys-only analysis was in UBE2DNL, which is expressed in testes but with unknown function. The most significant CpGs in the girls-only analysis were in EIF2S3 and AMOT. These three loci also displayed differential DNAm in the CHART cohort.ConclusionsGenes that co-localized with the significant CpGs and DMRs associated with ART are implicated in several key biological processes (e.g., neurodevelopment) and disorders (e.g., intellectual disability and autism). These connections are particularly compelling in light of previous findings indicating that neurodevelopmental outcomes differ in ART-conceived children compared to those naturally conceived.

BackgroundChildren born after assisted reproductive technologies (ART) differ in birthweight from those naturally conceived. It has been hypothesized that this might be explained by epigenetic mechanisms. We examined whether cord blood DNA methylation mediated the birthweight difference between 890 newborns conceived by ART (764 by fresh embryo transfer and 126 frozen thawed embryo transfer) and 983 naturally conceived newborns from the Norwegian Mother, Father, and Child Cohort Study (MoBa). DNA methylation was measured by the Illumina Infinium MethylationEPIC array. We conducted mediation analyses to assess whether differentially methylated CpGs mediated the differences in birthweight observed between: (1) fresh embryo transfer and natural conception and (2) frozen and fresh embryo transfer.ResultsWe observed a difference in birthweight between fresh embryo transfer and naturally conceived offspring of − 120 g. 44% (95% confidence interval [CI] 26% to 81%) of this difference in birthweight between fresh embryo transfer and naturally conceived offspring was explained by differences in methylation levels at four CpGs near LOXL1, CDH20, and DRC1. DNA methylation differences at two CpGs near PTGS1 and RASGRP4 jointly mediated 22% (95% CI 8.1% to 50.3%) of the birthweight differences between fresh and frozen embryo transfer.ConclusionOur findings suggest that DNA methylation is an important mechanism in explaining birthweight differences according to the mode of conception. Further research should examine how gene regulation at these loci influences fetal growth.

Background:The identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in children’s blood, using data from 832 children of the Human Early Life Exposome (HELIX) project.Methods:Blood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition.Results:We identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at <250 kb from eGene’s TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. In 40.4% of the eQTMs, the CpG and the eGene were both associated with at least one genetic variant. The overlap of autosomal cis eQTMs in children’s blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants.Conclusions:This catalogue of autosomal cis eQTMs in children’s blood can help the biological interpretation of EWAS findings and is publicly available at https://helixomics.isglobal.org/ and at Dryad (doi:10.5061/dryad.fxpnvx0t0).Funding:The study has received funding from the European Community’s Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative “A Healthy Diet for a Healthy Life” (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the projects PI17/01225 and PI17/01935, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, “A way to make Europe”) and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011-2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128).

Background: Maternal smoking is related to multiple adverse health outcomes in children. We previously reported an association between maternal smoking during pregnancy and differential DNA methylation in newborn cord bloods for CpG sites (CpGs) in genes involved in the aryl hydrocarbon receptor signaling pathway and key developmental processes. However, it remains unclear whether these modifications to the infant epigenome reflect in utero exposures only or the inheritance of epigenetic marks from the mother. Aims: We evaluated the association between maternal smoking before or during pregnancy and DNA methylation for 21 CpGs previously identified as differentially methylated in response to maternal smoking in 1,047 newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). Methods: Maternal self-report of smoking as well as maternal plasma cotinine measured during pregnancy were used to distinguish mothers into four groups (never smokers, former smokers, smokers who smoked during pregnancy but quit by 18 weeks, and active smokers throughout pregnancy). The association between smoking and methylation was assessed using linear regression. Results: Having a mother who formerly smoked was not associated with differential methylation in cord blood for any of the 21 CpGs (at FDR of 0.05). Quitting during pregnancy was also not significantly associated with differential methylation (at FDR of 0.05) although methylation differences were larger than those for former smokers compared to never smokers. Only active smoking throughout pregnancy was statistically significantly associated with differential methylation (at FDR of 0.05). Conclusions: Differential methylation in newborn cord blood at the 21 CpGs that we previously found to be related to maternal smoking in pregnancy was only observed where mothers smoked during pregnancy. These findings suggest that DNA methylation at these locations in the infant epigenome reflects in utero exposure rather than epigenetic inheritance of smoking-related modifications in the mother.

Study question Does the gestational weight gain trajectory differ across levels of time-to-pregnancy (TTP) for spontaneously conceived pregnancies and pregnancies conceived by assisted reproductive technology (ART)? Summary answer Women with ART pregnancies had lower weekly weight gain during the third trimester compared with women with spontaneous conceptions with TTP ≤3. What is known already Studies suggest that both prolonged TTP and conception by ART may increase the risk of pregnancy complications and affect perinatal outcomes such as birthweight. Suboptimal gestational weight gain might further increase the risk of adverse pregnancy outcomes. However, it remains unclear whether there may be differences in gestational weight gain according to TTP and ART, which contribute to the subsequent risk of adverse pregnancy outcomes. Study design, size, duration We studied 69,121 singleton planned pregnancies contributed by 60,847 women participating in the Norwegian Mother, Father and Child Cohort Study. Participants completed questionnaires at 15 weeks’ gestation (study entry), 30 weeks’ gestation, and six months postpartum on sociodemographic, reproductive, and behavioral factors. Participants reported their TTP, and up to four weight measurements and corresponding gestational age from preconception through delivery. We identified use of ART in the Medical Birth Registry of Norway. Participants/materials, setting, methods We fitted gestational weight gain trajectories using mixed-effects linear regression models and included an interaction term between time (gestational weeks) and the exposure groups, TTP ≤3 (reference); TTP 4-6; TTP 7-11, TTP ≥12 months, and ART, to assess differences across levels of TTP and conception by ART. The analyses were adjusted for maternal age at start of pregnancy, pre-pregnancy height, educational attainment, pre-pregnancy smoking, parity, and gestational age at birth. Main results and the role of chance The adjusted average weekly weight gain was -15g (95% CI: −19; −12) during the first trimester, 620g (95% CI: 615; 624) during the second trimester, and 491g (95% CI: 489; 494) during the third trimester. When we compared differences in average weekly weight change during the first trimester, we observed little difference between the exposure groups. During the second trimester, compared with TTP≤3 months women with TTP 4-6 months, TTP 7-11 months or TTP ≥12 months gained on average 7g (95% CI: −18; 3), 19g (95% CI: −33; −5) and 15g (95% CI: −29; −1) less per week, respectively, whereas women with ART pregnancies gained 11g more per week (95% CI: −13; 34). However, the results for TTP 4-6 and ART pregnancies were imprecise. During the third trimester, average weekly weight gain did not differ according to TTP for spontaneous conceptions, while women who conceived after ART gained 35g (95% CI: −49; −22) less than women with TTP≤3 each week. Limitations, reasons for caution Because TTP and gestational weight gain were self-reported, non-differential misclassification may have influenced our results. Wider implications of the findings Decelerated weight gain during the third trimester in ART pregnancies compared with women who conceived spontaneously within 3 months might be associated with adverse pregnancy outcomes. Although our findings are imprecise and need to be replicated, monitoring gestational weight gain trajectories might support identification of pregnancies at increased risk. Trial registration number Not applicable

After completing this article, readers should be able to: 1. Describe the outcomes of assisted reproductive technologies (ART) for singleton, twin, and other multiple births. 2. Describe the role of fertility in adverse outcomes seen with ART births. 3. Review the association of birth defects with ART. 4. Delineate the association of disease of genomic imprinting with ART. 5. Describe the relationship between ART and the subsequent incidence of neurodevelopmental sequelae. In the 1977 ruling “Carey v. Population Services International,” the United States Supreme Court ruled that the decision to bear children is constitutionally protected. (1) Significant interest already had been shown in the development and improvements of in vitro fertilization (IVF) for infertile couples. The first human pregnancy and human birth using IVF were reported by Steptoe and Edwards in the United Kingdom. (2) Their work resulted in the first baby born via reproductive technologies, Louise Brown, born on July 25, 1978, at Oldham General Hospital in Oldham, United Kingdom. (3) She was born via a planned cesarean section, and her birthweight was 2.61 kg. The first successful viable IVF in the United States was performed by Jones and Seager-Jones in 1981 in Norfolk, Virginia. (4) Assisted reproductive technologies (ART) have seen a recent surge in popularity. The Centers for Disease Control and Prevention (CDC) reported that 122,872 cycles of ART were initiated in 2003, resulting in the delivery of 48,756 neonates, (5) accounting for approximately 1% of all neonates delivered in the United States. The percentage is higher in many countries, including Denmark, where it is estimated that 5% of all deliveries are with the assistance of ART. (6) Couples pursue ART for myriad reasons, including tubal transport factors, ovulatory dysfunction, uterine factors, endometriosis, male- and female-specific factors, and when a cause of infertility is unknown. (5) It would be very …

BackgroundImpaired neurodevelopment is reported among children conceived by assisted reproductive technologies (ART). However, this might be explained by conditions underlying parental subfecundity, rather than the ART procedure.MethodsWe examined associations of parental time-to-pregnancy (TTP) and conception by ART with neurodevelopmental traits up to 8 years of age, including motor and language skills, social delays and difficulties, and inattention-hyperactivity, among 92 142 singletons participating in the Norwegian Mother, Father and Child Cohort Study (MoBa). Mothers reported TTP and neurodevelopmental traits through questionnaires. Mean differences in standardized neurodevelopmental traits were estimated using linear regression, adjusting for maternal age, parity, educational level, body mass index and smoking, and paternal age.ResultsA longer TTP was associated with decreased language skills and motor skills at 6, 18 and 36 months (P-values for trend ≤0.01), prosocial skills delay at 36 months (P-values for trend ≤0.001) and increased scores for inattention-hyperactivity traits at all ages up to 8 years (P-values for trend from 0.06 to 0.01). Effect sizes were small, ranging between 0.03 and 0.05 difference in the standardized neurodevelopmental scores. Estimates for ART were imprecise, but there were no differences between children conceived by ART and naturally conceived children of subfecund parents (TTP ≥12 months).ConclusionsLonger parental TTP is modestly but robustly associated with offspring neurodevelopmental delays and difficulties, with no added impact of ART. Future studies should investigate the underlying causes of—or aspects related to—parental subfecundity which might explain the association with offspring neurodevelopmental delays and difficulties.

ABSTRACTGenetic selection occurs at different stages before a successful birth. The genetic makeup of a couple may influence the likelihood of needing assisted reproductive technology (ART) to achieve conception. However, frequent early fetal losses may also be perceived as reduced couple fertility and may thus be a contributing factor to the need for ART treatment. As ART procedures may enhance early fetal survival, genes that impact fetal viability may have a different allele distribution in ART offspring than expected under Mendelian transmission, as well as compared with the general population. With genetic data available from the Norwegian Mother, Father, and Child Cohort Study, we defined fetal survival as the study outcome and analyzed 1336 case‐parent triads and dyads where the offspring were conceived by ART. Using log‐linear models implemented in the R package Haplin, we conducted genome‐wide scans to estimate fetal, maternal, and parent‐of‐origin effects and provided a detailed discussion on how these effects are estimated and interpreted. We detected fetal effects for single‐nucleotide polymorphisms (SNPs) located in CXXC4‐AS1, OPCML, and DYNLRB2‐AS1. Since these effects were not observed in a limited follow‐up analysis of non‐ART triads, the identified effects are unlikely caused by genetic selection before fertilization.

It is not known if children conceived through assisted reproductive technology (ART) might harbor subtle epigenetic disruptions which could lead to a predisposition to cancer either in childhood or adulthood. Evidence of an association between parental infertility treatment and childhood cancer has been inconsistent. However, a positive association between assisted reproductive technology (ART) and Beckwith-Wiedemann syndrome (BWS) has been seen in many studies. Disruptions of epigenetic mechanisms may be responsible for both the increase in imprinting disorders such as BWS and the suspected increased risk in childhood cancer. This clinical study examined potential differences in DNA methylation at genomic sites associated with BWS and certain cancers in non-syndromic children conceived either spontaneously or through ART. Mothers who conceived through ART and reported a live birth between March 2005 and December 2008 were recruited from the University of Minnesota Reproductive Medicine Center. Spontaneously conceived (SC) children born between March 2005 and December 2008 and their mothers were recruited through advertisement. Information about reproductive history, demographic factors, birth characteristics, and infertility treatment was obtained from maternal interview and medical records. Peripheral blood lymphocytes and buccal cell samples were collected from participating children. Methylation analysis was performed using pyrosequencing. Five specific gene regions were included (IGF2 DMR0, 3rd and 6th CTCF-binding sites of H19 DMR, IGF2R, and KvDMR). Statistical analysis of methylation differences was performed using linear regression with generalized estimating equations. Correlation between lymphocyte and buccal cell samples was assessed using Pearson's correlation coefficient. A total of 67 ART children and 31 SC children contributed samples. No statistically significant difference between groups was found at any loci examined in lymphocytes. However, possible differences were found in buccal cell samples for IGF2 DMR0 (Difference: 2.33; 95% CI: -0.08, 4.82; p = 0.06) and IGF2R (Difference: -2.83; 95% CI: -5.88, 0.22; p = 0.07). Subgroup analysis indicated potentially lower methylation among children whose parents had unexplained infertility. Notably, correlation between lymphocyte and buccal cell samples was very low for all loci. Observed differences in methylation between the ART and SC groups were very small in the two tissues examined so it is unlikely that important differences were missed. Although no significant differences were found in this study, possible changes in methylation patterns could still exist at other loci and in other tissues. Additional clinical and epidemiological studies are needed to more fully examine the possible association between ART and childhood cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2011-1897

STUDY QUESTIONWhat is the association between BMI and subfertility?SUMMARY ANSWERWe observed a J-shaped relationship between BMI and subfertility in both sexes, when using both a standard multivariable regression and Mendelian randomization (MR) analysis.WHAT IS KNOWN ALREADYHigh BMI in both women and men is associated with subfertility in observational studies and this relationship is further substantiated by a few small randomized controlled trials of weight reduction and success of assisted reproduction. Women with low BMI also have lower conception rates with assisted reproduction technologies.STUDY DESIGN, SIZE, DURATIONCohort study (the Norwegian Mother, Father and Child Cohort Study), 28 341 women and 26 252 men, recruited from all over Norway between 1999 and 2008.PARTICIPANTS/MATERIALS, SETTING, METHODSWomen (average age 30, average BMI 23.1 kg/m2) and men (average age 33, average BMI 25.5 kg/m2) had available genotype data and provided self-reported information on time-to-pregnancy and BMI. A total of 10% of couples were subfertile (time-to-pregnancy ≥12 months).MAIN RESULTS AND THE ROLE OF CHANCEOur findings support a J-shaped association between BMI and subfertility in both sexes using multivariable logistic regression models. Non-linear MR validated this relationship. A 1 kg/m2 greater genetically predicted BMI was linked to 18% greater odds of subfertility (95% CI 5% to 31%) in obese women (≥30.0 kg/m2) and 15% lower odds of subfertility (−24% to −2%) in women with BMI <20.0 kg/m2. A 1 kg/m2 higher genetically predicted BMI was linked to 26% greater odds of subfertility (8–48%) among obese men. Low genetically predicted BMI values were also related to greater subfertility risk in men at the lower end of the BMI distribution. A genetically predicted BMI of 23 and 25 kg/m2 was linked to the lowest subfertility risk in women and men, respectively.LIMITATIONS, REASONS FOR CAUTIONThe main limitations of our study were that we did not know whether the subfertility was driven by the women, men or both; the exclusive consideration of individuals of northern European ancestry; and the limited amount of participants with obesity or BMI values <20.0 kg/m2.WIDER IMPLICATIONS OF THE FINDINGSOur results support a causal effect of obesity on subfertility in women and men. Our findings also expand the current evidence by indicating that individuals with BMI values <20 kg/m2 may have an increased risk of subfertility. These results suggest that BMI values between 20 and 25 kg/m2 are optimal for a minimal risk of subfertility.STUDY FUNDING/COMPETING INTEREST(S)The MoBa Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Norwegian Ministry of Education and Research. This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement No 947684). It was also partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 262700. Open Access funding was provided by the Folkehelseinstituttet/Norwegian Institute of Public Health. D.A.L. is a UK National Institute for Health Research Senior Investigator (NF-SI-0611-10196) and is supported by the US National Institutes of Health (R01 DK10324) and a European Research Council Advanced Grant (DevelopObese; 669545). The funders had no role in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. D.A.L. receives (or has received in the last 10 years) research support from National and International government and charitable bodies, Roche Diagnostics and Medtronic for research unrelated to the current work. The rest of the authors declare that no competing interests exist.TRIAL REGISTRATION NUMBERN/A.

BackgroundGestational age is a useful proxy for assessing developmental maturity, but correct estimation of gestational age is difficult using clinical measures. DNA methylation at birth has proven to be an accurate predictor of gestational age. Previous predictors of epigenetic gestational age were based on DNA methylation data from the Illumina HumanMethylation 27 K or 450 K array, which have subsequently been replaced by the Illumina MethylationEPIC 850 K array (EPIC). Our aims here were to build an epigenetic gestational age clock specific for the EPIC array and to evaluate its precision and accuracy using the embryo transfer date of newborns from the largest EPIC-derived dataset to date on assisted reproductive technologies (ART).MethodsWe built an epigenetic gestational age clock using Lasso regression trained on 755 randomly selected non-ART newborns from the Norwegian Study of Assisted Reproductive Technologies (START)—a substudy of the Norwegian Mother, Father, and Child Cohort Study (MoBa). For the ART-conceived newborns, the START dataset had detailed information on the embryo transfer date and the specific ART procedure used for conception. The predicted gestational age was compared to clinically estimated gestational age in 200 non-ART and 838 ART newborns using MM-type robust regression. The performance of the clock was compared to previously published gestational age clocks in an independent replication sample of 148 newborns from the Prediction and Prevention of Preeclampsia and Intrauterine Growth Restrictions (PREDO) study—a prospective pregnancy cohort of Finnish women.ResultsOur new epigenetic gestational age clock showed higher precision and accuracy in predicting gestational age than previous gestational age clocks (R2 = 0.724, median absolute deviation (MAD) = 3.14 days). Restricting the analysis to CpGs shared between 450 K and EPIC did not reduce the precision of the clock. Furthermore, validating the clock on ART newborns with known embryo transfer date confirmed that DNA methylation is an accurate predictor of gestational age (R2 = 0.767, MAD = 3.7 days).ConclusionsWe present the first EPIC-based predictor of gestational age and demonstrate its robustness and precision in ART and non-ART newborns. As more datasets are being generated on the EPIC platform, this clock will be valuable in studies using gestational age to assess neonatal development.

Study question Does elementary school performance differ between children conceived through assisted reproductive technology (ART) compared to children born to subfertile couples not conceived by ART? Summary answer Children born after ART perform equally well at age 12 (end of elementary school) compared to children born to subfertile couples not conceived by ART. What is known already It is well established that pregnancies conceived by ART are at higher risk of adverse outcomes, such as pre-term birth and a lower birth weight, also when restricted to singleton pregnancies. However, whether ART also affects outcomes in later life, such as school performance, is less clear. Although some studies indicate that there might be subtle differences in the school performance of ART and spontaneously conceived children, it remains unclear whether this is due to the ART treatment, parental factors or a combination of both. Study design, size, duration Data were used from the OMEGA-cohort, a historical nationwide cohort with prospective follow-up in the Netherlands. The cohort comprises all offspring of women who were treated in one of the 13 IVF clinics or 2 regional fertility centers in 1983-2010. Of 89,249 live-born children, 54,417 were ART-conceived and 37,832 were not ART-conceived (conceived naturally with or without ovarian stimulation) by subfertile women. Participants/materials, setting, methods Data on type of fertility treatment and maternal risk factors were available from medical records of the mothers and through the national perinatal registry. The OMEGA-cohort was linked to the education dataset of Statistics Netherlands, including school performance data for the period 2006-2017, leaving 24,806 children in the analytical cohort. The overall test Z-score and Z-scores on the separate domains (Dutch language and mathematics) between ART and non-ART children were compared using multivariable linear regression. Main results and the role of chance The cohort comprises 14,958 ART-conceived children and 9,848 non-ART children with test scores around age 12 in 2006-2017. The mean overall score was 536,5 (SD = 9.7), with scores of 536.4 (SD = 9.9) in ART-conceived children and 536.6 (SD = 9.5) in non-ART children. After adjustment for maternal age and maternal and paternal educational level, in a multivariable linear regression model, the overall test Z-score was also not different among ART-children compared to non-ART children from subfertile parents (β=-0.02, 95% CI=-0.04-0.01). Overall test Z-scores were not different according to different ART treatment modalities, IVF (β=-0.02, 95%CI=-0.05-0.01), ICSI (β=-0.01, 95% CI=-0.05-0.02) and frozen embryo transfer (β = 0.07, 95%CI=-0.01-0.14) when compared to children born to subfertile couples not using ART. ART children performed equally well on mathematics and Dutch language compared to children born to subfertile couples not conceived by ART. Limitations, reasons for caution Although the current analyses include a large proportion of the ART children in the Netherlands with data on school performance and our study had sufficient power to address the research question, it is not clear to which extent our results are generalizable to other countries. Wider implications of the findings Reassuringly, based on first results, children born after ART had comparable test scores at the end of elementary school compared to children from subfertile couples not conceived by ART. Trial registration number Not applicable