Growth and other health outcomes of 2-year-old singletons born after IVM versus controlled ovarian stimulation in mothers with polycystic ovary syndrome.

In the IVM group, 92 out of 133 (69%) invited children were clinically examined at an average age of 2.3 years. Five families could not be reached and 36 families refused to participate because of the following: severe mental retardation of the child ( n  = 1), moribidity owing to prematurity ( n  = 1), long travel distance to the center ( n  = 4), no interest in another clinical visit after the previous (reassuring) one at 1 year of age ( n  = 14) or no reason specified ( n  = 16). From the 136 families approached in the COS group, 41 families refused participation, 11 families could not be reached and for 10 children an appointment was scheduled but canceled a few hours/days before the visit (mainly because of illness of the child). Finally, 74 (54.4%) children in the COS group were examined at our center. No differences were found between participating IVM children ( n  = 92) and the non-participating IVM children ( n  = 41) when comparing maternal characteristics (age, BMI and AMH levels), neonatal characteristics (birthweight and gestational age) and treatment characteristics (proportion of SET, vitrified embryo transfer and day 3/4 embryo transfer; data not shown). Likewise, when comparing maternal, neonatal and treatment characteristics, no differences were found between participating COS children ( n  = 74) and non-participating COS children (i.e those that were not selected during randomization or refused or were not reached; data not shown). In mothers of IVM children, day3/4 embryo transfers (IVM: 72.8% versus COS: 29.7%; P  < 0.001) and vitrified embryo transfers (IVM: 64.1% versus COS: 35.1%; P  < 0.001) were more prevalent in the IVM group than in the COS group. More IVM children were born after DET (in contrast to SET) when compared to those in the COS group (39.1% versus 23.0%; P  = 0.03). Maternal serum AMH level was significantly higher in the IVM mothers (11.5 ± 7.2 ng/mL) than in the COS mothers (7.7 ± 4.4 ng/mL; P   0.05, Table1) but IVM mothers were on average 1.7 years younger than COS mothers ( P  = 0.002) and less likely to smoke ( P  = 0.04). Comparable proportions of the mothers were diagnosed with a metabolically unfavorable PCOS phenotype (type A and C) in the two groups (in IVM: 52.1% and in COS: 45.9%) ( P  = 0.42; Table I ). None of the mothers had phenotype B. Parental and treatment characteristics of the study population. AMH: anti-Müllerian hormone; COS, controlled ovarian stimulation; HELLP, hemolysis elevated liver enzymes and low platelets; PCOS, polycystic ovary syndrome; SD, standard deviation; SET, single embryo transfer. °Hypertensive disorders including pregnancy-induced hypertension, (pre-)eclampsia and HELLP syndrome. * Continuous data: mean (SD) and categorical data: number (%). ** Chi-square test or Student’s t -test, as appropriate. Anthropometry of IVM and COS children at birth, at 4 months and at 2 years. AMH, anti-Müllerian hormone; CI, confidence interval; COS, controlled ovarian stimulation; IVM, In vitro maturation; PCOS, polycystic ovary syndrome. °Additional adjustment for birthweight z -score. * Adjusted for neonatal (birth order), treatment (number of embryos transferred, mode of transfer and day of embryo transfer), parental characteristics (maternal smoking during pregnancy, maternal age, maternal AMH level, maternal PCOS phenotype, gestational diabetes, hypertensive disorder, BMI of the mother and BMI of the father). Mean gestational age was comparable between the IVM (38.8 ± 1.7 weeks) and the COS children (38.9 ± 2.0 weeks) ( P  = 0.8) and the prematurity rate (<37 weeks) was also comparable (IVM: 15.4% versus COS: 8.1%) ( P  = 0.2). No children were born SGA and a comparable proportion of IVM and COS children was born LGA (7.7% versus 2.7% respectively; P  = 0.2). Major malformations were detected in four (4.3%) children born after IVM. Two major malformations (unilateral symbrachydactyly and hypospadias) were detected in the neonatal period and two were detected in the follow-up period (bilateral cryptorchidism and arachnoid cyst). Major malformations were detected in two (2.7%) children born after COS. One infant was diagnosed with a coarctatatio aortae and a single kidney and one child was diagnosed with Silver-Russell syndrome during the follow-up period. Weight, height and head circumference z -score were comparable between the two groups at birth, at 4 months and at 2 years ( Table II ). In addition, at 2 years of age, waist circumference and mid-upper arm circumference z -score were not significantly different between the IVM and COS group. Adjustment for neonatal, treatment and parental characteristics did not change this conclusion. Also, weight gain from birth ~2 years was comparable between the IVM and the COS group, even after adjustment for neonatal, treatment and parental characteristics (B: −0.5, 95% CI: −0.9; 0.1). At 2 years of age, the z -scores for mean weight, height and BMI were not statistically different from zero in the IVM and COS groups (all P  > 0.05). However, the mean z -score was significantly larger than zero for waist circumference in the IVM and COS group (both P  < 0.001), and for the MUAC in the IVM group ( P  = 0.01) but not in the COS group ( P  = 0.06). z -Scores of weight, height, BMI, waist circumference and MUAC at 2 years of age were comparable between IVM and COS children, either in the subgroup of children born after cleavage-stage embryo transfer (IVM: n  = 67, COS: n  = 22); after fresh embryo transfer (IVM: n  = 33, COS: n  = 48); and after SET (IVM: n  = 36, COS: n  = 17) or the subgroup of children born after blastocyst transfer (all P  > 0.05); after vitrified embryo transfer (all P  > 0.05); and after DET (all P  > 0.05). Surgery had been performed in 11 (12.0%) children born after IVM and in five (6.8%; P  = 0.3) children born after COS. Indications for surgical intervention were mostly minor, such as adenotonsillectomy and/or placement of grommets in 5/11 IVM children. Other interventions were as follows: hypospadias correction ( n  = 1), orchidopexy ( n  = 1), circumcision ( n  = 3) and surgery for symbrachydactyly ( n  = 1) in the IVM group; and circumcision ( n  = 2), placement of grommets ( n  = 1), cardiac surgery ( n  = 1) and a dermal cyst excision ( n  = 1) in the COS group. None of the IVM children was admitted to a Neonatal Intensive Care Unit (NICU) for >4 weeks compared with one child in the COS group ( P  = 0.26). Up to the age of 2.5 years, 23 children born after IVM (25%) had been hospitalized at least once (>1 day, NICU excluded), mainly for infectious diseases ( n  = 19), but also for allergy ( n  = 3) and monitoring after an apparent life-threatening event ( n  = 1). In total, 14 COS children (18.9%) were hospitalized at least once (>1 day, NICU excluded), also mainly for infectious diseases ( n  = 11) as well as for apnea ( n  = 2) and observation after an injury ( n  = 1). The rate of hospital admission was comparable between the two groups ( P  = 0.45).

The study design has been previously described by Mostinckx et al . (2019 ). In brief, included pregnancies were established after transfer of embryos generated by either IVM of oocytes or COS in women with PCOS. All singleton pregnancies beyond 20 weeks of gestation were included that resulted from an oocyte retrieval between January 2010 and December 2016. Only one offspring from each patient was included. The study included women between 18 and 36 years of age with PCOS, as defined by a follicle number per ovary ≥ 12 on ultrasound scan ( Balen et al ., 2003 ). All patients were diagnosed with a specific PCOS phenotype, i.e. A, B, C or D according to the recent National Institutes of Health 2012 extension of the ESHRE/ASRM 2003 diagnostic criteria. Patients with endometriosis grade III-IV were excluded. Furthermore, cycles with preimplantation genetic testing, with oocyte donation or with use of non-ejaculated sperm were excluded. As previously explained by Mostinckx et al . (2019 ), patients consented to undergo either IVM or COS after discussion with the fertility doctor at the outpatient clinic regarding the risks and benefits of each procedure. All IVM pregnancies were achieved using oocytes that had been retrieved without hCG priming and matured in vitro after 28–40 h of culture. Details of the IVM procedure are described in detail elsewhere ( Mostinckx et al ., 2019 ). Briefly, follicular aspirates were collected, supplemented with heparin and filtered. After collection, cumulus–oocyte complexes were washed and transferred to a dish containing IVM medium supplemented with highly purified-hMG, hCG and human serum albumin. Cumulus–oocyte complexes were cultured for 28–40 h at 37°C under 6% CO 2 and 20% O 2 , respectively. Insemination of all mature oocytes in this study was carried out using ICSI. In total, 393 ART pregnancies in patients diagnosed with PCOS were ongoing after 20 weeks. Of these, 164 pregnancies after IVM resulted in the birth of 160 liveborn singletons and 229 pregnancies after COS resulted in the birth of 225 liveborn singletons ( Mostinckx et al . 2019 ). For this study, singletons living in Belgium (a clinical visit to our center was required) and reaching the age of 2 years between November 2012 and June 2019 were eligible. All children were part of the neonatal cohort described by Mostinckx et al . (2019 ). IVM and COS families were invited for a clinical examination at the Center for Medical Genetics through telephone after they had received a letter explaining the study objectives and design. Overall, from the group of 160 IVM liveborns, 133 IVM children were contacted and 27 IVM children were not contacted, two children had died in the neonatal period due to prematurity, five families were living abroad, 12 children did not reach the age of 2 years during the follow-up period and from eight children the only available information was their live birth; without any details the latter were considered as lost to follow-up. From the 225 liveborns after COS, 17 were not eligible for follow-up (one child had died in the neonatal period, 16 families were living abroad). Of the eligible 208 COS children, 74 were participants in the standard longitudinal follow-up program. Owing to the large number of children conceived by ICSI in our center, only one in three liveborns are randomly selected, with replacement, for further follow-up until young adulthood. As for the IVM group, only children living in Belgium are eligible since a clinical visit in the Center for Medical Genetics is a prerequisite. A comprehensive follow-up program for children born after ART has been set up in our center since the introduction of IVF in clinical practice. Principally, children born after non-conventional techniques, including IVM, are closely monitored, including an additional physical assessment at the age of 1 year. Anthropometric measurements, collected at the age of 1 year in 74/92 IVM children, are not reported due to the lack of comparable data in COS children.

This study describes the general health status, including growth from birth ~2 years of age, in the largest cohort to date of singletons born after non-hCG triggered IVM of immature oocytes from mothers with PCOS. Anthropometry at birth, at 4 months and at 2 years, was found to be comparable in IVM and COS children even after adjustment for neonatal, treatment and parental characteristics. Furthermore, and reassuringly, the rates of surgical interventions and hospital admissions did not differ between IVM children and peers born after COS. z -Scores for anthropometric measurements at 2 years of age did not differ between the IVM and COS group, but waist circumference z -scores were significantly higher than zero (the population mean) in both groups. Whether this finding confirms the abundantly reported overall adverse effect of maternal PCOS on the cardiometabolic health of offspring remains unclear ( Sir-Petermann et al ., 2007 , 2009 ; Kent et al ., 2008 ; Recabarren et al ., 2008 ; Battaglia et al ., 2009 ; Maliqueo et al ., 2009 ; Hanem et al ., 2018 ; Vanky et al ., 2019 ), but it certainly calls for follow-up, especially because waist circumference is a widely accepted measure of central adiposity and hence a suitable screening tool for cardiometabolic risk factors in children ( Sardinha et al ., 2016 ). Our auxiological findings at the age of 2 years are in line with those from Roesner et al . (2017 ) who described development in 2-year-old IVM singletons, albeit in a very small group of only 12 IVM singletons and eight peers born after ICSI. However, Roesner et al . (2017 ) did not adjust for age or gender and did not provide (crucial) information regarding the maternal condition or the precise indication for IVM. This is a frequent drawback in studies on IVM outcome, together with the inclusion of comparison groups with largely different backgrounds ( Fadini et al ., 2012 ; Foix-L’Hélias et al ., 2014 ). Since IVM has been performed for distinct conditions, it is of utmost importance to include study and control groups that are comparable, apart from the IVM procedure itself. Indeed, describing the impact of infertility treatments on offspring health is complex since many factors, including infertility patients’ genetic makeup, the laboratory environment or procedures and maternal and paternal characteristics, can affect the outcome ( von Versen-Höynck et al ., 2019 ; Castillo et al ., 2019 ; Khandwala et al ., 2018 ). In most other studies an ‘hCG-primed’ approach has been adopted, the oocyte of origin ( in vitro versus in vivo matured, respectively) cannot unequivocally be assigned, and an unknown proportion of ‘IVM’ children have not been conceived using in vitro matured oocytes. We, on the contrary, did not use any hCG-trigger and hence all children in our study are born after IVM of immature oocytes. The current and previous findings obtained at the neonatal age provide reassurance regarding the practice of IVM of immature oocytes and offspring health ~2 years of age. Even though the stratification according to PCOS phenotype in the mothers is unique, which enabled us to compare study subjects and controls with a highly similar background, some limitations should be noted. The apparent differences in treatment characteristics (mainly the mode and type of embryo transfer) between the two groups are due to the dynamics in embryo transfer policy over the years in our center and could have affected the antropometric outcomes. However, while vitrified embryo transfer (versus fresh embryo transfer) as well as blastocyst (versus cleavage stage embryo) transfer are known to affect weight and LGA rate ( Beyer and Griesinger, 2016 ; Makinen et al ., 2013 ; Hann et al ., 2018 ), our stratified analysis, albeit in small subgroups, did not point to any impact of these characteristics on our results. A matched cohort study design that accounts for potential treatment and parental confounders would have strengthened our conclusions but was not feasible because matching for several parameters, including embryo freezing or not, number of embryos transferred, maternal BMI or PCOS phenotype, would require a number of candidate control children that largely exceeds the 208 that were available. Also, in case of drop-out of control children during the project, we would also have been forced to remove the study (case) child as well, or end up with an unbalanced design. Nevertheless, we believe that selection bias is unlikely since there were no differences in maternal, neonatal and treatment characteristics between the participating and non-participating IVM and COS groups. On the other hand, it should be acknowledged that the composition of the study group is a direct consequence of the fact that women were offered the choice to undergo either IVM or COS, although IVM as a treatment option was probably more frequently discussed with patients presenting with more severe symptoms. Furthermore, given the convincing evidence of the short- and long-term impact of maternal PCOS condition on the health of offspring (see review by Vanky et al ., 2019 ), research should be expanded to include cardiovascular and metabolic outcomes in children and teenagers. In view of this, the findings of a recently published study regarding cardiovascular and metabolic health in spontaneously conceived children from women with PCOS are appealing; although no differences were found in biometry, subtle cardiovascular and metabolic abnormalities were detected at an age as early as 2.5 years and also at 6–8 years in children whose mother was diagnosed with PCOS in comparison with a population-based reference cohort ( de Wilde et al ., 2018 ). Finally, by including a second control group of children born after spontaneous conception to mothers with PCOS, we could have investigated better the effect of the technique of IVM per se and this would have added to the generalisability of our findings. In addition, future studies should also focus on the follow-up of children born after IVM performed in patients in need of fertility preservation after a cancer diagnosis, which is another emerging indication for IVM. In conclusion, in our study of 166 children born to mothers with PCOS we did not find an adverse effect of IVM on body size and general health outcome parameters ~2 years of age when compared to COS. However, given the known cardiovascular and metabolic implications for children born to mothers with PCOS, continued follow-up of these children born after IVM is warranted.

In oocyte IVM, oocytes are collected from antral follicles and cultured, matured and fertilized in vitro ( Edwards, 1965 ). The avoidance of conventional stimulation of follicles to preovulatory sizes, the absence of an ovulatory trigger and thus the elimination of the risk of ovarian hyperstimulation syndrome makes IVM a suitable treatment option for women suffering from polycystic ovary syndrome (PCOS), which affects ~10% of women ( Smitz et al ., 2011 ). The widespread implementation of IVM is, however, hindered by lower live birth rates and higher miscarriage rates when compared to conventional ART ( Suikkari, 2008 ). Nevertheless, neonatal outcome data for children born after IVM are available. Unfortunately, studies on the health of IVM children are often flawed which impedes their comparability and makes it difficult to draw sound conclusions. Most studies are based on small sample sizes ( Cha et al ., 2005 ; Mikkelsen 2005 ; Soderstrom-Antilla et al ., 2006 ; Shu-Chi et al ., 2006 ), do not stratify outcomes regarding multiplicity ( Cha et al ., 2005 ; Foix-l’Hélias et al ., 2014 ) or do not include a comparison group ( Cha et al ., 2005 ; Mikkelsen 2005 ; Soderstrom-Antilla et al ., 2006 ). More troublesome is the fact that the IVM and comparison groups often have a different infertility background. For example, in the study of Fadini et al . (2012 ), >17% of the mothers of a singleton born after IVM were diagnosed with PCO (S) while this was only 5% in the intracytoplasmic sperm injection (ICSI) control group. In the study of Foix-l’Hélias et al . (2014 ), all mothers of children born after IVM had PCOS while the comparison consisted of children born after ICSI, mostly born to couples suffering from male infertility. Other limiting factors are the lack of health data beyond infancy and the lack of a uniform protocol of IVM, resulting in births derived from oocytes that were mature rather than immature at recovery owing to the use of an ovulation trigger. Although Roesner et al . (2017 ) investigated 2-year-old singletons born after IVM, ICSI and in vitro fertilization (IVF), only limited biometric data based on very few IVM ( n  = 12), IVF ( n  = 11) and ICSI ( n  = 8) singletons were reported. The procedure of IVM of immature oocytes gives rise to specific concerns regarding the health of offspring born following this technique. The prolonged culture time in vitro might induce epigenetic changes that affect growth, as has been shown both in animals ( McEvoy et al ., 2000 ; Kerjean et al ., 2003 ) and in humans ( Sunde et al ., 2016 ). Not only the prolonged culture but also the PCOS condition of the mother could have an impact on the linear growth of the offspring ( Doherty et al ., 2015 ). Although several studies reported a trend to a higher birthweight among children born after IVM ( Buckett et al ., 2007 ; Fadini et al ., 2012 ), it remains largely unknown whether IVM of oocytes affects skeletal growth and body composition beyond the neonatal period, particularly in children born to mothers diagnosed with well-defined PCOS phenotypes. In this study, we investigated if IVM of oocytes may have compromised the outcome of the offspring. Therefore, we compared anthropometry and health status of a unique cohort of children conceived following IVM with results from peers conceived by ICSI and conventional controlled ovarian stimulation (COS) in mothers with PCOS.

Body weight and (standing) height were measured using standard equipment. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m 2 ). Waist circumference and mid-upper arm circumference (MUAC) were measured using a non-stretchable tape. Waist circumference was measured at the narrowest point between the costal margin and the iliac crest, with the subjects standing upright. The MUAC was measured midway between the lateral projection of the acromion process of the scapula and the inferior margin of the olecranon process of the ulna. Weight, height, BMI, waist circumference and MUAC were converted to age and sex adjusted z -scores using Flemish reference curves ( Roelants et al ., 2009 ). Solely for the purpose of completeness, we report major congenital malformations, which are defined as malformations that generally cause functional impairment or require surgical correction ( Bonduelle et al ., 2002 ). Here, we report malformations from birth up to the age of 2.5 years. Malformations in neonates born after IVM are reported in detail elsewhere ( Mostinckx et al ., 2019 ). Parents were asked to complete a questionnaire covering a broad range of parameters related to their personal and their children’s health status. This included information on hospital admissions, illnesses and surgical interventions in the child and current parental height and weight. Written informed consent was obtained from all parents. The study was approved by the Ethics Committee of the UZ Brussel. Descriptive statistics are presented as mean and SD for continuous variables and as the frequency and percentage for categorical variables. Small-for-gestational age (SGA) was defined as birthweight z -score  2. Weight gain (delta weight z -score) from birth up to childhood was calculated as weight z -score at 2 years minus birthweight z -score. The IVM and COS groups were compared with a Student’s t -test for continuous variables and a chi-squared test for categorical variables. z -Scores of antropometric data at 2 years were additionally tested with a one-sample Student’s t -test. Data analysis was performed using SPSS Statistics version 25 (IBM, Chicago, IL, USA). Since the study was largely underpowered for comparison of expected congenital malformation rates, this outcome is only described and not tested for statistical significance. Multiple linear regression analysis was used to investigate differences in body measurements between the IVM and COS group, adjusted for covariates. Results are expressed as unstandardized regression coefficients (B) with a 95% confidence interval (CI). Covariates known to affect body size and composition and covariates that differed among groups were included in the final model; these include neonatal characteristics (birth order), treatment characteristics (fresh versus vitrified embryo transfer, day 3/4 versus day 5/6 embryo transfer, single embryo transfer (SET) or double embryo transfer (DET)) and parental characteristics (maternal smoking during pregnancy, maternal age, maternal anti-Müllerian hormone (AMH) level, maternal PCOS phenotype, gestational diabetes, hypertensive disorder, BMI of the mother and BMI of the father). Outcomes at 4 months and 2 years were additionally adjusted for birthweight z -score. In a second analysis, the data were stratified by day of embryo (day 3/4 versus day 5/6) and mode of transfer (fresh versus vitrified) in order to rule out the impact of treatment modalities other than IVM or COS. With an estimated sample of 75 children in each group, differences between group means will become statistically significant when they differ by >0.3 times the SD. Because the main outcome parameters are expressed as z -scores, which have a SD of approximately one, differences between groups will become statistically significant when they differ by >0.3 z -scores. Translated to the measurement scale in 2-year-old children, a difference of 0.3 z -scores corresponds to ~0.4 kg for weight, 1 cm for length and 0.4 kg/m 2 for the BMI ( Roelants et al ., 2009 ).