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Journal of Neonatal-Perinatal Medicine 6 (2013) 101–108 DOI 10.3233/NPM-1366812 IOS Press
Review Article
The impact of assisted reproductive technologies on the genome and epigenome of the newborn A. Kochanskia , T.A. Merrittb,c,∗ , J. Gadzinowskic and A. Jopekc a Neuromuscular b Division
Unit, Mossakowski Medical Center, Polish Academy of Science, Warsaw, Poland of Neonatology, Department of Pediatrics, Loma Linda University School of Medicine, Loma Linda,
CA, USA c Department of Neonatology, Poznan University of Medical Sciences, Poznan, Poland
Received 27 October 2012 Revised 28 December 2012 Accepted 08 January 2013
Abstract. The question of genetic alterations resulting from assisted reproductive technologies (ART) in humans is examined within the organization of the human genome. Increased rates of birth defects have been reported among children conceived using ART; however, questions remain and controversy exists regarding how “infertility” predisposes to birth defects. ART has been shown to be associated with an increased number of chromosomal alterations especially in the X chromosome. There is increased risk for embryonal tumors among ART conceived children, as well as, imprinting disorders (Beckwith-Wiedemann and Angelman Syndromes). Genetic studies of children conceived using ART reveal a larger (genome-wide) scale of methylation defects that encompass hundreds of genes. Genes involved in carcinogenesis and developmental pathways appear altered and may impact on later development of chronic illness, although these data are very preliminary. ART may create novel mutations by different chromosomal and molecular mechanisms; however, these techniques also enable propagation of pre-existing mutations that are associated with impaired fertility. While older maternal age is often associated with female infertility and chromosomal aneuploidy, sperm from older men have more new gene mutations. The prevalence of birth defects is increased when ART is used for conception. These data are summarized by large metaanalyses or from multi-year national registries. Whether the increased number of birth defects is due to ART procedures themselves or are a consequence of the impaired fertility of the parents is discussed. Long-term evaluation of children conceived using ART and/or ovarian hyper-stimulation is needed to determine whether alterations during embryonic development may increase the prevalence of chronic diseases in adulthood. Keywords: In vitro fertilization, intracytoplasmic sperm injection, imprinting disorders, birth defects, methylation defects, infertility
1. Introduction ∗ Corresponding
author: Dr. T. Allen Merritt, Division of Neonatology, 11175 Campus St, Coleman Pavilion 11121, Loma Linda University, Loma Linda, CA, 92354, USA. Tel.: +1 909 558 7448; Fax: +1 909 558 0298; E-mail: [email protected].
Louise Brown, the first and therefore oldest individual conceived using in vitro fertilization, is now 34 years old and has successfully given birth. This major
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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scientific breakthrough has resulted in broader application of assisted reproductive technologies (ART) especially among more affluent populations in the developed world. The percentage of ART-conceived children in the population varies by country, but in European countries and the USA births by ART contributes to about 1.4% of all live births annually [1]. Although ART registries exist in some countries, ART-conceived infants are not systematically examined throughout childhood to monitor for neoplasms, metabolic disorders, developmental or psychiatric diseases. Well-organized birth defect registries dedicated to evaluation of ART-conceived children for birth defects exist in Europe and Australia, but not in North America; the period of observation usually does not exceed 12 months [2]. The initial results of studies regarding the genetic safety of ART were published in 2002, however, appreciation of an increased prevalence of imprinting disorders and birth defects has emerged only in the last 3–4 years. Notably, the higher prevalence of Beckwith-Weidemann Syndrome (BWS) in ART conceptions was not detected through registries, but rather through focused studies on cases of BWS within the general population [3, 4] with subsequent recognition of the use of ART for their conception. Additionally, the increased rates of prematurity and multiple births associated with ART has had an impact on neonatal intensive care services by increasing the total utilization of health care resources for these infants [1]. In some NICUs infant’s conceived using these technologies represents a substantial portion (19%) of their admissions [5], and the associated morbidities of prematurity and the financial and emotional cost of NICU care is often unrecognized. Thus a review of the impact of ART on neonatal/perinatal medicine is warranted.
2. Potential effect on the human genome Genetic disturbances in children born of women undergoing ART have recently been identified using an epigenetic (factors influencing the regulation of the expression of an individual’s gene code) approach focused on DNA methylation [6–8], although these findings have not been universal [9]. Complete and reliable analyses of these studies have revealed the impact of ART on genes that are the “deciphered” by the human epigenome. Although the Human Genome Project sequenced the complete human genome in
2001, the International Human Epigenome Consortium (IHEC) launched in 2010 plans to map 4000 human “reference epigenomes” until 2020 [10]. While the majority of epigenetic studies concern the process of DNA methylation, disorders of methylation such as chromatin dys-regulation are only one of many factors, including histone deacylation and inhibitory messenger RNA that may modify the regulation of genetic expression, as well as, dietary factors and toxins within the environment [11]. There are limited studies on the impact of ART on the human genome and epigenome. In this review we use a hierarchical approach to review the effects of ART on the human genome, other epigenetic factors that may contribute to the increased prevalence of birth defects documented in infants conceived using these technologies.
3. ART technology and the transmission of Pre-existing mutations The pool of deleterious mutations in human genes and chromosomes abnormalities exists in a state of balance as some genetic defects are eliminated and novel mutations are introduced with each generation. Nearly 38% of early spontaneous miscarriages (through the 10th week of gestation) are associated with genetic factors [12, 13]. Thus the vast majority of chromosomal alternations are not transmitted to the next generation due to infertility and the natural process of pregnancy termination that occurs during early human development. In humans, many chromosomal defects are eliminated at the pre-zygotic or early post-zygotic state of development also as spontaneous miscarriages. Unbalanced chromosome translocation is the most common chromosomal anomaly found in fetuses of couples experiencing early pregnancy loss [12]. Chromosomal alterations are also detected in male infertility [13] with associated oligospermia and azospermia (24%). ART procedures are performed in both males and females who present with limited fertility facilitate the development of zygotes from gametes harboring chromosomal defects [14]. Similarly, ART may enable transmission of deleterious point mutations by enhancing fertility. For example, mutations that cause cystic fibrosis (CF), the most common autosomal recessive disorder among Caucasians especially those of Northern European descent [16], may be transmitted to the next generation when ART is used to treat the naturally
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occurring infertility in this population. CF in males is associated with infertility owing to congenital absence or obstruction of the vas deference. Among most males harboring mutations in the CFTR gene of the “classic description” (delta F508), males are often infertile [17]. In genetic terms, the infertile male harboring two mutations in the CFTR gene may transmit two different CTRF mutations to the next generation. Use of testicular fine needle sperm aspiration and/or microsurgical epididymal sperm aspiration permits capture of sperm that can be used in ART to achieve conception; thus when using these male ART procedures, the barrier or blockage of the vas deferens is bypassed. The question of whether an abnormal pattern of gene methylation in gametes alters embryonic growth remains unanswered. However, studies on methylation of the PgK-2, Apo-A1, and Oc-3/4 genes in mouse gametes residing in the epididymis and testicular tissues have showed that there are marked differences in their methylation profiles [18]. It is suspected, though not confirmed, that gametes obtained using MESA in CF affected males are disturbed by mutation of the CFTR gene, with an aberrant methylation profile of the gametes. The use of ART procedures to assist procreation among males with CF illustrates the potential for transmission of pre-existing mutations to subsequent generations. Thus it should be obligatory to inform infertile prospective fathers and mothers that using their own gametes could increase the risk of transmission of pre-existing mutations to their offspring.
4. ART and the creation of novel mutations Novel mutations may be created using ART especially the use of intracytoplasmic sperm injection (ICSI) techniques and these may not be predictable. A study of 1586 children conceived using ART found the rate of de novo chromosomal anomalies to be approximately1.6% [15], and higher than that reported among naturally conceived children. The majority of the chromosomal modifications among ART conceived children occur on the X chromosome [18], in contrast to autosomal abnormalities that are often lethal. The mechanism of X chromosomal alterations is unclear. One hypothesis suggests that the “accuracy” of meiosis is dependent on the methylation status of the repetitive DNA sequences disperse with the human genome in which methylation is conserved.
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Bourc’his and Bestor documented the meiotic consequences of demethylation of these repetitive sequences as a “meiotic catastrophe” [20, 21], and concluded that global demethylation of DNA sequences may result in a generalized disruption of chromosomal architecture during meiotic division. The findings of Bourc’his and Bestor [20] suggest a link between the higher rate of de novo chromosomal anomalies in ART conceived children and the observed increase in birth defects. To verify this hypothesis, studies of the level of methylation of repetitive DNA sequences are required in larger populations of ART-conceived children that identify de novo chromosomal changes. It is possible that de novo chromosomal modifications among ART conceived children are induced by molecular mechanisms apart from methylation or alternations of repetitive DNA sequences. Patsalis and coworkers found coexistence of deletions AZF regions on the Y chromosome and monosomy/mosaicism of the X chromosome (e.g. 45 X), 45 X)/46 XY) [22]. These authors provide evidence for ART-transmission of the Y chromosome with deletions and the X chromosome that may cause additional genetic defects of the X chromosome. The mechanism between AZF region deletions and X chromosome alterations are presently unknown. In contrast to “classical” mutations dispersed within genes, epimutations are not associated with alternations in the DNA sequence per se. Rather these epimutations result from chemical modifications of DNA that do not affect the DNA sequence. These can result from exposure to toxins (e.g. tobacco smoke) and other environmental toxins [23]. This is best understood by the mechanism of epimutation or the methylation of cytosine residues with the DNA sequence. Methylation of cytosine is catalyzed by DNA methyltransferases that are highly conserved. Epimutations may be transmitted forward through future generations. To illustrate this propagation, a Japanese family with Silver-Russell Syndrome offers this example [24]. The father of twins conceived using in vitro fertilization was shown to carry an epimutation with different methylated regions in the PEGi/MEST gene. Four cytosine residues were methylated, whereas in a normal population this region is demethylated [24]. Interestingly, when the epimutation occurs in the normally demethylated region of the PEGi/MEST gene and is transmitted during meiosis, four additional cytosine residues are methylated causing a new epimutation containing eight methylated cytosine residues.
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This is considered the etiology for the Silver-Russell Syndrome in the ART conceived child [25]. While it has been understood that new mutations occur that are not heritable, this can be quantified now by sequencing sperm DNA and comparing that to the germ-line DNA of the father. There have been multiple reports demonstrating the link between de novo mutations and intellectual disability, schizophrenia, and autism. This research comes from the Icelandic group DECODE which linked the father’s age with the incidence of such de novo mutations [26–29]. These new mutations are not frequent but if there is just one mutation per exome (or even in one exome) throughout the coding elements of the genome these can be quite damaging. Characteristically, de novo mutations that are expressed have been associated with adverse consequences [26]. While there are many more in a typical genome, somewhere around 70 or more of various types of mutations, whether they are single nucleotide polymorphisms or insertions/deletions most of do not fall within protein coding elements. However, current research suggests that they can be damaging [27, 28]. It is unclear whether these de novo mutations are related to our environment or paternal behaviors such as smoking. While it may be possible to screen sperm for de novo mutations, it seems unlikely that in the future all sperm used for either in vitro fertilization or ICSI (including the donor sperm) or sperm from older men will be evaluation for these epimutations. At this time there are data to suggest the high-risk age range is paternal age >40 years [27–29]. Future research will be needed to determine whether these de novo mutations will exert a specific phenotype. It is also possible that some de novo mutations could potentially have a beneficial role [28]. While previous attention has been focused on the mother’s “biological clock” as a cause for infertility, paternal de novo mutations require a new focus on the father’s biological clock and the potential paternal origin of birth defects and other disabilities [29].
5. ART and defects in genomic imprinting Geneticists have reported an increased incidence of selected genomic imprinting defects in children conceived using ART. For example, BeckwithWiedemann Syndrome (BWS) occurs in 1:14,000 in the general population, but occurs with a 4.2 greater frequency among ART conceived children [30]. Char-
acterized by large body mass, organ hypertrophy, and umbilical hernia and neonatal hypoglycemia these infants are prone to develop embryonal tumors (e.g. Wilm’s tumor) [31] in the naturally conceived population. BWS is caused by loss of methylation of the KvDMR gene in 50%, paternal uniparental disomy at 11p15.5 (20–30%) and rare mutations occur in another 10% [31]. In 13–15% of BWS the molecular defect is unknown. However, among ART conceived infants nearly 95% of those with BWS have demethylation of the KvDMR gene [31]. The apparent lack of genetic heterogeneity of BWS among ART conceived infants suggest the existence of a molecular mechanism associated with ART that is not limited to demethylation of 11p15.5, but to more generalized hypomethylation with associated unique clinical features [31]. Angelman Syndrome (AS) is another imprinting defect located at 15 q11-13. Most cases of AS are associated with maternal deletions located in 15q11-13 or paternal uniparental disomy of this region of 15q [32, 33]. AS has been reported in higher frequency among ART conceived infants from sub-fertile couples. In the ART conceived infants hypomethylation of 15q11-13 and not uniparental disomy is found with greater more frequency than in the naturally conceived population [34].
6. ART and genome-wide methylation disorders Recent studies comparing ART-conceived and naturally conceived children have reported significant differences in blood pressure, distribution of adipose tissue, and thyroid function [35, 36]. Whether ART conceived children will later manifest the metabolic syndrome is an area of current study. Genes that may be influenced by ART related embryonic culture and manipulation may not be vulnerable simply to focal areas of genetic imprinting but may be at risk for more widely spread hypomethylation. Indeed, methylation of DNA is not limited to specific sets of genes, rather involves thousands of CpG sequences or CpG “islands” that are dispersed throughout the genome but highly localized within regulatory regions. The pattern of methylation of CpG “islands” is not stable and may change throughout development from embryo through adulthood. A pattern of global genome-wide methylation disturbances has been verified in ART conceived children compared to those naturally conceived [36].
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To test whether demethylation/hypomethylation process of the genes was global or more localized 736 genes were studied and important methylation differences between ART children were contrasted with naturally conceived children [37]. However, more recent studies find contradictory results [38].
7. Birth defects and ART Controversy exists regarding whether ART procedure themselves are associated with higher rates of birth defects or whether the increased rates reported are primarily due to the infertility of the parents or other factors such as ovarian stimulation used by most fertility specialists prior to oocyte “harvest” for either in vitro fertilization or ICSI. Hansen and coworkers report that infants conceived using ART have a doubling of major birth defects [39]. The literature from the 1990 s dismissed these observed differences in rates of birth defects because the samples sizes were small and some studies failed to report a control group [40]. Green reported from a population based, multicenter, case-control study of birth defects and found that malformations occurred more often in infants conceived using ART [41]. Rimm and coworkers found an increased odds ratio of 1.29 and 1.31, respectively (or about a 30% increase) compared to the general population among infants conceived using ART [42]. Wen and coworkers reported in an extensive meta-analysis in 2012 that compared 56 separate studies showing the increase in birth defects conceived by either in vitro fertilization or ICSI [43]. Their pooled risk estimation was 1.37 (95% confidence intervals 1.26–1.48), and they further found no difference in the increase risk by ART modality (in vitro versus ICSI). A report of the Australian registry in 2012 [44], noted an unadjusted odds ratio of 1.47 (95% confidence interval CI = 1.33–1.62) for birth defects among infants born using ART and found a greater risk associated with ICSI. When this increased risk was adjusted using multivariate analysis, the odds ratio was reduced to 1.28 (95% confidence interval 1.07–1.28). These authors suggest that a parent’s history of infertility was also associated with a slightly increased risk for birth defects as previously reported from a Danish cohort [45]. Hansen and coworkers report from three ART clinics in Western Australia that ART mothers were older, less likely to have had a previous child, and less likely to smoke tobacco than non-ART mothers; however, their infants
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were more likely to have low birth weight and born prematurely whether born as singletons or twins [46]. When two periods of ART were compared, 1994–1998 vs. October 1998 to 2002, the rate of birth defects was 8.7% compared to 5.4% among singletons conceived. The odds ratios decreased slightly when adjusted for maternal age (OR = 1.53; 95% CI = 1.30–1.79). These investigators found little difference in birth defect risk when ICSI or in vitro singletons were compared (OR = 1.09; 95% CI = 0.77–1.54). These authors compared the two time frames and they found a decrease in birth-defect prevalence for both in vitro and ICSI (11.4%–6.2%, p = 0.001) and to a lesser degree among twins irrespective of whether fresh or frozen-thawed embryos were used. In addition, termination of pregnancy for fetal anomaly in non-assisted reproductive technology singletons was 5.8/1000 births contrasted to 9.7/1000 births among ART conceived singletons. These authors summarize that that multiple changes in the ART procedures and embryo incubation conditions or use of recombinant gonadotropins for ovarian stimulation may explain the reduction in birth defects over time, although the exact cause remains unexplained [46]. Overall, women who conceive using ART have a higher proportion of infants with birth defects, although some argue that this increased rate is inherent among couples with a history of infertility. Undoubtedly this will remain an area of controversy, but one worthy of discussion with parents whose aspirations are for a “normal baby born at term” when discussing the risks and benefits of ART [46]. A significant impact of ART, especially in the U.S.A., has been the rising multiple gestation birth rate. In 2008, according to the Centers for Disease Control and Prevention, twin births rose to the unprecedented rate of 32.2 twins per 1000 total birth. Triplet and higher order births have risen more than 400% [47–49]. The longer-term outcomes of children conceived using ART have been carefully reviewed by Willson and coworkers [50]. Their review found that the physical and psychosocial health outcomes in adolescents and young adults are generally comparable to those to those conceived naturally. However, higher blood pressures, elevated fasting blood glucose, increased bone age, elevated serum luteinizing hormone and dehydroepiandrosterone sulphate, as well as, increased growth velocity during infancy was found in cohorts of infants and children conceived using ART [51, 52]. As carefully noted by these authors longer term followup will be needed to determine whether there are
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negative “downstream” effects of ART, with a special focus on rates of neurologic dysfunction, occurrence of the “metabolic syndrome”, or cancers suggestive of concerns regarding fetal origins of adult chronic disease [53]. Encouraging data regarding the psychological well-being of children conceived using ART have been reported; although, as emphasized by Willson et al. the numbers and duration of longitudinal follow-up are small and too short to arrive at definitive conclusions. Kallen et al. [54] report among Swedish children conceived using in vitro fertilization there is an elevated rate of asthma that has been suggested to result from alterations in maternal immune function in infertile women by Davies [55].
8. Conclusion The influence of ART on specific genes, the genome, and the epigenome in humans is now without question. However, more research needs to be done to understand the full impact of these technologies on birth outcomes and the overall health of children born after ART. Indeed, whether fetal programming is affected by the method of conception is presently unknown. ART has increased the prevalence of imprinting disorders and some data from large meta-analysis confirm an increased risk of some birth defects. The present state of knowledge cannot determine whether alternations in single genes, small groups of genes, hypomethylation of genes, or selective epigenetic factors or a combination of these factors may influence the outcome of pregnancies conceived using various forms of ART. The finding of selective morbidities of infants conceived using ART suggest that there are maternal and paternal factors that are emerging as modifiers of fetal epigenetic status which may have implications for long-term health of these infants, in addition to the morbidities associated with being low birth weight and more frequently premature. The 34-year period of observation after the first “in vitro” baby has been too short to fully understand the changes that may manifest themselves in later life. Application of these technologies has expanded exponentially over the last 3 decades. Registries designed to enroll infants conceived using these technologies and collect data on long-term follow-up will yield meaningful results for both physicians providing these ART services, neonatologists caring for many of these infants, geneticists who counsel parents. Parents with
infertility need to fully understand the benefits, as well as, the risks of these procedures when seeking treatment for infertility using ART. Acknowledgments The co-authors acknowledge that Poznan University of Medical Sciences and Loma Linda University School of Medicine have Cooperative Agreement under which this review was prepared by faculty from both institutions. The author’s appreciate the comments of the reviewers and acknowledge the careful editing of this manuscript by Robin Clark, M.D., Associate Professor of Medical Genetics at Loma Linda University Children’s Hospital, Loma Linda, California, USA. Financial disclosure The authors have nothing to disclose and have no conflicts of interest and further attest that they have no financial interest in the subject matter discussed within this manuscript.
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Willson CL, Fisher JR, Hammarberg K, Amor DJ, Halliday JL. Looking downstream: A review of the literature on physical and psychosocial health outcomes in adolescents and young adults who were conceived by ART. Hum Reprod 2011;26(5):1209-19. Ceelen M. Growth during infancy and early childhood in relations to blood pressure and body fat measure at age 8–18 of IVF children and spontaneously conceived controls born to subfertile parents. Hum Reprod 2009;24:2788-8 Ceelen M, van Weissenbruch M, Vermeiden J, van Leeuwen F, Delemarreo van de Waal H. Pubertal development in children and adolescents born after IVF and spontaneous conception. Hum Reprod 2008;23:2791-8. Gluckman PD, Hanson MA. The developmental origins of the metabolic syndrome. Trends Endocrinol Metab 2004;15(4):183-7. K¨all´en B, Finnstro¨om O, Nygren K-G, Otterblad P. Asthma in Swedish children conceived by in vitro fertilization. Arch Dis Child 2013;98:92-6. Davies MJ. Infertility treatment at the edge: Discovery and risk converge at the limits of knowledge. Arch Dis Child 2013;98:89-90.
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Journal of Neonatal-Perinatal Medicine 6 (2013) 101–108 DOI 10.3233/NPM-1366812 IOS Press
Review Article
The impact of assisted reproductive technologies on the genome and epigenome of the newborn A. Kochanskia , T.A. Merrittb,c,∗ , J. Gadzinowskic and A. Jopekc a Neuromuscular b Division
Unit, Mossakowski Medical Center, Polish Academy of Science, Warsaw, Poland of Neonatology, Department of Pediatrics, Loma Linda University School of Medicine, Loma Linda,
CA, USA c Department of Neonatology, Poznan University of Medical Sciences, Poznan, Poland
Received 27 October 2012 Revised 28 December 2012 Accepted 08 January 2013
Abstract. The question of genetic alterations resulting from assisted reproductive technologies (ART) in humans is examined within the organization of the human genome. Increased rates of birth defects have been reported among children conceived using ART; however, questions remain and controversy exists regarding how “infertility” predisposes to birth defects. ART has been shown to be associated with an increased number of chromosomal alterations especially in the X chromosome. There is increased risk for embryonal tumors among ART conceived children, as well as, imprinting disorders (Beckwith-Wiedemann and Angelman Syndromes). Genetic studies of children conceived using ART reveal a larger (genome-wide) scale of methylation defects that encompass hundreds of genes. Genes involved in carcinogenesis and developmental pathways appear altered and may impact on later development of chronic illness, although these data are very preliminary. ART may create novel mutations by different chromosomal and molecular mechanisms; however, these techniques also enable propagation of pre-existing mutations that are associated with impaired fertility. While older maternal age is often associated with female infertility and chromosomal aneuploidy, sperm from older men have more new gene mutations. The prevalence of birth defects is increased when ART is used for conception. These data are summarized by large metaanalyses or from multi-year national registries. Whether the increased number of birth defects is due to ART procedures themselves or are a consequence of the impaired fertility of the parents is discussed. Long-term evaluation of children conceived using ART and/or ovarian hyper-stimulation is needed to determine whether alterations during embryonic development may increase the prevalence of chronic diseases in adulthood. Keywords: In vitro fertilization, intracytoplasmic sperm injection, imprinting disorders, birth defects, methylation defects, infertility
1. Introduction ∗ Corresponding
author: Dr. T. Allen Merritt, Division of Neonatology, 11175 Campus St, Coleman Pavilion 11121, Loma Linda University, Loma Linda, CA, 92354, USA. Tel.: +1 909 558 7448; Fax: +1 909 558 0298; E-mail: [email protected].
Louise Brown, the first and therefore oldest individual conceived using in vitro fertilization, is now 34 years old and has successfully given birth. This major
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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scientific breakthrough has resulted in broader application of assisted reproductive technologies (ART) especially among more affluent populations in the developed world. The percentage of ART-conceived children in the population varies by country, but in European countries and the USA births by ART contributes to about 1.4% of all live births annually [1]. Although ART registries exist in some countries, ART-conceived infants are not systematically examined throughout childhood to monitor for neoplasms, metabolic disorders, developmental or psychiatric diseases. Well-organized birth defect registries dedicated to evaluation of ART-conceived children for birth defects exist in Europe and Australia, but not in North America; the period of observation usually does not exceed 12 months [2]. The initial results of studies regarding the genetic safety of ART were published in 2002, however, appreciation of an increased prevalence of imprinting disorders and birth defects has emerged only in the last 3–4 years. Notably, the higher prevalence of Beckwith-Weidemann Syndrome (BWS) in ART conceptions was not detected through registries, but rather through focused studies on cases of BWS within the general population [3, 4] with subsequent recognition of the use of ART for their conception. Additionally, the increased rates of prematurity and multiple births associated with ART has had an impact on neonatal intensive care services by increasing the total utilization of health care resources for these infants [1]. In some NICUs infant’s conceived using these technologies represents a substantial portion (19%) of their admissions [5], and the associated morbidities of prematurity and the financial and emotional cost of NICU care is often unrecognized. Thus a review of the impact of ART on neonatal/perinatal medicine is warranted.
2. Potential effect on the human genome Genetic disturbances in children born of women undergoing ART have recently been identified using an epigenetic (factors influencing the regulation of the expression of an individual’s gene code) approach focused on DNA methylation [6–8], although these findings have not been universal [9]. Complete and reliable analyses of these studies have revealed the impact of ART on genes that are the “deciphered” by the human epigenome. Although the Human Genome Project sequenced the complete human genome in
2001, the International Human Epigenome Consortium (IHEC) launched in 2010 plans to map 4000 human “reference epigenomes” until 2020 [10]. While the majority of epigenetic studies concern the process of DNA methylation, disorders of methylation such as chromatin dys-regulation are only one of many factors, including histone deacylation and inhibitory messenger RNA that may modify the regulation of genetic expression, as well as, dietary factors and toxins within the environment [11]. There are limited studies on the impact of ART on the human genome and epigenome. In this review we use a hierarchical approach to review the effects of ART on the human genome, other epigenetic factors that may contribute to the increased prevalence of birth defects documented in infants conceived using these technologies.
3. ART technology and the transmission of Pre-existing mutations The pool of deleterious mutations in human genes and chromosomes abnormalities exists in a state of balance as some genetic defects are eliminated and novel mutations are introduced with each generation. Nearly 38% of early spontaneous miscarriages (through the 10th week of gestation) are associated with genetic factors [12, 13]. Thus the vast majority of chromosomal alternations are not transmitted to the next generation due to infertility and the natural process of pregnancy termination that occurs during early human development. In humans, many chromosomal defects are eliminated at the pre-zygotic or early post-zygotic state of development also as spontaneous miscarriages. Unbalanced chromosome translocation is the most common chromosomal anomaly found in fetuses of couples experiencing early pregnancy loss [12]. Chromosomal alterations are also detected in male infertility [13] with associated oligospermia and azospermia (24%). ART procedures are performed in both males and females who present with limited fertility facilitate the development of zygotes from gametes harboring chromosomal defects [14]. Similarly, ART may enable transmission of deleterious point mutations by enhancing fertility. For example, mutations that cause cystic fibrosis (CF), the most common autosomal recessive disorder among Caucasians especially those of Northern European descent [16], may be transmitted to the next generation when ART is used to treat the naturally
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occurring infertility in this population. CF in males is associated with infertility owing to congenital absence or obstruction of the vas deference. Among most males harboring mutations in the CFTR gene of the “classic description” (delta F508), males are often infertile [17]. In genetic terms, the infertile male harboring two mutations in the CFTR gene may transmit two different CTRF mutations to the next generation. Use of testicular fine needle sperm aspiration and/or microsurgical epididymal sperm aspiration permits capture of sperm that can be used in ART to achieve conception; thus when using these male ART procedures, the barrier or blockage of the vas deferens is bypassed. The question of whether an abnormal pattern of gene methylation in gametes alters embryonic growth remains unanswered. However, studies on methylation of the PgK-2, Apo-A1, and Oc-3/4 genes in mouse gametes residing in the epididymis and testicular tissues have showed that there are marked differences in their methylation profiles [18]. It is suspected, though not confirmed, that gametes obtained using MESA in CF affected males are disturbed by mutation of the CFTR gene, with an aberrant methylation profile of the gametes. The use of ART procedures to assist procreation among males with CF illustrates the potential for transmission of pre-existing mutations to subsequent generations. Thus it should be obligatory to inform infertile prospective fathers and mothers that using their own gametes could increase the risk of transmission of pre-existing mutations to their offspring.
4. ART and the creation of novel mutations Novel mutations may be created using ART especially the use of intracytoplasmic sperm injection (ICSI) techniques and these may not be predictable. A study of 1586 children conceived using ART found the rate of de novo chromosomal anomalies to be approximately1.6% [15], and higher than that reported among naturally conceived children. The majority of the chromosomal modifications among ART conceived children occur on the X chromosome [18], in contrast to autosomal abnormalities that are often lethal. The mechanism of X chromosomal alterations is unclear. One hypothesis suggests that the “accuracy” of meiosis is dependent on the methylation status of the repetitive DNA sequences disperse with the human genome in which methylation is conserved.
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Bourc’his and Bestor documented the meiotic consequences of demethylation of these repetitive sequences as a “meiotic catastrophe” [20, 21], and concluded that global demethylation of DNA sequences may result in a generalized disruption of chromosomal architecture during meiotic division. The findings of Bourc’his and Bestor [20] suggest a link between the higher rate of de novo chromosomal anomalies in ART conceived children and the observed increase in birth defects. To verify this hypothesis, studies of the level of methylation of repetitive DNA sequences are required in larger populations of ART-conceived children that identify de novo chromosomal changes. It is possible that de novo chromosomal modifications among ART conceived children are induced by molecular mechanisms apart from methylation or alternations of repetitive DNA sequences. Patsalis and coworkers found coexistence of deletions AZF regions on the Y chromosome and monosomy/mosaicism of the X chromosome (e.g. 45 X), 45 X)/46 XY) [22]. These authors provide evidence for ART-transmission of the Y chromosome with deletions and the X chromosome that may cause additional genetic defects of the X chromosome. The mechanism between AZF region deletions and X chromosome alterations are presently unknown. In contrast to “classical” mutations dispersed within genes, epimutations are not associated with alternations in the DNA sequence per se. Rather these epimutations result from chemical modifications of DNA that do not affect the DNA sequence. These can result from exposure to toxins (e.g. tobacco smoke) and other environmental toxins [23]. This is best understood by the mechanism of epimutation or the methylation of cytosine residues with the DNA sequence. Methylation of cytosine is catalyzed by DNA methyltransferases that are highly conserved. Epimutations may be transmitted forward through future generations. To illustrate this propagation, a Japanese family with Silver-Russell Syndrome offers this example [24]. The father of twins conceived using in vitro fertilization was shown to carry an epimutation with different methylated regions in the PEGi/MEST gene. Four cytosine residues were methylated, whereas in a normal population this region is demethylated [24]. Interestingly, when the epimutation occurs in the normally demethylated region of the PEGi/MEST gene and is transmitted during meiosis, four additional cytosine residues are methylated causing a new epimutation containing eight methylated cytosine residues.
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This is considered the etiology for the Silver-Russell Syndrome in the ART conceived child [25]. While it has been understood that new mutations occur that are not heritable, this can be quantified now by sequencing sperm DNA and comparing that to the germ-line DNA of the father. There have been multiple reports demonstrating the link between de novo mutations and intellectual disability, schizophrenia, and autism. This research comes from the Icelandic group DECODE which linked the father’s age with the incidence of such de novo mutations [26–29]. These new mutations are not frequent but if there is just one mutation per exome (or even in one exome) throughout the coding elements of the genome these can be quite damaging. Characteristically, de novo mutations that are expressed have been associated with adverse consequences [26]. While there are many more in a typical genome, somewhere around 70 or more of various types of mutations, whether they are single nucleotide polymorphisms or insertions/deletions most of do not fall within protein coding elements. However, current research suggests that they can be damaging [27, 28]. It is unclear whether these de novo mutations are related to our environment or paternal behaviors such as smoking. While it may be possible to screen sperm for de novo mutations, it seems unlikely that in the future all sperm used for either in vitro fertilization or ICSI (including the donor sperm) or sperm from older men will be evaluation for these epimutations. At this time there are data to suggest the high-risk age range is paternal age >40 years [27–29]. Future research will be needed to determine whether these de novo mutations will exert a specific phenotype. It is also possible that some de novo mutations could potentially have a beneficial role [28]. While previous attention has been focused on the mother’s “biological clock” as a cause for infertility, paternal de novo mutations require a new focus on the father’s biological clock and the potential paternal origin of birth defects and other disabilities [29].
5. ART and defects in genomic imprinting Geneticists have reported an increased incidence of selected genomic imprinting defects in children conceived using ART. For example, BeckwithWiedemann Syndrome (BWS) occurs in 1:14,000 in the general population, but occurs with a 4.2 greater frequency among ART conceived children [30]. Char-
acterized by large body mass, organ hypertrophy, and umbilical hernia and neonatal hypoglycemia these infants are prone to develop embryonal tumors (e.g. Wilm’s tumor) [31] in the naturally conceived population. BWS is caused by loss of methylation of the KvDMR gene in 50%, paternal uniparental disomy at 11p15.5 (20–30%) and rare mutations occur in another 10% [31]. In 13–15% of BWS the molecular defect is unknown. However, among ART conceived infants nearly 95% of those with BWS have demethylation of the KvDMR gene [31]. The apparent lack of genetic heterogeneity of BWS among ART conceived infants suggest the existence of a molecular mechanism associated with ART that is not limited to demethylation of 11p15.5, but to more generalized hypomethylation with associated unique clinical features [31]. Angelman Syndrome (AS) is another imprinting defect located at 15 q11-13. Most cases of AS are associated with maternal deletions located in 15q11-13 or paternal uniparental disomy of this region of 15q [32, 33]. AS has been reported in higher frequency among ART conceived infants from sub-fertile couples. In the ART conceived infants hypomethylation of 15q11-13 and not uniparental disomy is found with greater more frequency than in the naturally conceived population [34].
6. ART and genome-wide methylation disorders Recent studies comparing ART-conceived and naturally conceived children have reported significant differences in blood pressure, distribution of adipose tissue, and thyroid function [35, 36]. Whether ART conceived children will later manifest the metabolic syndrome is an area of current study. Genes that may be influenced by ART related embryonic culture and manipulation may not be vulnerable simply to focal areas of genetic imprinting but may be at risk for more widely spread hypomethylation. Indeed, methylation of DNA is not limited to specific sets of genes, rather involves thousands of CpG sequences or CpG “islands” that are dispersed throughout the genome but highly localized within regulatory regions. The pattern of methylation of CpG “islands” is not stable and may change throughout development from embryo through adulthood. A pattern of global genome-wide methylation disturbances has been verified in ART conceived children compared to those naturally conceived [36].
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To test whether demethylation/hypomethylation process of the genes was global or more localized 736 genes were studied and important methylation differences between ART children were contrasted with naturally conceived children [37]. However, more recent studies find contradictory results [38].
7. Birth defects and ART Controversy exists regarding whether ART procedure themselves are associated with higher rates of birth defects or whether the increased rates reported are primarily due to the infertility of the parents or other factors such as ovarian stimulation used by most fertility specialists prior to oocyte “harvest” for either in vitro fertilization or ICSI. Hansen and coworkers report that infants conceived using ART have a doubling of major birth defects [39]. The literature from the 1990 s dismissed these observed differences in rates of birth defects because the samples sizes were small and some studies failed to report a control group [40]. Green reported from a population based, multicenter, case-control study of birth defects and found that malformations occurred more often in infants conceived using ART [41]. Rimm and coworkers found an increased odds ratio of 1.29 and 1.31, respectively (or about a 30% increase) compared to the general population among infants conceived using ART [42]. Wen and coworkers reported in an extensive meta-analysis in 2012 that compared 56 separate studies showing the increase in birth defects conceived by either in vitro fertilization or ICSI [43]. Their pooled risk estimation was 1.37 (95% confidence intervals 1.26–1.48), and they further found no difference in the increase risk by ART modality (in vitro versus ICSI). A report of the Australian registry in 2012 [44], noted an unadjusted odds ratio of 1.47 (95% confidence interval CI = 1.33–1.62) for birth defects among infants born using ART and found a greater risk associated with ICSI. When this increased risk was adjusted using multivariate analysis, the odds ratio was reduced to 1.28 (95% confidence interval 1.07–1.28). These authors suggest that a parent’s history of infertility was also associated with a slightly increased risk for birth defects as previously reported from a Danish cohort [45]. Hansen and coworkers report from three ART clinics in Western Australia that ART mothers were older, less likely to have had a previous child, and less likely to smoke tobacco than non-ART mothers; however, their infants
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were more likely to have low birth weight and born prematurely whether born as singletons or twins [46]. When two periods of ART were compared, 1994–1998 vs. October 1998 to 2002, the rate of birth defects was 8.7% compared to 5.4% among singletons conceived. The odds ratios decreased slightly when adjusted for maternal age (OR = 1.53; 95% CI = 1.30–1.79). These investigators found little difference in birth defect risk when ICSI or in vitro singletons were compared (OR = 1.09; 95% CI = 0.77–1.54). These authors compared the two time frames and they found a decrease in birth-defect prevalence for both in vitro and ICSI (11.4%–6.2%, p = 0.001) and to a lesser degree among twins irrespective of whether fresh or frozen-thawed embryos were used. In addition, termination of pregnancy for fetal anomaly in non-assisted reproductive technology singletons was 5.8/1000 births contrasted to 9.7/1000 births among ART conceived singletons. These authors summarize that that multiple changes in the ART procedures and embryo incubation conditions or use of recombinant gonadotropins for ovarian stimulation may explain the reduction in birth defects over time, although the exact cause remains unexplained [46]. Overall, women who conceive using ART have a higher proportion of infants with birth defects, although some argue that this increased rate is inherent among couples with a history of infertility. Undoubtedly this will remain an area of controversy, but one worthy of discussion with parents whose aspirations are for a “normal baby born at term” when discussing the risks and benefits of ART [46]. A significant impact of ART, especially in the U.S.A., has been the rising multiple gestation birth rate. In 2008, according to the Centers for Disease Control and Prevention, twin births rose to the unprecedented rate of 32.2 twins per 1000 total birth. Triplet and higher order births have risen more than 400% [47–49]. The longer-term outcomes of children conceived using ART have been carefully reviewed by Willson and coworkers [50]. Their review found that the physical and psychosocial health outcomes in adolescents and young adults are generally comparable to those to those conceived naturally. However, higher blood pressures, elevated fasting blood glucose, increased bone age, elevated serum luteinizing hormone and dehydroepiandrosterone sulphate, as well as, increased growth velocity during infancy was found in cohorts of infants and children conceived using ART [51, 52]. As carefully noted by these authors longer term followup will be needed to determine whether there are
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negative “downstream” effects of ART, with a special focus on rates of neurologic dysfunction, occurrence of the “metabolic syndrome”, or cancers suggestive of concerns regarding fetal origins of adult chronic disease [53]. Encouraging data regarding the psychological well-being of children conceived using ART have been reported; although, as emphasized by Willson et al. the numbers and duration of longitudinal follow-up are small and too short to arrive at definitive conclusions. Kallen et al. [54] report among Swedish children conceived using in vitro fertilization there is an elevated rate of asthma that has been suggested to result from alterations in maternal immune function in infertile women by Davies [55].
8. Conclusion The influence of ART on specific genes, the genome, and the epigenome in humans is now without question. However, more research needs to be done to understand the full impact of these technologies on birth outcomes and the overall health of children born after ART. Indeed, whether fetal programming is affected by the method of conception is presently unknown. ART has increased the prevalence of imprinting disorders and some data from large meta-analysis confirm an increased risk of some birth defects. The present state of knowledge cannot determine whether alternations in single genes, small groups of genes, hypomethylation of genes, or selective epigenetic factors or a combination of these factors may influence the outcome of pregnancies conceived using various forms of ART. The finding of selective morbidities of infants conceived using ART suggest that there are maternal and paternal factors that are emerging as modifiers of fetal epigenetic status which may have implications for long-term health of these infants, in addition to the morbidities associated with being low birth weight and more frequently premature. The 34-year period of observation after the first “in vitro” baby has been too short to fully understand the changes that may manifest themselves in later life. Application of these technologies has expanded exponentially over the last 3 decades. Registries designed to enroll infants conceived using these technologies and collect data on long-term follow-up will yield meaningful results for both physicians providing these ART services, neonatologists caring for many of these infants, geneticists who counsel parents. Parents with
infertility need to fully understand the benefits, as well as, the risks of these procedures when seeking treatment for infertility using ART. Acknowledgments The co-authors acknowledge that Poznan University of Medical Sciences and Loma Linda University School of Medicine have Cooperative Agreement under which this review was prepared by faculty from both institutions. The author’s appreciate the comments of the reviewers and acknowledge the careful editing of this manuscript by Robin Clark, M.D., Associate Professor of Medical Genetics at Loma Linda University Children’s Hospital, Loma Linda, California, USA. Financial disclosure The authors have nothing to disclose and have no conflicts of interest and further attest that they have no financial interest in the subject matter discussed within this manuscript.
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