Importance:The VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal defects, and limb defects) pattern of birth defects is a complex congenital condition with largely unknown causes. Studies suggest a link between in vitro fertilization (IVF) and birth defects, including VACTERL, but it remains unknown whether IVF has a unique association with VACTERL. Objective:To characterize the association between IVF and VACTERL. Design, Setting, and Participants:This cohort study included 1 555 936 live births in 4 US States (Massachusetts, New York, North Carolina, and Texas) between January 1, 2004, and December 31, 2018. A total of 175 161 births were IVF conceived, and 1 380 775 naturally conceived children were selected as a 10:1 comparison group. Births were linked to IVF cycle data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System. Data were analyzed from August 2 to October 30, 2025. Exposures:Method of conception (IVF vs natural) and IVF treatment parameter information. Main Outcomes and Measures:VACTERL, defined as 3 or more of the defects included in the pattern. Multivariable logistic regression models were used to estimate adjusted odds ratios and 95% CIs. Results:A total of 1 555 936 live births were included in the analysis (796 892 males [51.2%]). The naturally conceived group included 1 380 775 children; the IVF-conceived group, 175 161. Approximately half of children were male in both groups, while IVF-conceived children were more often born to older mothers and those with a higher educational level and were from multiple-gestation pregnancies. Two hundred children with VACTERL (41 IVF and 159 non-IVF) were identified. The prevalence of VACTERL among IVF-conceived births was 2.3 (95% CI, 1.7-3.2) per 10 000 live births. After adjusting for demographic, socioeconomic, and pregnancy-related factors, IVF conception was associated with increased odds of VACTERL (adjusted odds ratio, 1.86; 95% CI, 1.16-2.98). Conclusions and Relevance:This population-based prospective cohort study identified an association of IVF with the multiple congenital anomaly pattern VACTERL. Although the absolute risk among live births remains low, these findings highlight the need for further investigations of child health outcomes by method of conception.

Objective:To assess neonatal outcomes in siblings born after frozen embryo transfer (FET) in comparison with siblings born after fresh embryo transfer. Design:Prospective cohort study. Linear mixed models and generalized linear mixed models were used to study the association between outcomes after FET vs. fresh embryo transfer, considering the correlation of observations from siblings and controlling for treatment and maternal characteristics. Subgroup analysis according to the day of fresh embryo transfer and according to the FET protocol was also performed. Subjects:Sibling pairs of singletons born to mothers who had both fresh and vitrified embryos transferred between 2008 and 2021, irrespective of the order (fresh-frozen or frozen-fresh) from a single center were included. Sibling pairs born after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) and after embryo biopsy were considered. Exposure:Women who had singleton deliveries after both fresh (cleavage stage or blastocyst stage) and vitrified (cleavage stage or blastocyst stage) embryo transfers. Main outcome measures:Measurements at birth, as well as neonatal outcomes including prematurity, small-for-gestational age, large-for-gestational age (LGA), perinatal death and congenital malformations. Results:Data were available for 1,185 sibling pairs born after IVF/ICSI and for 155 sibling pairs born after embryo biopsy. Higher mean birthweight standard deviation scores were found in FET siblings compared with siblings born after fresh embryo transfer, even after adjustment for treatment and maternal characteristics. This finding was confirmed in the subgroup analysis according to the day of fresh embryo transfer and according to the FET protocol. Frozen embryo transfer was associated with a lower risk of prematurity and a higher risk of LGA after adjustment for treatment and maternal characteristics. The rates of perinatal deaths and congenital malformations were comparable between siblings born after FET and fresh embryo transfer. Like the outcomes for IVF/ICSI sibling pairs, higher LGA rates and higher mean birthweight standard deviation scores were found for FET siblings born after embryo biopsy when compared with siblings born after fresh embryo transfer. Conclusion:Our results in siblings born after IVF/ICSI and after embryo biopsy, indicated that FET is associated with higher birthweight, even after controlling for confounders.

(Abstracted from Nat Commun 2024;15:1232 Assisted reproductive technology (ART) has enabled many couples before considered untreatably infertile to have children, though there have been concerns that children born using these technologies are at risk for adverse outcomes. These adverse outcomes vary, and many of them stem from unknown causes.

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

Breast cancer-related lymphedema(BCRL)is a debilitating disorder affecting an estimated 1 in 5 women and men treated for breast cancer.Fortunately,super microsurgical techniques have advanced in recent years and now provide better options for the treatment of lymphedema,allowing timely surgical intervention that can delay or even prevent lymphatic degeneration.Lymphovenous anastomosis(LVA),a physiologic procedure that restores lymphatic drainage by connecting functioning lymphatic vessels with nearby veins,has been shown to be both minimally invasive and highly effective.The authors describe innovative approaches to LVA that will help optimize outcomes for patients with BCRL.

Abstract Study question Do children born in oocyte donation families perceive their relationships with their parents differently than children conceived trough ICSI? Summary answer No significant differences were found in the family relationships between children born in oocyte donation families (OACC) compared with controls (ICSI). What is known already Majority of the previous research concerning relationships in families using oocyte donation focused on the mothers’ perspective. Little is known about how children in these families view their family relationships. Studies investigating young children’s perspectives in donor-families (5-10 years old) indicate good parent-child relationships (Imrie, et al., 2021; Blake, et al., 2013; Casey, et al., 2013). Few of these studies involve young children conceived using anonymous oocyte donation. Study design, size, duration This study included 17 children born through anonymous oocyte donation in heterosexual parents and a comparison group of 13 children conceived ICSI using the heterosexual parents’ own gametes. Data were collected between August 2021 and January 2023. The sample is part of a larger ongoing case-control-study investigating family relationships and the wellbeing of both parents and their children in families created by anonymous oocyte donation. Participants/materials, setting, methods Children were 5 to 7 years old (M = 6.13; SD = 0.5) and had been born after assisted reproduction (ICSI with or without using anonymous oocytes) with their two heterosexual parents. All children were invited to the hospital to undergo biomedical and psychological testing, including the Family Relationship Test (FRT; English version: Bene and Anthony, 1985; Dutch version: Baarda and van Londen, 1985). Multiple blind evaluators were used. Main results and the role of chance No significant differences were found between children born in oocyte donation families (OACC) compared with controls (ICSI). Two-sampled-T-tests show no significant differences for (1) positive feelings (towards and received by children) to their mothers (OACC: M = 6.65, SD = 4; ICSI:M=8.85, SD = 4), fathers (OACC: M = 5.06, SD = 0; ICSI: M = 5.08, SD = 0) or themselves (OACC: M = 0.35, SD = 0.5; ICSI: M = 0.77, SD = 0), (2) negative feelings (towards and received by children) to their mothers (OACC: M = 1.47, SD = 0.5; ICSI: M = 1.92, SD = 4), fathers (OACC: M = 2.06, SD = 0; ICSI: M = 2.31, SD = 0.5) or themselves (OACC: M = 0.76, SD = 0; ICSI: M = 0.38, SD = 0) and (3) dependency to their mothers (OACC: M = 4.29, SD = 3; ICSI: M = 4.54, SD = 0), fathers (OACC: M = 2.82, SD = 1; ICSI: M = 2.69, SD = 2) or themselves (OACC: M = 0.53, SD = 0.5; ICSI: M = 0.85, SD = 0.5). Limitations, reasons for caution This study used multiple evaluators, which may induce a potential evaluators bias. The small sample size limits the validity of our findings to a whole population. As the study is still ongoing, the data to be presented will expand. Wider implications of the findings The findings are relevant to clinics offering anonymous oocyte donation, to (future) parents who used or who considerate using anonymous oocyte donation to create a family. Trial registration number not applicable

Breast cancer-related lymphedema (BCRL) is a debilitating disorder affecting an estimated 1 in 5 women and men treated for breast cancer. Fortunately, super microsurgical techniques have advanced in recent years and now provide better options for the treatment of lymphedema, allowing timely surgical intervention that can delay or even prevent lymphatic degeneration. Lymphovenous anastomosis (LVA), a physiologic procedure that restores lymphatic drainage by connecting functioning lymphatic vessels with nearby veins, has been shown to be both minimally invasive and highly effective. The authors describe innovative approaches to LVA that will help optimize outcomes for patients with BCRL.

OBJECTIVE:To assess health outcomes, including growth up to 2 years of age, in children born after embryo vitrification in comparison with children born after fresh embryo transfer. DESIGN:A prospective cohort study. SETTING:A single-center university hospital. PATIENT(S):Singletons born after the transfer of vitrified or fresh embryos, either at the cleavage or blastocyst stage between 2014 and 2018, were included. INTERVENTION(S):Multiple linear and logistic regression analyses were used to study the association between outcomes after vitrified versus fresh embryo transfer, controlling for neonatal, treatment, and maternal characteristics. Subgroup analysis according to cycle protocol (hormone replacement vs. natural cycle) and strategy (freeze-all vs. previous fresh cycle) was also performed. MAIN OUTCOME MEASURE(S):Measurements at birth and growth in infancy and childhood, as well as health outcomes, including congenital malformations, interventions, medication use, and hospitalizations are reported. RESULT(S):Birth characteristics were available for 1237 and 2063 children born after embryo vitrification and fresh embryo transfer, respectively. Follow-up data were available for 582 and 757 children at infancy and for 233 and 296 children at 2 years, respectively. Birthweight, height, and head circumference SD scores of children born after embryo vitrification were higher than children born after fresh embryo transfer, even after adjustment for neonatal, treatment, and maternal characteristics. In infancy, weight and height SD scores were larger for children born after embryo vitrification, but not after adjustment for covariates. In childhood, no differences in anthropometry were observed between the groups. Weight and height gain from birth to infancy and from infancy to early childhood were comparable between the groups. Comparable rates of severe developmental problems, hospital admissions, surgical interventions, and of chronic medication use were observed up to the age of 2 years. Subgroup analysis showed that growth parameters were not affected by the cycle protocol or strategy at any age. CONCLUSION(S):Our study indicated that embryo vitrification is associated with higher birthweight, even after controlling for confounders. However, in early childhood, anthropometry and weight and height gain was not different in children born after vitrified or fresh embryo transfer.

Studies show conflicting results on neonatal outcomes following embryo biopsy for PGT, primarily due to small sample sizes and/or heterogeneity in the timing of embryo biopsy (day 3; EBD3 or day 5/6; EBD5) and type of embryo transfer. Even fewer data exist on the impact on children’s health beyond the neonatal period. This study aimed to explore outcomes in children born after EBD3 or EBD5 followed by fresh (FRESH) or frozen-thawed embryo transfer (FET). This single-centre cohort study compared birth data of 630 children after EBD3, of 222 EBD5 and of 1532 after non-biopsied embryo transfers performed between 2014 and 2018. Follow-up data on growth were available for 426, 131 and 662 children, respectively. Embryo biopsy, either at EBD3 or EBD5 in FET and FRESH cycles did not negatively affect anthropometry at birth, infancy or childhood compared to outcomes in non-biopsied FET and FRESH cycles. While there was no adverse effect of the timing of embryo biopsy (EBD3 versus EBD5), children born after EBD3 followed by FET had larger sizes at birth, but not thereafter, than children born after EBD3 followed by FRESH. Reassuringly, weight and height gain, proportions of major congenital malformations, developmental problems, hospital admissions and surgical interventions were similar between comparison groups. Our study indicated that neither EBD3 nor EBD5 followed by FRESH or FET had a negative impact on anthropometry and on health outcomes up to 2 years of age.

Abstract Children born using assisted reproductive technologies (ART) have an increased risk of a lower birth weight, the cause of which remains unclear. As a causative factor, we hypothesized that variants in the mitochondrial DNA (mtDNA) that are not associated with disease, may explain changes in birth weight. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or cycles with ovarian stimulation (OS). The mtDNA genotypes were compared across groups and logistic regression and discriminant analysis were used to study the impact of the different factors on birth weight percentile. ART individuals more frequently carried variants with higher heteroplasmic loads in protein and rRNA-coding regions. These differences in the mitochondrial genome were also predictive of the risk of a lower birth weight percentile, irrespective of the mode of conception but with a sex-dependent culture medium effect. The higher incidence of these variants in ART individuals results both from maternal transmission and de novomutagenesis, which we found not to be caused by OS but to be associated to maternal ageing. MtDNA variants in protein and rRNA coding regions are associated with a lower birth weight and are more frequently observed in ART children. We propose that these non-disease associated variants can result in a suboptimal mitochondrial function that impacts birth weight. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

Abstract 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

Lymphoedema is caused by an imbalance between fluid production and transport by the lymphatic system. This imbalance can be either caused by reduced transport capacity of the lymphatic system or too much fluid production and leads to swelling associated with tissue changes (skin thickening, fat deposition). Its main common complication is the increased risk of developing cellulitis/erysipelas in the affected area, which can worsen the lymphatic function and can be the cause of raised morbidity of the patient if not treated correctly/urgently. The term primary lymphoedema covers a group of rare conditions caused by abnormal functioning and/or development of the lymphatic system. It covers a highly heterogeneous group of conditions. An accurate diagnosis of primary lymphoedema is crucial for the implementation of an optimal treatment plan and management, as well as to reduce the risk of worsening. Patient care is diverse across Europe, and national specialised centres and networks are not available everywhere. The European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN) gathers the best expertise in Europe and provide accessible cross-border healthcare to patients with rare vascular diseases. There are six different working groups in VASCERN, which focus on arterial diseases, hereditary haemorrhagic telangiectasia, neurovascular diseases, lymphoedema and vascular anomalies. The working group Paediatric and Primary Lymphedema (PPL WG) gathers and shares knowledge and expertise in the diagnosis and management of adults and children with primary and paediatric lymphoedema. The members of PPL WG have worked together to produce this opinion statement reflecting strategies on how to approach patients with primary and paediatric lymphoedema. The objective of this patient pathway is to improve patient care by reducing the time to diagnosis, define the best management and follow-up strategies and avoid overuse of resources. Therefore, the patient pathway describes the clinical evaluation and investigations that lead to a clinical diagnosis, the genetic testing, differential diagnosis, the management and treatment options and the patient follow up at expert and local centres. Also, the importance of the patient group participation in the PPL WG is discussed.

Intracytoplasmic sperm injection (ICSI), developed 30 years ago in our Center, allowed men with low to extremely low sperm quality to become the genetic father of their child. Overall, ICSI is now applied in up to 70% of all ART cycles and has resulted in the birth of millions of children worldwide. ICSI circumvents natural sperm selection mechanisms and concerns related to transgenerational inheritance of male infertility following ICSI could not be fully answered till today, given the young age of the offspring born after ICSI. Since genetic abnormalities account for one third of all cases of male factor infertility, it is essential to identify these genetic abnormalities because of the potential risk of their transmission to the offspring through the use of ART. As ICSI is nowadays performed even in couples with normal semen parameters, the disentanglement of procedure-related and parental background-related factors on the health of the offspring is important. Due to the safety concerns regarding the health of children born after ART, a longitudinal follow-up program was set up in our Center in the 80’s, resulting in data of more than 50 000 pregnancies. To date, more than 25 000 children are conceived after ICSI in our Center. We have not only the worldwide eldest cohort of ICSI offspring but also a uniform cohort of children and young adults conceived by ICSI because of (severe) male factor infertility. We present the results of these prospective studies with focus on the reproductive health in ICSI children between 8 and 22 years and compare these with available data from others. First of all, we will show data on the gonadal function assessed both clinically and by means of biomarkers at pre-pubertal and pubertal ages in ICSI boys. Secondly, we will present findings on the reproductive status of young adult men and women conceived after ICSI between 1992 and 1996: results of hormonal work-up, physical examination and semen parameters for ICSI men and results of hormone levels and antral follicle counts for ICSI women. Finally, we will discuss the findings in these cohorts of children and young adults conceived because of male infertility in view of the available literature regarding reproductive function in offspring conceived following ICSI.

Little is known about the overall prevalence of lymphoedema in children and the types of paediatric lymphoedema seen by specialist centres. Therefore, this study was aimed to provide a profile of children with primary or secondary lymphoedema seen by the expert centres of the paediatric and primary lymphoedema working group (PPL-WG) of VASCERN and to compare the profile between the different countries. A retrospective review of all children (aged up to 18 years) seen for the first time by the expert centres over one year (2019) was carried out. Lymphoedema-, patient- and genetics-related data was collected and described for the whole group and compared between the different European countries/UK. In 2019, a total of 181 new children were seen by eight expert centres. For primary lymphoedema, the phenotype was based on the St George's classification of lymphatic anomalies. The percentages diagnosed according to each category were: 7.2% for syndromic lymphoedema, 2.8% for systemic/visceral involvement, 30.4% for congenital, 35.9% for late-onset lymphoedema and 19.3% for vascular/lymphatic malformations. 4.4% had secondary lymphoedema. Nearly 10% of all children had had at least one episode of cellulitis. The median delay from onset of symptoms to being seen by an expert centre was 2.4 years. In 44.4% of the children with primary lymphoedema a genetic test was performed, of which 35.8% resulted in a molecular diagnosis. Across the different centres, there was a wide variety in distribution of the different categories of paediatric lymphoedema diagnosed and the frequency of genetic testing. In conclusion, this paper has demonstrated that there is a large delay between the onset of paediatric lymphoedema and the first visit in the expert centres and that an episode of cellulitis is a relatively common complication. Diagnostic variation across the centres may reflect different referral criteria. Access to genetic testing was limited in some centres. It is recommended that these issues are addressed in the future work of the PPL-WG to improve the referral to the expert centres and the consistency in service provision for paediatric lymphoedema in Europe.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

Abstract Study question Does embryo vitrification affect children’s health including growth, up to 2 years of age when compared to fresh embryo transfer? Summary answer While embryo vitrification had an impact on birth parameters, no differences in growth or health outcomes were found up to 2 years of age. What is known already Vitrification has become the preferred cryopreservation method for embryos. Frozen embryo transfer has been repeatedly associated with altered health outcomes when compared with fresh transfer including a decreased risk for small-for gestational age (SGA) and an increased risk for large-for-gestational-age (LGA) and macrosomia. Not only there is uncertainty which factors are responsible for the observed differences, also the heterogeneity among studies limits overall conclusions. Notwithstanding the observed differences at birth, little is known about growth and health of children born after embryo vitrification beyond birth while aberrant growth trajectories have been linked to cardiometabolic morbidity later in life. Study design, size, duration This single-center cohort study compared anthropometry and health outcomes in singletons conceived after cleavage-stage or blastocyst-stage embryo vitrification with results after fresh embryo transfer between 2014 and 2018. Pregnancies after PGT, IVM, oocyte vitrification or oocyte/embryo donation were excluded. Eligible singletons living in Belgium and randomly selected for continued follow-up were invited for examination in our center at 2 months (infancy) and 2 years of age (early childhood). Participants/materials, setting, methods Birth characteristics were available for 1237 and 2063 children born after embryo vitrification and fresh embryo transfer, respectively. Follow-up data were available for 582 and 757 children at 2 months and for 233 and 296 children at 2 years. Growth parameters were adjusted for neonatal, treatment and maternal characteristics. Subgroup analysis according to cycle regimen (HRT versus NC) and strategy (freeze-all versus previous fresh cycle) was performed. In addition, outcomes restricted to blastocysts are presented. Main results and the role of chance Mothers giving birth to a child conceived after embryo vitrification presented more often with pregnancy-induced hypertensive disorders than controls (P < 0.001). Birthweight, height and head circumference SDS of children born after embryo vitrification were higher than for children born after fresh embryo transfer (all P < 0.001) even after adjustment for neonatal, treatment and maternal characteristics. Embryo vitrification was also associated with a decreased risk of SGA (AOR 0.48; 0.00, 0.44) and an increased risk of macrosomia and LGA (AOR 3.59; 1.12, 11.59)(all P < 0.05). Restricting the sample to blastocysts (n = 1795), we found a higher birthweight SDS and increased risks of LGA, macrosomia and pregnancy-induced hypertensive disorders after vitrification (all P < 0.05). At infancy, weight and height SDS were larger for children born after embryo vitrification, but not after adjustment for co-variates. At childhood, no differences in anthropometrics were found between the groups. Weight and height gain from birth to infancy and from infancy to early childhood were comparable between the groups. Until 2 years, comparable rates of severe developmental problems, hospital admissions, surgical interventions and of chronic medication intake were found between the groups. Subgroup analysis showed that growth parameters at all ages were not affected by cycle regimen or cycle strategy. Limitations, reasons for caution Participation rate at 2 years was lower than expected in both groups, probably due to cancellation/postponement of the visit related to the corona pandemic. Furthermore, although cycle strategy was not found to affect growth parameters, the sample size of the subgroup analysis remains rather small to draw firm conclusions. Wider implications of the findings When adjusted for co-variates including birthweight, the observed differences in anthropometrics at birth in children born after embryo vitrification attenuated by 2 years of age. This suggests that outcomes in early childhood are determined by size at birth. Trial registration number Not applicable

ABSTRACTBackgroundChildren born using assisted reproductive technologies (ART) have an increased risk of a lower birth weight, the cause of which remains unclear. As a causative factor, we hypothesized that variants in the mitochondrial DNA (mtDNA) that are not associated with disease, may explain changes in birth weight.MethodsWe deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or cycles with ovarian stimulation (OS). The mtDNA genotypes were compared across groups and logistic regression and discriminant analysis were used to study the impact of the different factors on birth weight percentile.ResultsART individuals more frequently carried variants with higher heteroplasmic loads in protein and rRNA-coding regions. These differences in the mitochondrial genome were also predictive of the risk of a lower birth weight percentile, irrespective of the mode of conception but with a sex-dependent culture medium effect. The higher incidence of these variants in ART individuals results both from maternal transmission and de novo mutagenesis, which we found not to be caused by OS but to be associated to maternal ageing.ConclusionsMtDNA variants in protein and rRNA coding regions are associated with a lower birth weight and are more frequently observed in ART children. We propose that these non-disease associated variants can result in a suboptimal mitochondrial function that impacts birth weight. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.