Are imprinting disorders more prevalent after human in vitro fertilization or intracytoplasmic sperm injection?

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1 Are imprinting disorders more prevalent after human in vitro fertilization or intracytoplasmic sperm injection? Jan P. W. Vermeiden, Ph.D. and Rob E. Bernardus, M.D., Ph.D. Nij Barrah^us Fertility Center, Wolvega, the Netherlands Objective: To review the literature and present original data to answer the question of whether in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is associated with an increase in imprinted diseases in offspring. If the answer is positive, to investigate whether there is a causal relationship between IVF or ICSI and the imprinted diseases. Design: Review study. Result(s): Eight epidemiologic studies were suitable to calculate the weighted relative risk for the birth of a child with Beckwith- Wiedemann syndrome following IVF or ICSI compared with the risk in the normal population. This relative risk was 5.2 (95% CI ). In one study the relative risk was corrected for parents fertility problems and no significant association was found. Data on the Silver-Russell syndrome are too sparse to draw conclusions, but a positive association with IVF or ICSI treatment is probable. No significant associations were found between the incidences of the Angelman and Prader-Willi syndromes and IVF or ICSI treatments. Children with Prader-Willi syndrome or Angelman syndrome are more likely to be born to parents with fertility problems. All retinoblastomas in children born after IVF or ICSI could be explained by de novo mutations in the RB1 gene and were not associated with imprinted genes. Imprinted diseases result from methylation errors already present in sperms or oocytes. There is no proof of a causal relationship between imprinted diseases and IVF or ICSI treatments. Conclusion(s): Imprinting disorders are more prevalent after human IVF or ICSI. Future studies should correct for fertility problems in the affected and comparison groups. It is highly improbable that assisted reproduction technologies cause imprinted diseases in humans. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman syndrome, Prader-Willi syndrome, retinoblastoma, IVF, ICSI Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/vermeidenjp-ivf-icsi-imprinted-diseases/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. There are reports that in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is associated with imprinting disorders, and it is even presumed that IVF or ICSI can cause imprinted diseases. In the present paper we aimed to evaluate the published evidence on these problems. Imprinted genes are transcribed from only one specific parental allele, either the maternally derived copy or Received November 21, 2012; revised January 15, 2013; accepted January 22, J.P.W.V. has nothing to disclose. R.E.B. has nothing to disclose. Reprint requests: Jan P. W. Vermeiden, Ph.D., Nij Barrah^us Fertility Center, Heerenveenseweg 99b, 8471 ZA Wolvega, the Netherlands ( j.vermeiden@xs4all.nl). Fertility and Sterility Vol. 99, No. 3, March 1, /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. the paternally derived copy. This monoallelic expression of imprinted genes is associated with epigenetic modifications and DNA methylation and is parent-of-origin specific. This parentspecific modification pattern present in the germlines leads to differentially methylated regions (DMRs) after fertilization. These stay more or less intact after fertilization and escape the genome-wide demethylation that occurs in the zygote as well as the de novo methylation during differentiation of somatic cells through as yet mostly unknown mechanisms. In early embryonic germ cell development, the parental patterns are erased, to be set anew in adult life. The paternal imprinting pattern is set during spermatogenesis. The maternal pattern is set before, during, or after fertilization. This timing is gene specific. The DMRs originating in the germ line are called primary DMRs. If paternal genes are silenced due to a primary DMR, maternal genes are predominantly or exclusively expressed, or vice versa. This distinguishes primary DMRs from secondary DMRs, which arise only after de novo 642 VOL. 99 NO. 3 / MARCH 1, 2013

2 Fertility and Sterility methylation in the postimplantation embryo. Secondary DMRs would be developmental stage, cell, and tissue specific, and would in general not show allelic-specific methylation (1). In imprinted diseases, the monoallelic expression is disturbed by aberrant methylation of the imprinting control region of a specific gene causing the disease. This can be either hypomethylation or hypermethylation. The situation can be complex. For example, imprinting of H19 (maternally expressed) is reciprocal with the imprinting of IGF2 (paternally expressed). Hypermethylation of H19 is associated with the Beckwith-Wiedemann syndrome (BWS) and hypomethylation with the Silver-Russell syndrome (SRS). IVF and ICSI are well accepted assisted reproduction techniques (ART) for subfertile couples. ART does not heal the causes of subfertility but rather makes it possible for subfertile couples to conceive. The first IVF baby was born in 1978, and now, 35 years later, optimal culture conditions have not been determined and we do not know the effect of the treatment on the children; we do not know whether and how the treatments effect the epigenome. Studies following the health of children born after IVF or ICSI are reassuring, but the mean values of several variables differ from the general population: Slightly higher fasting glucose and higher blood pressure have been observed compared with those found in spontaneously conceived children. The individual values of these variables are mostly within normal range, indicating slight difference among IVF/ICSI children (2). Children conceived and born in periods of famine and stress also showed higher fasting glucose and increased blood pressure. Before mechanisms behind these phenomena were delineated the term programming was used (3, 4). Today, programming can be explained by adaptations of the epigenome, as demonstrated by differential methylation of various DMRs of IGF2/H19 (5). These epigenomic adaptations are normal physiologic variations and are not imprinted diseases. Imprinted diseases are real diseases, as opposed to physiologic adaptations to a changing environment. The first encounter of ART with imprinted diseases was the large offspring syndrome. Because of certain culture conditions (high concentration of serum and coculture with somatic cells), in vitro maturation of bovine oocytes and in vitro fertilization of these oocytes resulted in large calves (compared with 45 kg normal weight, up to 90 kg for the large calves). It appeared that the imprinting control center of Igf2r was hypomethylated, resulting in the overgrowth of the calves (6). From 2002 on, alarming papers were published on an increase of imprinted diseases because of IVF and ICSI (7), which raised the question of whether IVF and ICSI caused the imprinted diseases BWS, SRS, Angelman syndrome(as), Prader-Willi syndrome (PWS), and retinoblastoma. However, because of the rarity of these diseases in the general population as well as in children conceived by IVF or ICSI, and confusion in understanding the implications of programming and imprinted diseases, and finally because studies were performed with differing methodologies that produced conflicting results, the associations between imprinted diseases and ART are not clear. BECKWITH-WIEDEMANN SYNDROME AND ART BWS is a fetal overgrowth syndrome. The diagnosis is based on patient physical characteristics; in 80% of all cases confirmation by genetic molecular tests is possible. The genetic errors are found on chromosome 11. The major causes are: uniparental disomy (UPD) of H19 (20% of cases; UPD involves both imprinting centers 1 and 2); hypomethylation of LIT1 (maternally methylated, 50% of cases); hypermethylation of H19 (paternally methylated, 5% of cases; and microdeletions, translocation, inversion, duplications (each <1% of cases). In 20% of cases the cause is unknown (8). In ARTassociated cases, almost all BWS was due to hypomethylation of LIT1. BWS is a rare disease, with population prevalence estimated to be 1:14,500 13,700 in Western Europe and North America (8, 9). A prevalence of 1:287,000 has been reported in Japan (10). In the literature, the standard expression for hypomethylation of imprinted genes such as LIT1 is loss of methylation. Loss refers to a formerly methylated gene that subsequently lost this methylation. However, it is very possible that the gene was never methylated. Therefore neutral terms, such as absence of methylation or hypomethylation, are preferred. This absence can be caused either by loss or by failure of methylation. The term hypomethylation is applied after assessment of the methylation index and indicates that this index is low. Hypomethylation is a neutral term that describes the concrete methylation status of a gene in a tissue or in a cell suspension. Epidemiologic Studies of BWS and ART Several papers have been published with original data on BWS and IVF/ICSI. Most of the papers used data from voluntary registries. Only three were based on complete registries (11 13). The differences in methodology did not allow for proper meta-analysis, but we calculated a weighted relative risk (RR) based on all available data. DeBaun et al. (14) used the data of two voluntary registries of BWS. They estimated that BWS is at least 6 (95% confidence interval [CI] ) more prevalent in children born after IVF/ICSI than in the general population. They described the molecular basis of seven BWS children born after ART: Five had hypomethylation of LIT1, one had normal LIT1 and normal H19, and one had methylation errors in both LIT1 and H19. They did not report the incidence of BWS in other forms of ART, such as intrauterine insemination (IUI), donor insemination, and the use of fertility drugs. In the United Kingdom several papers with new data were published on BWS related to IVF/ICSI (9, 15 17). These papers were all based on voluntary registries. These papers repeated some data from the same patients. For that reason, only one paper was selected for the calculation of the weighted RR (16). A questionnaire was sent to 213 families and a response was received from 83. Of these, 79 had sporadic BWS, six (7.6%) of these individuals were conceived with IVF or ICSI. After correction for nonresponders at least 2.9% (95% CI 1.4% 6.3%) of the BWS children were born after IVF or ICSI (expected rate 0.8%; RR 3.6 (95% CI ). Thus, even after correction for nonresponders, there was a significant VOL. 99 NO. 3 / MARCH 1,

3 VIEWS AND REVIEWS association between BWS and IVF/ICSI. Using the original data, we calculated a RR of 9.5 (95% CI ). We used this figure to calculate the weighted RR of all epidemiologic studies, because in all other studies no correction for nonresponders was made. The cited paper (16) reported the molecular data of 8/11 patients. All eight had hypomethylation of LIT1, but the type of treatment (IVF/ICSI or just fertility drugs) was not indicated. Of the same group, the molecular data of 13 post-icsi and 12 post-ivf BWS patients were reported (17): 24/25 had hypomethylation of LIT1. It is presumed that the data of the six BWS IVF/ICSI patients of Sutcliffe et al. were incorporated into these data (16). Bowdin et al. (9) sent a questionnaire to 2,492 families with IVF/ICSI children living in Ireland and central England; 1,542 responded, and one BWS child was reported. If the expected number of patients was calculated, using both the RR of 9.5 and the corrected RR of 3.6 (16), the incidence of BWS in this group would have been between 1:4,000 1,500 and BWS child could be expected. Thus the results are within expected values, but the study lacked sufficient power for a solid conclusion. Gicquel et al. (11) based their work on a complete nationwide registry (France) of all BWS patients. The study period was not defined, but six BWS patients were born after IVF or ICSI; nationwide, 149 BWS children were born in the same period, meaning that 4% of the BWS children were born after IVF/ICSI, giving an RR of 3.2 (95% CI ) compared with the normal population. Rossignol et al. (from the same group as Gicquel) published the molecular results of new BWS patients after ART (18). Together the result of 12 BWS patients after ART were published. All 12 patients had hypomethylation of LIT1, and two of them also had hypomethylation of H19. Halliday et al. (19) published the results of BWS and IVF/ ICSI in the state of Victoria, Australia. Among 1,316,500 live births in Victoria from 1983 to 2003, 37 people with BWS were detected, giving an overall BWS prevalence of 1:35,580 live births for this period. In the same period, 14,894 children were born by IVF/ICSI in the State of Victoria. The incidence of BWS in these children was 4 out of 14,894 (1:3,723; RR 9.6, 95% CI ). No figures of other fertility treatments were given. K allen et al. (12) reviewed BWS registries in Sweden. In the cohort of children born after IVF/ICSI, one child had PWS and one had SRS. In this period ( ) 16,280 children were born after IVF/ICSI. It was concluded that in Sweden, IVF/ICSI was not associated with an increased incidence of imprinted diseases. No epidemiologic data on BWS births in the Swedish population were available, but 1 (2) out of 16,280 could be expected (assuming that the incidence in Sweden equals that of Western Europe [1:13,700]). Lidegard et al. (13) reviewed the registries in Denmark. All ART children and all congenital birth defects are centrally registered in Denmark. In the 6,052 children born after IVF/ ICSI no BWS child was reported. It was concluded that ART was not associated with an increased incidence of imprinted diseases in Denmark. In fact, no BWS children were reported in the database of all children born in Denmark. Because at least 21 BWS children could have been expected (number of children born [442,349] divided by the incidence of BWS [13,700], minus 2 SD ¼ ¼ 21), these findings are difficult to understand. Doornbos et al. (20) published the figures of imprinted diseases and ART in The Netherlands during the period The study was based on a voluntary registry. Taking the calculated incidence of BWS in the Netherlands, 24% of the families with BWS children participated in the study. Seventy-one families with a BWS child participated in the study, of whom four children were born after IVF or ICSI. The RR was 6.1 (95% CI 2.5.9) compared with the normal population. Fifteen of these 71 families reported fertility problems (21.1%, compared with 5.6% in the Dutch population; RR 4.6, 95% CI ). After correction for fertility problems in the group of parents with BWS children and for children born in families with fertility problems of any kind in the normal population, there was no significant increase of BWS births in the group of IVF/ICSI children. Hiura et al. (10) published a paper on the association between ART and imprinted diseases in Japan. That paper reported an incidence of BWS of 1:287,000. The figure was based on a questionnaire sent to 3,158 pediatric departments in Japan. The published incidence of BWS in Western Europe varies from 1:14,500 to 1:13,700. This could mean that the incidence of BWS is approximately 20 lower in Japan than in Western Europe. However, the authors indicated in the discussion of their manuscript that the response rate to the questionnaire was not optimal (10). They reported on six BWS patients out of 70 conceived by IVF/ICSI, or 8.6%. Because 0.64% 0.98% of the total number of babies born in Japan were a result of IVF and ICSI, the RR of BWS after ART was estimated to be 12.5 (95% CI ). To calculate the RR of the association between IVF/ICSI and the incidence of BWS, we assumed that in Denmark and in Sweden the incidence of BWS would be equal to other Western European countries (1:13,700). We included the assumed data of these countries, because no ART-related BWS were reported and we did not want to overestimate the RR. The weighted RR of the eight epidemiologic studies was 5.2 (95% CI ), indicating a significant positive association between IVF/ICSI treatment and the incidence of BWS. Other Studies on ART and BWS Kobayshi et al. (21) analyzed 78 aborted conceptuses conceived by IVF or ICSI and 38 aborted spontaneously or IUIconceived conceptuses. The age of the conceptuses was from 6 to 9 weeks. They assessed the methylation index of LIT1, and in all groups low methylation indices were found. The IVF/ICSI group did not differ from the other groups. The notion that IVF/ICSI causes imprinted diseases in humans is supported by differences in the proportion of the various molecular causes of BWS observed in the normal population and in the population of IVF/ICSI children. The molecular data of 46 BWS IVF/ICSI children are reported in the papers discussed above: 44/46 had hypomethylation of LIT1, and in 3/44 abnormal methylation of H19 was also found, i.e., in 95.7% (44/46) of the children with BWS born after IVF/ICSI the disease was associated with hypomethylation 644 VOL. 99 NO. 3 / MARCH 1, 2013

4 Fertility and Sterility of LIT1. In the general population this percentage is 50%. Reductio ad absurdum led to the conclusion that IVF/ICSI treatment is the cause. However, a similarly high percentage of hypomethylation of LIT1 (89.5%; 17/19) was found in naturally conceived discordant BWS monozygotic twins (22 24). Therefore, the high proportion of hypomethylation of LIT1 of BWS children born after IVF/ICSI does not support a causal relationship. Monozygotic twinning is increased in the IVF/ICSI population (25). One can argue that the BWS children with hypomethylation of LIT1 are the twins that survived while the others would have been categorized as vanished twins. However, most of the monozygotic twins discordant for BWS are female. In reported BWS ART children, the male-female ratio appeared to be balanced. In spontaneous conception, monozygotic twinning with BWS has to be explained by a different mechanism than in the BWS children born after ART. It was presumed that in the discordant monozygotic twins hypomethylation of LIT1 occurs after cleaving in the BWS twin (24). Geuns et al. (26) assessed the methylation of LIT1 in 16 human oocytes and reported one human oocyte with absent methylation of LIT1. The other 15 were normally methylated. These observations suggest that BWS can be explained by absence of methylation of LIT1 in the oocyte. Experimental work with nuclear transfer from immature murine oocyte nuclei into mature enucleated murine oocytes resulted in pups with hypomethylation of imprinting genes in Igf2r, Snrp, and Mest, demonstrating that hypomethylation can originate in oocytes (27). Aberrant methylation can also be explained by dysfunction of methylation enzymes. Bestor (28) suggested that aberrant function of DNMT1 (DNA methyltransferase 1) is associated with BWS. Kobayashi et al. (21) reported DNA sequence variations in the gene encoding DNMT3L (DNA methyltransferase 3L), which was associated with the abnormal paternal DNA methylation. There are several arguments to support the idea that IVF/ ICSI can induce imprinted diseases in humans. One of the strongest arguments is based on results from bovine and ovine IVF. IVF of oocytes of these species can give rise to the large offspring syndrome. A rich culture system was used (surplus of serum and coculture with fibroblasts). After changing the culture system, large offspring syndrome disappeared. Large offspring syndrome can be explained by hypomethylation of the ovine imprinted Igf2r gene (6). We agree that analogue reasoning is a methodologically sound way to design experiments, but the finding that large offspring syndrome is caused by in vitro culture and IVF and in vitro growth of embryos is not proof that BWS has to be caused by IVF. It must also be noted that timing of methylation differs in these species (29). Another question is whether ART other than IVF/ICSI, such as IUI in a stimulated cycle or use of fertility drugs and natural conception, are associated with an increase of the incidence of BWS. Doornbos et al. (20) reported a nonsignificant RR of 2.4 (95% CI ). Sutcliffe et al. (16) reported five BWS children born after fertility drug treatment without IVF/ICSI. The number of children born in the relevant period in the United Kingdom after ART treatment other than IVF/ICSI was not reported. To estimate risk, we assumed that in the United Kingdom the number of these treatments is pro rata similar to that of The Netherlands. In our estimate the RR was 5.6 (95% CI ). The combined data of Doornbos et al. (20) and Sutcliffe et al. (16) gave an RR of 4.0 (95% CI ). These results indicate a positive association between fertility treatments other than IVF/ICSI and BWS. Apparently the in vitro phase is not necessary to increase the incidence of BWS. Further research is needed to investigate this question. Another concern raised by the above studies is the use of a suitable comparison group. In almost all studies, the normal population is used as control group. However, in the normal population only a small proportion of the children are born to parents with a fertility problem (5.6% in the Dutch population [20]), whereas 100% of the children conceived after IVF/ ICSI are born to parents with fertility problems. Controlling for fertility problems would result in a truer picture of the associations between fertility problems, fertility treatments, and imprinted diseases. Another difficulty presented by rare diseases is the sample size needed to reach significant and meaningful conclusions. Rancourt et al. (30) compared the difference in methylation of six DMRs in placentas or cord blood from 61 spontaneously conceived children, 57 children conceived by IVF, and 21 children conceived by ovulation induction. No meaningful differences were found. Puumala et al. (31) compared imprinting in lymphocytes and buccal smears in 67 ART and 31 spontaneously conceived children and found no meaningful differences. If we assume that in the group of children born after IVF/ICSI the prevalence of BWS is 1:2,700 and that the prevalence in the normal population is 1:13,700, study groups of >45,000 would be necessary to demonstrate a statistically significant difference with an a of 5% and b of 20% (see: Association of BWS and ART: Conclusions There is a significant positive association between IVF/ICSI treatment and BWS, with an RR of 5.2 (95% CI ). With the population prevalence of 1:13,700, this suggests that among every 2,700 IVF/ICSI births one BWS child will be born. One study found that after correcting for fertility problems among the parents with BWS children and in the control group, the association was no longer significant. This indicates that the IVF/ICSI treatment in itself was likely not the cause of the increased incidence of BWS in these children, but rather the (epi)genetic background of the parents was. Many causal relationships are possible, but none have been proven in humans. Possible causes are the culture system, ovarian hyperstimulation, monozygotic twinning, genetic background of the parents, such as genetic variation in DNMT1 and DNMT3L, and aberrant methylation in both oocytes and sperm. SILVER-RUSSELL SYNDROME AND ART SRS is a disease associated with imprinted genes. The main characteristics of SRS are severe intrauterine and postnatal growth restriction, relative macrocephaly, and a small triangular face (32). Approximately one-half of children with SRS have significant cognitive impairment (33). In 50% of VOL. 99 NO. 3 / MARCH 1,

5 VIEWS AND REVIEWS the patients with clinical diagnosis of SRS, molecular confirmation or explanation of the disease is not found. Prevalence rates from 1:392,000 to 1:3,000 have been reported, supporting the statement that the prevalence of SRS is not known (32). This makes discussions of an increased incidence of SRS following ART problematic. SRS is the only known imprinted disease that can be caused by two different genetic mechanisms on two different chromosomes: hypomethylation of H19 (chromosome 11), and UPD of maternal chromosome 7 (mudp7). Hypomethylation of H19 is often associated with other epimutations. Hypomethylation of H19 seems to represent the more severe forms of SRS than the mupd7 form, however this is questioned by some investigators (32). We will discuss the results of four case studies (34 37) and four epidemiologic studies. A study from Denmark reported no SRS birth in the ART cohort (13), the study from Sweden reported one child (12), from Japan five children (10), and from The Netherlands three SRS children born after IVF/ICSI (38). Together these papers report 13 SRS children born after IVF/ICSI. The molecular data of 11 patients were reported: ten hypomethylation of H19 and one normal methylation of H19 but hypermethylation of PEG1/MEST DMR (37). Case Studies Three of the case studies reported on the birth of a child with SRS; all three children were born after ICSI. Two had hypomethylation of H19 (34, 36). No molecular data were included for the third child (35). The fourth case study was of special interest (37). It reported on a girl with SRS of dizygotic twins conceived by IVF. Diagnosis of SRS was based on clinical features. Genetic and molecular diagnosis revealed no mupd7, normal methylation of H19 and partial hypermethylation at the DMR of PEG1/MEST. Her father also showed aberrant hypermethylation at the DMR of PEG1/MEST, though to a lesser degree than the girl, suggesting that the aberrant methylation of the father was heritable. We do not know whether PEG1/MEST is of relevance in SRS, but the observation that aberrant imprinting patterns can be heritable is of importance for our discussion on the association of imprinted diseases and ART. Epidemiologic Studies One child with SRS was reported in Sweden (12). The child was born after an ICSI treatment. The molecular data were not reported. Lidegard et al.(13) concluded from the Danish data that there was no increase of imprinted diseases in association with ART. In a study in which all Danish children born January 1, 1995 December 31, 2001, were included (total 442,349 singleton non-ivf children and 6,052 IVF/ICSI children). In the non-ivf group, two SRS and three PWS cases were registered. In the IVF/ICSI group, no children with imprinted diseases were found. If we presume that in Western Europe and in North America each of the four imprinted diseases has an average incidence of R1:16,000, the combined incidence would be 1:4,000. This means that a minimum of 90 children with BWS, SRS, AS, or PWS should have been registered in the non-ivf group. The results of the Danish registry study are therefore puzzling. Hiura et al. (10) reported five patients with SRS, all born after IVF. In a nationwide questionnaire, the birth of 42 SRS children was reported, of which four had been born after IVF or ICSI. If the average ART birthrate is 0.75% in Japan, the RR of SRS associated with ART would be 12.6 (95% CI ). While interpreting these data we have to keep in mind that the authors had indicated that the response rate to their questionnaires was not optimal. Lammers et al. (38) had access to a complete Dutch SRS registry containing 38 SRS children. The parents of these children were asked to participate in a study and 23 agreed. Three had mupd7 and 20 had hypomethylation of H19. Two of these 23 children were born after IVF and one after ICSI. In the relevant period, 2% of all children in The Netherlands were born after IVF/ICSI. This gives an RR of 6.7 (95% CI ). These two papers (10, 38) indicate a positive significant association between ART and SRS. Methylation Studies A methylation index of H19 can inform us of the time period in which the (de)methylation occurred. A methylation index of 0% indicates no gain of demethylation at any time, or that demethylation occurred before fertilization (34, 39). Hiura et al. (10) did not present the figures of the methylation indices, but reported that 4/5 of the ART SRS patients were mosaic for methylation of H19, suggesting that the methylation errors were established after fertilization and cleavage divisions. Kobayashi et al. (21) analyzed the methylation of seven autosomal imprinted loci and the XIST locus of 78 aborted fetuses conceived by ART and 38 aborted fetuses conceived spontaneously. Although not reported, serious complications or diseases of the conceptuses had to be involved to have abortion performed by women conceived by ART, so it is questionable how representative these conceptuses were of live-born ART children. Molecular analyses were performed on fetal tissue, placental tissue was carefully removed. Hypomethylation of H19 was found in six ART fetuses (methylation index 4% 23.5%). In none of the spontaneously conceived conceptuses were similarly low values of the methylation index of H19 found. This could suggest that hypomethylation of H19 occurred after fertilization. However, Kobayashi et al. (21) also found, with the use of DNA polymorphisms at the H19 and GTL2 loci, seven cases where exactly the same DNA methylation error was present in the sperm of the fathers as in their conceptuses, suggesting that the methylation errors were preexisting and were not a consequence of ART. In other words, the ART procedure had not affected the methylation status of these genes. We conclude that imprinted diseases can be caused by aberrant methylation of the genes in the sperm. Obata et al. (27) reported that in murine oocytes aberrant methylation patterns may be present and passed on to the offspring, as judged by their presence in the pups. Although this does not prove that this also can happen in humans, it is proof 646 VOL. 99 NO. 3 / MARCH 1, 2013

6 Fertility and Sterility of the principle that aberrant methylation patterns are present in the gametes and can be transferred into the conceptuses. Association of SRS and ART: Conclusions Of 13 SRS children born after ART, five were born after ICSI and eight after IVF. There seemed to be no prevalence for IVF or ICSI, but the numbers are too small for other conclusions. Two epidemiologic studies, with low power, did not show an association between ART and the incidence of SRS. Two other epidemiologic studies reported a positive significant association between the incidence of SRS and IVF/ICSI (RRs 12.6 [95% CI ] [10] and 6.7 [95% CI ] [38]). We would like to conclude that there is probably a significant positive association between the incidence of SRS and IVF/ ICSI treatment, but the number of published cases is small. Many of the SRS children, both spontaneous or IVF/ICSI conceived were mosaic, suggesting that in these children the aberrant methylation patterns were established in the early differentiation phase of the embryos (10, 39). Because of the low number of observations we do not know whether the mosaic methylation pattern is associated with IVF/ICSI. Aberrant methylation patterns detected in sperm were also detectable in offspring. This indicated that aberrant methylation can occur during spermatogenesis, suggesting that in vitro procedures such as ICSI and IVF facilitate the transfer of the aberrant sperm but are not the intrinsic cause of the methylation errors and imprinted diseases. ANGELMAN SYNDROME AND ART AS is a neurogenetic disorder characterized by severe mental retardation, delayed motor development, poor balance accompanied by jerky movements, absence of speech, and happy disposition. It is caused by the absence of function of the maternal allele of the UBE3A gene on chromosome 15, resulting from a deletion, a point mutation, UPD, or an imprinting defect. Only in 5% of the cases is there absence of imprinting of the maternal allele. In 5% 10% of the cases that appear to have the major clinical phenotypic features, there are no identifiable genetic abnormalities. AS is a rare disease. The reported incidence varies from 1:20,000 to 1:12,000 in the West (41) and from 1:134,000 in Japan (10). A recent review was published by Dagli et al. (40). Case Studies In the years two case studies (7, 41) and four epidemiologic papers with original data (10, 16, 20, 42) were published on this disease and its association with subfertility and fertility treatments. Cox et al. (7) described two patients, both born after ICSI. Both patients had absence of methylation of the maternal allele. According to Cox et al. (7), absence of methylation occurs only in 1:300,000 AS patients. These two cases (probability of 1:300,000 2 ), evidence that maternal methylation at chromosome 15 is established at or during fertilization, and that ICSI and embryo culture is a process outside the body, as well as the observations of Young et al. (4) that IVF of ovine oocytes can result in hypomethylation of the imprinting control element of the maternal Igf2r allele, led Cox et al. (7) to conclude that there are arguments that ICSI might interfere with the establishment of the maternal imprint (impaired gain of methylation) in the oocyte or preembryo and increases the risk of imprinting defects. Ørstavik et al. (41) reported a third AS patient with hypomethylation after ICSI treatment, reinforcing the conclusions of Cox et al. (7). Epidemiologic Studies Ludwig et al. (42) performed a large nationwide study in Germany. The authors contacted all the members of the German AS Support Group. Seventy-nine valid questionnaires were received from 270 members. Twenty of the 79 children were born to couples with fertility problems (25.3%). In Germany the proportion of children born in families with fertility problems of any kind is 5.3% (44). This resulted in an RR of 4.8 (95% CI ). We conclude that the incidence of AS is significantly associated with fertility problems. Three of the 79 AS children were born after ICSI, none after IVF. If we compared this with the German population, assuming that 0.9% of the children were born after IVF/ICSI in the relevant period, we calculated an RR of 4.1 (95% CI ). We conclude that in Germany, in the period before the year 2005, AS is significantly associated with ART. We also concluded that the incidence of AS is significantly associated with fertility problems of the parents. Ludwig et al. (43) published the genetic data of 16 patients. They found three patients with imprinting defects, ten with deletions, and three with unknown defects. The three patients with imprinting defects were born after time to pregnancy >2 years, time to pregnancy >2 years and hormonal treatments, and after time to pregnancy >2 years and hormonal treatments and ICSI. One AS patient had absence of methylation of the maternal allele after ICSI, and two AS patients without ICSI treatment also displayed absence of methylation of the maternal allele. The rare form of AS (absence of methylation), incidence of 1:300,000, observed by Cox et al. (7) in each of two ICSI patients, was here also observed in the non-icsi AS children. This seems to indicate that absence of methylation of the maternal allele is associated with fertility problems and not with the fertility treatment. These numbers are far too low for final conclusions, but the simplest and most likely explanation is that parents with fertility problems have a higher chance to have a child with AS than parents without fertility problems. It was also concluded that ovarian hyperstimulation is a variable positively and independently associated with AS (43). But again, that conclusion is based on small numbers. Sutcliffe et al. (16) sent questionnaires to 384 families with AS children and received 81 usable responses: Six had familial AS, 75 were sporadic. Three of these 75 (4%) were born to families with fertility problems, but no IVF or ICSI was involved. Assuming that the percentage of families with fertility problems and children was similar to those of Germany and The Netherlands (5.3% and 5.6%, respectively), then there was no association between fertility problems and AS in the United Kingdom. VOL. 99 NO. 3 / MARCH 1,

7 VIEWS AND REVIEWS In 2007 Doornbos et al. (20) also performed a study based on results from questionnaires sent to members of AS support groups. Sixty-three valid questionnaires were received. Results similar to the findings of Ludwig et al. (43) were achieved. Although none of the 63 children were born after IVF or ICSI, there was a significant association between fertility problems of the parent and the incidence of AS (RR 3.4, 95% CI ). In the AS group, four (6.3%) children were born after any form of ART. This percentage was 2.1% in the Dutch population (RR 3.1, 95% CI ), giving a significant association between any form of ART and the incidence of AS. Hiura et al. (10) reported the results of an epidemiologic study on imprinted diseases and IVF/ICSI. They found no association between AS and IVF/ICSI. The four discussed epidemiologic studies reported five AS cases born after IVF or ICSI, in a total of 340 cases (10, 16, 20, 42). Considering the number of IVF/ICSI births and the total number of births in the four countries in the relevant periods, the weighted RR was 1.7 (95% CI ). We conclude that there was no significant association between AS and IVF or ICSI treatments. AS and ART: Conclusions Taking all the published evidence together, we conclude that there is probably a positive association between fertility problems and the incidence of AS. As a consequence of this association we expected a positive association between IVF/ICSI and AS, but that was not demonstrated. The imprinting of the AS imprinting center (AS-IC) is established during or after fertilization (7). It was presumed that because of this, AS-IC would be vulnerable to methylation errors associated with ICSI or IVF. In the literature discussed here, there were seven cases of AS reported with methylation errors: Four were born after ICSI treatment and three after hormonal treatment or in families with a history of fertility problems. Because of these observations and because of the absence of association between AS and IVF/ICSI, we conclude that it is highly improbable that ICSI or IVF prevents proper methylation of AS-IC. Studies investigating (micro)deletions in the gene methylation enzymes, such as the DNMT3L gene, will be much more fruitful in explaining these methylation errors (21). From a clinical standpoint, AS has no impact on ART owing to its low incidence. PRADER-WILLI SYNDROME AND ART PWS is a complex neurodevelopmental disorder resulting from errors in genomic imprinting with loss of methylation of imprinted genes that are paternally expressed in the chromosome 15q11 q13 region, the same region that is responsible for AS. PWS is characterized by severe hypotonia and feeding difficulties in early infancy, followed in later infancy or early childhood by excessive eating and gradual development of morbid obesity. All individuals have some degree of cognitive impairment. The worldwide prevalence is 1:30,000 10,000. Approximately 70% of PWS cases stem from de novo deletion in the paternally derived chromosome 15q11 q13 region. About 25% of the cases can be explained by maternal disomy of chromosome 15. In about 3% of cases PWS can be explained either by genomic imprinting defects due to microdeletions or epimutations of the imprinting center located in the 15q11 q13 region or by chromosome 15 translocations or rearrangements (44, 45). There are only a few studies in which original data are reported on the association between IVF/ICSI and PWS (10, 12, 16, 20). All of the studies concluded that there was no association between ART and PWS. In the period the birth of 16,280 IVF/ICSI children was reported in Sweden, and one of these had PWS (12). Sutcliffe et al. (16) contacted 522 families with PWS children, 169 replied, and six families had a history of PWS. Two children (1.2%) were born after ICSI, none after IVF. These numbers did not differ from that of the normal population. Seven children (4.3%) were born after the use of fertility drugs. The authors indicated that 2% of the children in the United Kingdom are born after any type of fertility treatment. In the PWS population 5.5% were born after fertility treatment (RR 2.8, 95% CI ). This figure indicates that there is an association between fertility problems of the parents and an increased incidence of PWS. Doornbos et al. (20) performed a similar study. They received replies from 86 families with PWS children. Twelve of those families had fertility problems of any kind (RR 2.5, 95% CI , compared with the normal population). There was no significant association between IVF/ICSI and the incidence of PWS (RR 2.5, 95% CI ). Hiura et al. (10) reported the birth of four IVF/ICSI children in a group of 261 PWS children (1.5%). This proportion did not differ from that of IVF/ICSI children born in the normal population. PWS and ART: Conclusions Children with PWS are more likely to be born to parents with a fertility problem (RR ), however, there is no significant association between PWS and IVF/ICSI. The same paradox was observed in the group of children with AS. RETINOBLASTOMA Retinoblastoma is a cancer originating from the retina of one or both eyes in young children. There are several reports of associations between IVF/ICSI and retinoblastoma. Moll et al. (46) studied the incidence in the period and calculated an RR of 4.9 (95% CI ) or 7.2 (95% CI ) (4.9 if it was assumed that 1.5% of the children in The Netherlands were born after IVF/ICSI and 7.2 if this assumed percentage was 1.0%). The same group published a new study in 2009 in which the incidence of retinoblastoma was studied over the period (47). The RR in this period was 1.29 (95% CI ), and it was concluded that there was no significant association between IVF/ICSI and retinoblastoma. In an abstract of another study, an RR of 4.5, without confidence interval, was reported; no follow-up of that result was published (48). Retinoblastoma can be heritable and can be caused by two mutations in the RB1 gene or by hypermethylation of the promoter of the RB1 gene. If the retinoblastomas observed in IVF/ICSI children is caused by hypermethylation of this 648 VOL. 99 NO. 3 / MARCH 1, 2013

8 Fertility and Sterility promoter, a relationship between the fertility treatment and the occurrence of retinoblastoma can not be excluded. In 2012 a paper was published in which the molecular origin of the tumors was delineated. For all tumors, two causative RB1 mutations were found. None of the tumors showed hypermethylation of the RB1 promoter. This demonstrated that an association between IVF or ICSI and retinoblastoma through this epigenomic mechanism is highly unlikely (49). DISCUSSION Alarming studies on the association between imprinted diseases and IVF/ICSI were published in recent years. The incidences of BWS was reported to be up to 12 times higher in children born after IVF/ICSI than in the normal population. Also, the birth of calves and lambs with the large offspring syndrome after IVF with both ovine and bovine gametes, and the observation that this could be explained by demethylation of the Igf2r gene, raised the question of whether in vitro culture of oocytes and embryos could cause methylation errors and imprinted diseases. Imprinted diseases are rare, with reported incidences of 1:30,000 10,000. An incidence as low as 1:392,000 for SRS has been reported in Japan (10), although that study was based on questionnaires sent to departments of pediatrics with suboptimal response rate. It can be expected that the reported prevalence in Japan will be adjusted. However, it has not been established whether prevalence of imprinted diseases differs among different human ethnic backgrounds. A consequence of the rarity of these diseases is the difficulty to perform studies with sufficient power. If we assume that in the group of children born after IVF/ICSI the rate of BWS may reach 1:2,700 compared with 1:14,000 in the normal population, study groups of >45,000 are needed to demonstrate a statistically significant difference with an a of 5% and a b of 20%. Studies of the association between ART and retinoblastoma illustrate the problem of imprinted disease and studies lacking sufficient statistical power. The highly significant association first reported vanished over time as greater numbers were collected. Subsequently it was reported that for all tumors two causative RB1 mutations were found and none of the tumors showed hypermethylation of the RB1 promoter. It is concluded, in light of these additional data, that an association between IVF or ICSI and retinoblastoma through this epigenetic mechanism is unlikely (49). Keeping these pitfalls in mind, we examined the existence of a statistical relationship between the incidence of an imprinted disease and fertility treatments. If such a relationship exists, the possible etiology must be investigated before we can conclude that there is a causal relationship between imprinted diseases and ART. However, because it would be highly unethical to try to induce imprinted diseases by experimental studies in human embryos, the question of causality will be very difficult to answer. Based on the extant literature, it was possible to calculate a weighted RR for BWS. We included eight studies: two studies in which no association was found (12, 13) and six (10, 11, 14, 16, 19, 20) with a positive association. The weighted RR was 5.2 (95% CI ). We concluded that there is a significant positive association between IVF/ICSI treatment and risk of BWS. Four epidemiologic studies examined the relationship between SRS and IVF/ICSI. Two studies with low power found no association, whereas two other studies that included eight children with SRS after IVF/ICSI treatment reported a significant positive association (RRs 12.6 [95% CI ] [10] and 6.7 [95% CI ] [38]). The incidence of SRS is not known. Many children born small for gestational age may have some form of SRS. Improvement in the diagnosis will be essential for studying the putative association between SRS and IVF/ICSI. We did not find a positive association between the risk of AS and IVF/ICSI. Two studies found a positive association between AS and other fertility treatments (20, 42). We also did not find a positive association between the incidence of PWS and IVF/ICSI. However, we concluded that there is a significantly higher risk for a child with AS or PWS to be born to parents with fertility problems (16, 20). The apparent paradox of increased fertility problems but no increased numbers of IVF/ICSI treatments can be explained by the rarity of the diseases. Another problem of investigation into the statistical relationships between ART and imprinted diseases is the comparison group. In all of the studies but one (20), the normal population was used as the comparison group. Couples undergoing IVF or ICSI have fertility problems, whereas in the general population the vast majority of children are conceived spontaneously. Valid comparison between IVF/ICSI conceived children and control subjects must account for fertility problems in both groups. Applying this principle, the statistical relationship between imprinted diseases and IVF/ICSI disappeared in the aforementioned study. Thus the increased incidence of imprinted diseases after IVF/ICSI can be explained by the fertility problem of the parents. Apparently the increased incidence of imprinted diseases in children born after IVF or ICSI treatment is not an intrinsic effect of the treatments they received, and we must search for other causes. Several publications reported methylation errors in sperm and oocytes that could be traced in the offspring, explaining BWS or SRS (21, 26, 36). It may be that absence of methylation and mosaic patterns of methylation present in SRS patients are caused by improper enzyme functions of the methylation enzyme DNMT3L caused by microdeletions (21, 36). This would indicate that the imprinted error is not caused by the culture system, but that the in vitro system is only permissive for the transfer and propagation of aberrantly methylated genes. In conclusion, retinoblastoma, AS, and PWS are not associated with IVF/ICSI, BWS is significantly associated with IVF/ICSI, and the number of cases of SRS is small but a positive association is probable. The incidences of AS and PWS are positively associated with families with fertility problems, but not with IVF/ICSI. This paradox can be explained by the low power of the studies due to the rarity of the diseases. There are no indications for a causal relationship between ART and imprinted diseases in humans. That does not imply that the IVF/ICSI procedures do not affect the epigenome (31). Children conceived during stressful situations can display VOL. 99 NO. 3 / MARCH 1,

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