In Vitro Fertilization Improves Childhood Growth and Metabolism

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The Journal of Clinical Endocrinology & Metabolism 92(9):3441–3445 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2465

In Vitro Fertilization Improves Childhood Growth and Metabolism Harriet L. Miles, Paul L. Hofman, John Peek, Mark Harris, Dyanne Wilson, Elizabeth M. Robinson, Peter D. Gluckman, and Wayne S. Cutfield The National Research Centre for Growth and Development and Liggins Institute (H.L.M., P.L.H., M.H., D.W., P.D.G., W.S.C.) and Department of Community Health (E.M.R.), University of Auckland, Auckland 1010, New Zealand; and Fertility Associates (J.P.), Auckland 1051, New Zealand Background: There is limited information regarding the long-term outcome of children born after in vitro fertilization (IVF), although an increase in rare imprinted gene disorders such as Beckwith-Wiedemann syndrome has been reported. Methods: We recruited healthy, prepubertal children born at term after singleton pregnancy. The children in the study group were conceived using IVF with fresh embryo transfer, whereas controls were naturally conceived. Anthropometric measurements, bone age, dual-energy x-ray absorptiometry, fasting serum glucose, insulin, lipid profile, IGF-I and -II, and IGF-binding proteins 1, 2, and 3 were performed. Results: There were 69 IVF children aged 5.9 ⫾ 0.2 yr and 71 control children aged 6.9 yr. IVF children were taller than controls when corrected for parents’ heights (height SD score of 1.05 ⫾ 0.1 vs. 0.51 ⫾

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N VITRO FERTILIZATION (IVF) has had a major impact on the successful treatment of infertility. Since the birth of the first IVF baby in 1978, this technique has grown in popularity, accounting for 1–3% of births in Western countries with more than 2 million babies born worldwide (1). IVF is a relatively new technology and largely first generational. Follow-up studies are limited and relate to pregnancy and neonatal outcome. IVF children have an increased risk of major malformation detected in infancy (2). There is a known increase in low birth weight in singleton IVF pregnancies (3, 4). Recently, an increased risk of imprinting disorders including the overgrowth disorder Beckwith-Wiedemann syndrome and the neurodevelopmental disorder Angelman syndrome has been reported in children born after IVF (1, 5). Beckwith-Wiedemann and Angelman syndromes are due to abnormal methylation patterns of imprinted genes. Methylation of cytosine in islands of cytosine paired with guanine switches off gene transcription (6). Imprinted genes are methylated according to whether they are inherited from the mother or the father. There are over 50 known imprinted genes in the human, and many of these genes are involved First Published Online June 12, 2007 Abbreviations: BMI, Body mass index; CV, coefficient of variation; DEXA, dual-energy x-ray absorptiometry; HDL, high-density lipoprotein; ICSI, intracytoplasmic sperm injection; IGFBP, IGF-binding protein; IVF, in vitro fertilization; LDL, low-density lipoprotein; SDS, sd score. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

0.11, P ⫽ 0.001) with higher levels of serum IGF-II (850 ⫾ 24 vs. 773 ⫾ 24 ␮g/liter, P ⫽ 0.03), higher IGF-I to IGF-binding protein 3 ratio (P ⫽ 0.04), and a trend toward higher IGF-I (105 ⫾ 4 vs. 92 ⫾ 4 ␮g/liter, P ⫽ 0.06). IVF children had higher high-density lipoprotein (1.67 ⫾ 0.04 mmol/liter vs. 1.53 ⫾ 0.04 mmol/liter, P ⫽ 0.02), lower triglycerides (0.65 ⫾ 0.04 mmol/liter vs. 0.78 ⫾ 0.04 mmol/liter, P ⫽ 0.02), and a lower total to high-density lipoprotein cholesterol ratio (2.58 vs. 2.86, P ⫽ 0.01). There were no differences in body composition. Conclusions: IVF children are taller with higher IGF-I and IGF-II levels and have a slightly more favorable lipid profile. We speculate that IVF results in epigenetic change through altered methylation of genes involved in growth and metabolism. IVF programs should consider long-term longitudinal follow-up of IVF offspring. (J Clin Endocrinol Metab 92: 3441–3445, 2007)

in cellular proliferation and growth. Methylation of nonimprinted genes can also occur, altering gene expression. Culture of mammalian embryos results in a change in the expression of several imprinted genes (1, 7). Furthermore, nutritional manipulation early in fetal life has been shown to lead to reduced methylation and overexpression of nonimprinted genes such as the glucocorticoid receptor (8). Phenotypic features seen in children with BeckwithWiedemann syndrome are variable, with the full syndrome the extreme end of a continuum (9). We hypothesize that subtle differences may exist in the previously under-investigated IVF population manifested as measurable changes in phenotype and hormonal profile in mid-childhood. Subjects and Methods Subjects We recruited healthy prepubertal children between the ages of 4 and 10 yr, born at term (⬎36 wk gestation) after singleton pregnancy. Subjects were recruited into IVF and control groups. IVF subjects living in Auckland were recruited from Fertility Associates, the largest IVF provider in New Zealand. All IVF children were conceived using IVF with fresh embryo transfer between January 1995 and December 2000. All IVF fathers were documented as the sperm donors for their IVF child. Exclusion criteria for all subjects included multiple pregnancy identified on early scan, a known medical syndrome, chronic illness, or receiving regular medications. Although breastfeeding rates were not collected, virtually all subjects are likely to have been breastfed. Rates of breastfeeding in New Zealand are high at 92% of Caucasian infants (the ethnicity of almost all subjects studied), with the highest rates seen in mothers in the 30- to 40-yr age group (which matches the maternal age range of the subjects we studied) (from the “Report on maternity: maternal and newborn information 2002”; www.nzhis.govt.nz/publications/maternity/report.pdf).

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J Clin Endocrinol Metab, September 2007, 92(9):3441–3445

Miles et al. • IVF Improves Childhood Growth and Metabolism

Commercial embryo culture was sourced from Medicult (52 subjects) or Scandinavian IVF (17 subjects); the protein supplement was human serum albumin. Embryos were transferred on d 2 with two to six cells per embryo. Fertility Associates annual pregnancy rates, corrected for percentage of eggs retrieved progressively, increased through the study period from 15 to 40%, placing them within the top five New Zealand and Australian centers (10). Controls were matched as closely as possible for socioeconomic background using school decile and residential address, age, sex, and ethnicity. Body mass index was recorded for both groups of parents, and all children were asked to complete a 3-d dietary diary before the study for later comparison. In addition to peers recruited by the IVF subjects, a small number of naturally conceived siblings of IVF children were also included in the control group. Ethics approval was provided by the Auckland Ethics Committee, and written informed consent was obtained for all subjects.

Methods Demographic information was collected including mode of conception and birth data. Supplementary data were collected for children conceived using IVF, including protocol for ovulation induction, mode of conception [conventional IVF or intracytoplasmic sperm injection (ICSI), defined as the direct injection of sperm into oocytes], and embryo culture medium. Parental height and weight were recorded and midparental height calculated and corrected to a sd score (SDS) (11). Standing height was measured using a Harpenden stadiometer and head circumference recorded as an SDS (12). Weight and body composition were assessed using both body mass index (BMI) [weight (kg)/height (m)2] and whole-body dual-energy x-ray absorptiometry (DEXA) (Lunar Prodigy 2000, General Electric, Madison, WI). Bone age was assessed by a single blinded pediatric radiologist using standards established by Greulich and Pyle (13). A fasting blood sample was obtained for assessment of metabolic and growth factors. Plasma glucose was measured using an automated random-access analyzer (model 911; Hitachi, Tokyo, Japan) with an interassay coefficient of variation (CV) of 1.2%. Insulin levels were determined by enzyme immunoassay (IMX microparticle assay; Abbott Laboratories, Chicago, IL) with an interassay CV of less than 5%. Total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglyceride were measured using a Roche (Indianapolis, IN) modular analyzer. Low-density lipoprotein (LDL)-cholesterol was calculated using the Friedwald formula. IGF-I was measured using a Diagnostic Products Corp. (Los Angeles, CA) Immulite analyzer (interassay CV 9.3%). Commercially available ELISAs (Diagnostic Systems Laboratories, Webster, TX) were used to evaluate plasma IGF-II (DSL-10-9100, intraassay CV 1.7%, interassay CV 6.2%), IGF-binding protein (IGFBP)-1 (DSL-10-7800, intraassay CV 1.7% interassay CV 6.2%), IGFBP-2 (DSL-10-7100, intraassay CV 1.5%, interassay CV 3.4%), IGFBP-3 (DSL-10-6600, intraassay CV 7.3%, interassay CV 8.2%) and GH-binding protein (DSL-10-48100, intraassay CV 4.8%, interassay CV 5.1%).

Statistical analysis Linear regression analyses were used to investigate the differences in baseline characteristics and anthropometric, endocrine, and metabolic measures between the study groups of children. Gestation, birth weight, midparental height, maternal age, bone age, age, and sex were included in models of anthropometric data in the calculation of adjusted height data. Gestation, birth weight, midparental height, maternal age, standing height, weight, age, and sex were included in models of lipid and

insulin data and age, sex, BMI, DEXA percent fat, and height for the remaining endocrine data. Means and sem are reported for baseline data, means and se adjusted for other variables in the model are reported for other measures. SAS version 9.1 was used for all statistical analyses. A P value of ⬍ 0.05 was used to define significance.

Results

In total, 140 children were enrolled in the study, 69 children conceived using IVF (34 ICSI) and 71 naturally conceived controls (65 friends and 6 sibling pairs). Eighty-two IVF children were eligible for the study, of which 84% were recruited. There were no differences in gestation, birth weight SDS, or maternal age between the children in the study and the children who did not take part. The cause of infertility and proportion conceived using ICSI were also similar. We are therefore confident that IVF children recruited for this study are representative of IVF children born during this time period. Birth and parental clinical characteristics of the two groups are summarized in Table 1. Parental anthropometry was comparable between the two groups. Mothers of the IVF children were older than mothers of control children; however, maternal age did not influence any of the outcome parameters measured. There was no difference in midparental heights between the groups; in particular, there was no difference in maternal height SDS values between IVF and control groups (0.55 vs. 0.4, P ⫽ 0.45). All families in the study were from higher socioeconomic groups. Dietary diaries were similar for IVF children and controls, and over 50% of control children were firstborn or singleton children. Although the IVF group had slightly lower birth weight, only two children fulfilled the criteria for being small for gestational age (SGA) (14). Predictably, IVF children were more likely to be firstborn; however, birth order did not influence any of the outcome measures, including height SDS, bone age, and BMI, or any of the serum hormone measurements. Anthropometric, endocrine, and metabolic data are summarized in Tables 2 and 3. The control group was older than the IVF group; however, all children were prepubertal. Bone maturation of the hand and wrist, an index of biological maturity, measured as the difference between chronological age and bone age, was not different between the two groups. IVF children were taller than controls and remained so when adjustment was made for age and parents’ heights. In both groups, girls (height SDS 1.82 ⫾ 0.17 in IVF vs. 1.09 ⫾ 0.16 in control girls) were taller than boys (height SDS 0.3 ⫾ 0.17 in IVF vs. ⫺0.07 ⫾ 0.17 in control boys); thus, IVF girls were the tallest group. The distribution of heights of subjects are shown in Fig. 1 and are expressed as height corrected for parents’ heights (height SDS ⫺ midparental height SDS) for

TABLE 1. Comparison of parental and birth characteristics of control children and children born after IVF Group

Control

IVF

P value

Midparental height SDS Midparental BMI SDS Maternal age at delivery (yr) Gestation (wk) Birth weight SDS Firstborn percentage

0.85 (0.10) 1.16 (0.13) 31.24 (0.64) 39.56 (0.18) 0.39 (0.11) 54% (50% male)

0.65 (0.11) 1.07 (0.09) 34.13 (0.46) 39.3 (0.19) ⫺0.1 (0.15) 91% (50% male)

0.2 0.49 0.0003 0.35 0.004 ⬍0.05

Values are expressed as means (SEM).



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TABLE 2. Comparison of anthropometric characteristics of control children and children born after IVF

Sex (male/female) Age (yr) Height SDS Sitting height SDS Leg length SDS Bone age ⫺ chronological age (yr) BMI (kg/m²) DEXA % fat BMD SDS Female head circumference (cm) Male head circumference (cm)

Control (n ⫽ 71)

IVF (n ⫽ 69)

P value, ANOVA

37/34 6.94 (0.24) 0.51 (0.11) ⫺0.33 (0.14) 1.14 (0.13) ⫺0.06 (0.08) 16.47 (0.23) 18.60 (0.82) 0.20 (0.08) 51.4 (0.23) 52.9 (0.26)

33/36 5.88 (0.16) 1.05 (0.11) 0.18 (0.14) 1.63 (0.12) 0.03 (0.08) 15.68 (0.23) 18.15 (0.80) 0.07 (0.08) 51.93 (0.23) 53.1 (0.25)

0.87 0.0003 0.001 0.02 0.01 0.48 0.03 0.71 0.30 0.18 0.61

Values are expressed as means (SEM). BMD, Bone mineral density.

the IVF and control groups. Sixty-two percent of IVF subjects had a height that either matched or exceeded genetic height potential (height ⫺ midparental height SDS of ⱖ0), whereas 44% of control subjects had a height ⫺ midparental height SDS of ⱖ0 (P ⬍ 0.03). Similar increases in sitting and standing heights of IVF children indicate that this increase in stature is proportionate. There were no other sex differences between IVF and control groups found for any other variables examined. The corrected BMI in the IVF group was lower, but there was no difference in percent fat assessed by DEXA. There was a trend toward higher serum IGF-I levels in the IVF group. In modeling IGF-I, the difference between IVF and control groups increased with age (P ⫽ 0.05), with older children (⬎7 yr of age) born after IVF having the highest IGF-I levels (P ⬍ 0.05). Similarly, the serum IGF-I to IGFBP-3 ratio, an index of free IGF-I, was higher in the IVF group (0.022 vs. 0.019, P ⫽ 0.04). Serum IGF-II levels in the IVF group were higher than in the control group; however, IGF-II levels did not become progressively higher than the control group with increasing age. For all children, there was an association between taller stature and higher serum IGF-I (r2 ⫽ 0.10; P ⬍ 0.001) and also serum IGF-II (r2 ⫽ 0.11; P ⬍ 0.001). IVF children had a more favorable lipid profile with higher HDL (P ⫽ 0.02), lower triglyceride level (P ⫽ 0.02), and lower total to HDL-cholesterol ratio (P ⫽ 0.01). There was no difference in fasting insulin or glucose between the two groups. There were no differences between children conceived by conventional IVF and those conceived by ICSI, nor did ovar-

ian stimulation protocol or the type of culture media influence any of the outcome measures. Discussion

We found that children conceived after IVF were taller than expected with this difference most evident in girls. In addition, serum IGF-II levels were higher, and there was a trend toward higher serum IGF-I values in the IVF group. The increased height in IVF children equated to approximately 3 cm and was corrected for parental height, removing the influence of genetic growth potential. It is unlikely that IVF children were taller because of more rapid biological maturation that could lead to earlier puberty and normal adult height. The IVF subjects had estimated bone ages, an index of biological maturity, that matched chronological age and were no different from control children. There are several differences in our IVF cohort compared with that reported by Kai et al. (15), who found that at 5 yr of age, IVF children were the same height as normal control children. The same authors found that a separate longitudinal cohort of children at 3 yr of age conceived by ICSI were shorter than their target height (SDS) compared with controls, which was not evident in the cohort at 5 yr of age (15). Conversely, we found that IVF children conceived after ICSI were just as tall as non-ICSI IVF children and taller than control children. However, Kai et al. (15) included twins, SGA, and prematurely born children in their IVF group, which accounted for 53% of the group. It is well established that SGA children and

TABLE 3. Comparison of fasting serum endocrine and metabolic parameters of control children and children born after IVF

IGF-I (␮g/liter) IGFBP-3 (␮g/liter) IGF-II (␮g/liter) IGFBP-1 (␮g/liter) IGFBP-2 (␮g/liter) GH-binding protein (pmol/liter) Insulin (mU/liter) Glucose (mmol/liter) Total cholesterol (mmol/liter) HDL (mmol/liter) LDL (mmol/liter) Triglycerides (mmol/liter) Total to HDL-cholesterol ratio Values are expressed as means (SEM).

Control

IVF

P value, ANOVA

92.4 (4.3) 4225.1 (125.1) 772.8 (24.0) 121.5 (5.8) 329.3 (23.1) 970.6 (57.0) 5.2 (0.3) 4.8 (0.1) 4.31 (0.08) 1.53 (0.04) 2.42 (0.07) 0.78 (0.04) 2.86 95% CI 关2.72,3.013兴

104.9 (4.4) 4504.2 (124.7) 850.3 (24.2) 104.4 (5.9) 296.7 (23.4) 984.7 (57.4) 5.1 (0.3) 4.7 (0.1) 4.33 (0.08) 1.67 (0.04) 2.37 (0.07) 0.65 (0.04) 2.58 95% CI 关2.44,2.73兴

0.06 0.14 0.03 0.05 0.35 0.87 0.78 0.71 0.86 0.02 0.61 0.02 0.01

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FIG. 1. Distribution of subjects’ heights corrected for parents’ heights [height SDS ⫺ midparental height (MPH) SDS] in control (dashed line) and IVF (solid line) children. SDS values are rounded to 0.5.

prematurely born children are short for their genetic height potential (16 –18); therefore, the IVF subjects in Kai et al. (15) would be predicted to have been shorter than controls rather than the same height as reported. We actively excluded twins and prematurely born children from our cohort, and only one IVF subject was SGA, to remove any confounding effects of these three subgroups (14). In addition, Kai et al. (15) acknowledge that their low IVF participation rate (33% of eligible subjects) could have created an inadvertent selection bias. We achieved an 84% participation rate in our IVF cohort to remove any selection bias. In our IVF cohort, we observed higher serum IGF-II and increasingly higher serum IGF-I with age, without any differences between the ICSI and non-ICSI IVF children. Conversely, Kai et al. (15) found lower serum IGF-I levels in ICSI singleton males and non-ICSI IVF females at 3 months of age; however, these differences were not present in their cohort of 5-yr-old children. The slightly lower birth weight and taller childhood stature in our IVF group suggests a postnatal rather than prenatal growth effect. In a cross-sectional study such as ours, it is difficult to ascertain whether the higher serum IGF-II or pattern of higher IGF-I with increasing age played a role in promoting taller stature in the IVF group. More precise clarification of the role of IGF-I or IGF-II as potentially causative in growth promotion in IVF would require a longitudinal study with detailed auxology and sequential serum hormone measurements. Although all children studied had lipid parameters within the normal range, the IVF children displayed a slightly more favorable lipid profile with higher levels of HDL and lower triglyceride levels. The long-term significance of subtle improvement in lipid profile in IVF children is unclear but warrants further evaluation of adults born after IVF. There are many frequently cited examples of maladaptive responses to an adverse fetal environment; however, the presence of taller stature in IVF children may be the first described example of a better outcome as a result of events during fetal life (19 –21). There are several possible etiological mechanisms that could explain taller stature with hormonal and biochemical differences in IVF children, which include programmed endocrine changes due to the IVF process, selection of the best quality embryos for implantation, or selection bias in the


control population. The alterations in growth and lipid profile suggest that manipulation during the periconceptual period can result in persistent later differences. Observations in animal studies lend support to this concept with both nutritional manipulation at the time of conception or in vitro culture affecting later outcome (1, 7, 22–24). Thus, we propose that in vitro manipulation has resulted in persistent and possibly permanent metabolic alterations in the IVF children we studied. Although the etiology of the growth and metabolic differences in IVF children is yet to be determined, it is possible that the IVF process leads to epigenetic alteration of imprinted and nonimprinted genes. Beckwith-Wiedemann syndrome has been reported to occur up to nine times more commonly after IVF conception than in the general population (5, 25–27). In a metaanalysis, 23 of the 24 cases of Beckwith-Wiedemann syndrome after IVF were found to be due to hypomethylation of the Beckwith-Wiedemann syndrome imprinted region KvDMR1 (1). Given that our cohort was taller, it is possible that these children have more subtle alterations in DNA methylation patterns in imprinted genes associated with growth such as IGF-II at H19 or KvDMR1. Mild phenotypic features, such as taller stature in childhood with normal birth weight, have been described in relatives of children with Beckwith-Wiedemann syndrome (9). A potential risk to drawing conclusions in any study of this kind is the applicability of the control group. Potential confounders influencing growth are genetic potential (or parental heights), socioeconomic background, nutritional status, and pubertal stage. We have tried to address this in our study design by attempting to ensure the two groups are similar in all respects. IVF children in New Zealand tend to be born to parents of higher socioeconomic background. To ensure appropriate matching, control subjects were of the same school decile code and similar residential address to remove any influence of socioeconomic status. Parental heights between the groups were similar as was nutritional status (based on body composition). All children were confirmed to be prepubertal, eliminating the effects of sex steroids on stature and body composition. We are aware that the IVF group represents a cohort of much wanted children and that parental care may reflect this; however, we are not aware of any studies suggesting that stature is affected by increased parental attention in families of higher socioeconomic status. The number of single-parent families was the same in both groups (one per group). In those firstborn, a small increase in adult height in women of 2 mm and in men 7 mm has been reported (28). These differences in height may be due to other stronger influences such as differences in parental heights and environmental factors. The authors stated that birth order was powerfully associated with all other environmental ratings and encapsulated much of their influence. Conversely, we found that birth order did not influence any of the parameters measured within either the IVF or control groups. Dietary records revealed similar eating patterns in both IVF and control children. Collectively, the extensive exclusion criteria used and lack of effect with the few different characteristics between the groups suggests that our findings do not reflect selection bias. Although every effort has been made to match IVF and control subjects, it remains


conceivable that unrecognized potential confounders may still exist that could have influenced postnatal growth. The ideal study design would be to randomize couples to IVF or naturally conceived pregnancies and study the growth patterns of the offspring. However, this would be impossible to achieve; thus, all studies of IVF children may be confounded by unrecognized factors. Furthermore, the availability of cross-sectional rather than longitudinal growth data from birth prevents us from fully understanding the early growth pattern of IVF children. In summary, we have shown that prepubertal children born after IVF are taller when adjusted for parents’ heights with higher serum IGF-II, IGF-I, and IGF-I to IGFBP3 ratio and more favorable lipid profile than naturally conceived control children. We propose that the differences in stature, growth factors, and lipid metabolism observed in our study may be due to subtle epigenetic alteration of imprinted genes or other genes that undergo epigenetic modification that are involved in growth and development. Despite the birth of 2 million IVF children and substantial data from birth registries, little is known about IVF children beyond the risk of congenital abnormalities. Comparisons of our data with other groups using different IVF technology will be important to define whether the changes we observed are modified with different methodologies. This study demonstrates the need to monitor the health of IVF offspring through childhood into adulthood. Acknowledgments We thank the staff at Fertility Associates, Auckland; Nathalie Billett, our research nurse; and Dr. Sally Vogel, Pediatric Radiologist, Starship Hospital, Auckland, for their help with this study. Received November 9, 2006. Accepted June 6, 2007. Address all correspondence and requests for reprints to: Associate Professor Wayne Cutfield, The Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: [email protected]. This work was supported by a National Research Centre for Growth and Development grant. H.L.M., P.L.H., M.H., D.W., E.M.R., P.D.G., and W.S.C. have nothing to declare. J.P. is employed as scientific director for Fertility Associates. None of the authors have a competing interest to declare.

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.