Assisted reproduction treatment and epigenetic inheritance.

In order to gain information on transgenerational epigenetic inheritance and epigenetic effects induced by ART, literature databases (Pubmed, Medline) were thoroughly searched for papers on this topic by using relevant keywords.

A.P.A.vM., L.H., P.dS., S.V., J.P.M.G. and P.dB. played a role in conception and design. A.P.A.vM., L.H., P.dS., S.V., J.P.M.G. and P.dB. were involved in acquisition, analysis and interpretation of the data. A.P.A.vM., L.H. and P.dB. drafted the manuscript. A.P.A.vM., L.H., P.dS., S.V., J.P.M.G. and P.dB. contributed to the revision of the manuscript and approval of the final version.

In all publications on epigenetic and physiological, behavioural readouts after ART in the mouse (Table  I ), effects have been noted. Most experiments address the effects of ovulation induction and preimplantation embryo culture on maintenance of imprinting up to mid-gestation. Imprinting status is followed using the methylation status of the DMR, in which surveys the paternally imprinted H19 is often studied. Imprinted gene expression has been determined as well. Examples of unaffected ( Fauque et al. , 2007 ) or slightly affected methylation (Market-Velker et al. , 2010a , b ) followed by affected expression, are given. Hence, all details of regulation by CpG methylation of imprinted gene expression are not yet available, which is a lacuna in our understanding. Table I Survey of the mouse experiments aimed at testing imprinting and physiological parameters after ART. Reference Genotype Conditions Readouts In vivo Superov in vivo Superov in vitro cleavage Superov IVF + cleavage ET Media comparison Blastocyst gDMR methyl Blastocyst Impr expr Mid gest Embryo gDMR methyl Mid gest Embryo Impr expr Mid gest Placenta gDMR methyl Mid gest Placenta Impr expr Market-Velker et al. (2010a ) B6(CAST7) x x x x x Market-Velker et al. (2010b ) B6(CAST7) x x x Doherty et al. (2000 ) B6(CAST7) x x x x x Mann et al. (2004 ) B6(CAST7) x x x x x x x x x x Khosla et al. (2001 ) B6CBA F1 x x x x Fauque et al. (2007 ) B6CBA F1 x F1 x x x x x x x Fauque et al. (2010a ) B6CBA F1 x F1 x x x x x x x x x Fauque et a l. (2010b ) B6CBA F1 x F1 x x x a Rivera et al. (2008 ) B6(CAST7) x x x x x x x x Fortier et al. (2008 ) CD1 x B6(CAST7) x x x x x x x Li et al. (2005 ) B6 x Spretus x x x Adult readout Morgan et al. (2005 ) FVB/N x B6 A vy /a x x x Phenotype epiallele Fernandez-Gonzalez et al. (2010 ) B6CBA F1 x 129/Rr Axin1 fu x x x Phenotype epiallele Ecker et al. (2004 ) 129Sv x B6 x b x x Behavioural testing Fernandez-Gonzalez et al. (2004 ) B6CBA F1 x x x Behavioural testing Watkins et al. (2007 ) CBAB6 F1 x MF1 x x x Blood pressure, serum angiotensin converting enzyme, hepatic phosphoenolpyruvate carboxykinase Scott et al. (2010 ) B6C3 F1 x x c x Glucose parameters Superov, superovulation; ET, embryo transfer; gDMR, germline differentially methylated region (DMR); Impr expr, Imprinted expression; Mid gest, mid gestation. a Transcriptome analysis. b In vivo fertilization was followed by in vivo and in vitro development. c Culture to the 2-cell stage. Survey of the mouse experiments aimed at testing imprinting and physiological parameters after ART. Superov, superovulation; ET, embryo transfer; gDMR, germline differentially methylated region (DMR); Impr expr, Imprinted expression; Mid gest, mid gestation. a Transcriptome analysis. b In vivo fertilization was followed by in vivo and in vitro development. c Culture to the 2-cell stage. There are clear indications for both an effect of ovulation induction and of in vitro embryo culture on maintenance of DMR methylation. Also, the physiological status of the maternal tract after a ovulation induction procedure adds to deregulation of imprinting. As the only paper devoted to DMR methylation in maturing and mature oocytes after ovulation induction did not find an effect of hormonal priming ( Sato et al. , 2007 ), a maternal ‘zygotic’ effect and/or an effect of the female tract after ovulation induction, and (and should not be removed) an effect of the in vitro environment, is indicated. In the experiments reported upon, inbred strains or hybrids between inbred strains have been predominantly used. No experiments with oocytes from random bred stocks have been reported in the literature. So principally, the oocyte inbred genotype could already confer cell biological stress ( Nadeau, 2009 ) that exacerbates after hormonal priming and during in vitro culture. In general the placenta is much more vulnerable than is the embryo proper, which is an illustration of the great significance of imprinting regulation for placental gene expression ( Kawahara et al. , 2009 ). One reason for the increased sensitivity of the placenta for lack of maintenance of DMR methylation might be related to the overall lower level of 5methylCpG ( Monk et al. , 1987 ; Fig.  5 ). In the mouse at least, effects on placental imprinted gene expression of H19 , the fine regulator of prenatal growth, translate into deregulation of the imprinted gene network ( Gabory et al. , 2009 , 2010 ; Fauque et al. , 2010a , b ) most likely also influencing gene expression among non-imprinted genes. Effects of in vitro culture have also been observed in the A vy an Axin fu genetic systems, leading to hypomethylation of the IAP cryptic promoters. In the mouse, ART also affects physiological parameters at adult age, such as insulin sensitivity and blood pressure. Effects on adult behaviour have been reported too. For more information regarding the effects in the mouse, the reader is referred to the Supplementary Data (available online) . Since 2002, a number of reports have shown an association between ART and the frequency of imprinting disorders, notably BWS (Table  II ). Risk, expressed as the relative abundance of ART in the BWS cases compared with relative abundance of ART in the general population, was estimated at between 3.1 and 16.1 (Table  II ). Cases occurred irrespective of cause of infertility and are reported after IVF and ICSI, after transfer of fresh and frozen embryos, after transfer on Day 2, 3 or Day 5 and after different levels of hormonal stimulation, with in vitro culture being the common denominator ( Gicquel et al. , 2003 ; Chang et al. , 2005 ; Sutcliffe et al. , 2006 ; Doornbos et al. , 2007 ). However, also after intrauterine insemination that often involves gonadotrophin stimulation, or the use of fertility drugs alone, BWS cases have been reported ( Chang et al. , 2005 ; Sutcliffe et al. , 2006 ; Doornbos et al. , 2007 ), which led Doornbos et al. to speculate that not ART practice but subfertility is at the heart of this increase in BWS. Indications for a link between ART and epigenetic regulation are that in the general population BWS is caused by a DMR CpG methylation error in 50-60% of the cases ( Manipalviratn et al. , 2009 ), while after ART almost all cases are related to hypomethylation of the maternal KCNQ1OT1 DMR (Table  II ). Also, more often other maternally methylated regions are hypomethylated in ART-BWS than in non-ART BWS children ( Lim et al. , 2009 ). To investigate whether children born after ART might have subclinical forms of BWS, Bowdin et al. (2007) analysed 1524 probands for clinical features linked to BWS. Four children had at least one of these signs, of which one was already diagnosed as having BWS. None of the other three children showed loss of methylation at KCNQ1OT1 , suggesting that no milder forms of BWS have been missed in previous ART-BWS reports. Table II Reports on the incidence of imprinting disorders after human IVF. Reference Type of study N cases % IVF in cases % IVF in ref Estimated risk Type of IVF Molecular defect Beckwith–Wiedemann syndrome   DeBaun et al. (2003 ) Case series 65 4.6 0.76 6.1 IVF and ICSI 5/6 LOM KCNQ1OT1 gDMR c 1/6 GOM H19 DMR 1/6 no imprint defect   Maher et al. (2003 ) Case series 149 4.0 0.997 4.0* IVF ( n = 3) and ICSI ( n = 3) 2/6 LOM KCNQ1OT1 gDMR 4/6 not analysed   Gicquel et al. (2003 ) Case series 149 4.0 1.3 3.1* IVF ( n = 4) and ICSI ( n = 2) 6/6 LOM KCNQ1OT1 gDMR   Halliday et al. (2004 ) Case control 37 10.8 0.67 16.1* IVF ( n = 3) and ICSI ( n = 1) 3/4 LOM KCNQ1OT1 gDMR 1/4 not analysed   Chang et al. (2005 ) Case series 341 5.6 a – IVF ( n = 5) and ICSI ( n = 5) a NA   Sutcliffe et al. (2006 ) Survey 209 2.9–7.6 b 0.8 3.6–9.5 b * IVF ( n = 1) and ICSI ( n = 5) 6/6 LOM KCNQ1OT1 gDMR   Doornbos et al. (2007 ) Survey 71 5.6 0.92 6.1* IVF ( n = 4) 4/4 LOM KCNQ1OT1 gDMR Angelman Syndrome   Cox et al. (2002 ) Case series 2 – – – ICSI ( n = 2) 2/2 LOM SNRPN   Orstavik et al. (2003 ) Case report 1 – – – ICSI ( n = 1) 1/1 LOM SNRPN   Ludwig et al. (2005 ) Survey 79 3.8 – – ICSI ( n = 3) 1/3 LOM SNRPN 2/3 maternal deletion 15q11   Sutcliffe et al. (2006 ) Survey 75 0 0.8 – – –   Doornbos et al. (2007 ) Survey 63 0 0.92 – – – LOM, loss of methylation; GOM, gain of methylation; –, not analysed. a All 19 ART cases are included, 10 after IVF (and ICSI), 2 after hormonal stimulation and insemination and 7 for which no data on type of ART were available. b Range takes into account the large number of lost to follow-up by assuming that all non-responders conceived naturally. c Six ART- Beckwith–Wiedemann syndrome cases were identified in a database. Three patients were from before 2001, when use of ART was not systematically assessed. This period was excluded from the risk assessment. *Risk is significantly increased in IVF compared with non-IVF pregnancies. Reports on the incidence of imprinting disorders after human IVF. LOM, loss of methylation; GOM, gain of methylation; –, not analysed. a All 19 ART cases are included, 10 after IVF (and ICSI), 2 after hormonal stimulation and insemination and 7 for which no data on type of ART were available. b Range takes into account the large number of lost to follow-up by assuming that all non-responders conceived naturally. c Six ART- Beckwith–Wiedemann syndrome cases were identified in a database. Three patients were from before 2001, when use of ART was not systematically assessed. This period was excluded from the risk assessment. *Risk is significantly increased in IVF compared with non-IVF pregnancies. Angelman syndrome (AS) is caused by a shortage of maternal UBE3A expression in the SNRPN imprinting cluster. Less than 5% of cases are caused by an imprinting defect. Six cases of AS have been reported after ICSI (Table  II ; Cox et al. , 2002 ; Orstavik et al. , 2003 ; Ludwig et al. , 2005 ), of which the unexpected high number of four shows a methylation defect. In two other studies covering more than 400 AS cases, none were conceived by IVF or ICSI. Instead, seven AS cases originated from ovulation induction and/or intrauterine insemination ( Sutcliffe et al. , 2006 ; Doornbos et al. , 2007 ). To date, five cases of Silver–Russell Syndrome (SRS) have been published in children born after IVF or ICSI ( Svensson et al. , 2005 ; Kagami et al. , 2007 ; Galli-Tsinopoulou et al. , 2008 ; Kallen et al. , 2010b ). In one, hypermethylation of the paternal MEST DMR was reported ( Kagami et al. , 2007 ). Generally, around 44% of the SRS cases is caused by H19 DMR hypomethylation and 5–10% by maternal uniparental disomy of chromosome 7. Thus far, no imprinted candidate gene on chromosome 7 could be identified ( Binder et al. , 2011 ). The number of cases involving ART is too small for a relation to be indicated. Retinoblastoma (RB) and Prader–Willi syndrome (PWS) are two (epi)genetic disorders involving imprinting. In most cases the underlying molecular mechanism is a (point)mutation or a deletion and not an epimutation, just as in three reported PWS-ART cases ( Sutcliffe et al. , 2006 ; Doornbos et al. , 2007 ) and two out of seven RB-ART cases ( Marees et al. , 2009 ). For the other five cases no gene defect was found and methylation was not analysed. In the PWS/AS region, methylation was normal in 92 children born after ICSI ( Manning et al. , 2000 ). In a large follow-up study of children born after IVF in Sweden, one BWS, two SRS and four PWS patients were found ( n = 31 850) ( Kallen et al. , 2005 , 2010b ). In the Danish National Cohort study ( Lidegaard et al. , 2005 ) among 6052 children there were none with a genomic imprinting disease. Recently, the French follow-up association reported 6 BWS cases (and no PWS, AS or SRS) in a cohort of 15 162 IVF children ( Viot et al. , 2010 ). With a spontaneous BWS incidence of 1 out of 13 700 ( Amor and Halliday, 2008 ), the results of this study follow the tendency of the case series towards an increased risk after ART. In humans, studies on imprinting directed epigenetic reprogramming during oogenesis are very limited for ethical reasons. Only one study used immature oocytes from growing follicles in non-stimulated fertile patients after laparoscopy [( Sato et al. , 2007 ) Table  III ]. At the primary follicle stage almost 50% of the maternally imprinted MEST , KCNQ1OT1 and PLAGL1 DMR alleles was methylated. This level gradually increased in growing preantal follicles and at the antral follicle stage, almost all alleles were methylated ( Sato et al. , 2007 ), just as in mice ( Obata and Kono, 2002 ; Lucifero et al. , 2004 ). The paternal imprint of the H19 DMR was partially erased at the primary follicle stage. A remnant of around 10% methylation was found at the antral follicle stage, which is different from the expectation based on mice, where in E13.5 PGCs the imprint is already removed ( Hajkova et al. , 2002 ). Table III DNA CpG methylation in human oocytes at different stages of development with and without ovarian stimulation and IVM. Reference gDMR Methylation No ovarian stimulation Ovarian stimulation, in vivo maturation Ovarian stimulation, IVM of GV Primary follicle Pre-antral follicle Antral follicle GV MI MII Arrested GV Arrested MI MII Sato et al. (2007 ) MEST M 50.0 57.0 91.6 55.6 (5/9) 71.4 (5/7) PLAGL1 M 40.0 10.4 83.3 KCNQ1OT1 M 55.5 66.6 87.5 Geuns et al. (2007b ) KCNQ1OT1 M 100 (6/6) 83.3 (5/6) 100 c (4/4) Khoueiry et al. (2008 ) KCNQ1OT1 M 67.8 (GV) a 62.5 66.7 89.5 61.2 65.2 78.3 70.3 (MI) a Geuns et al. (2003 ) SNRPN M 100 (7/7) 100 (3/3) 100 c (3/3) El-Maarri et al. (2001 ) SNRPN M 0 b NDN M 33.3 b Sato et al. (2007 ) H19 P 28.2 33.4 12.5 0 (0/3) 66.7 (2/3) Borghol et al. (2006 ) H19 P 0 (0/5) 24.3 13.8 16.7 Geuns et al. (2007a ) DLK1 P 0 (0/4) 0 (0/6) 0 c (0/6) Methylation is depicted as the percentage of methylated alleles (greater-than two-third of DMR is methylated) of the total number of alleles analysed. M, maternal; P, paternal. If known, the number of methylated oocytes and the total number of oocytes are put in parentheses. a GV and MI are obtained from non-stimulated patients with polycystic ovaries and after culture arrested in GV and MI. b Unfertilized oocytes. c MII obtained after culture of both GV and MI. DNA CpG methylation in human oocytes at different stages of development with and without ovarian stimulation and IVM. Methylation is depicted as the percentage of methylated alleles (greater-than two-third of DMR is methylated) of the total number of alleles analysed. M, maternal; P, paternal. If known, the number of methylated oocytes and the total number of oocytes are put in parentheses. a GV and MI are obtained from non-stimulated patients with polycystic ovaries and after culture arrested in GV and MI. b Unfertilized oocytes. c MII obtained after culture of both GV and MI. Table  III gives a numerical overview of the data on genomic imprinting in oocytes from ovulation induction in women. The analysis of a possible effect of hormonal priming on imprinting can be confounded with maternal age and/or general suboptimal oogenesis. Proper control oocytes are scarce but, nevertheless, important information can be obtained. In contrast to the almost complete methylation at the antral follicle stage of non-stimulated oocytes, after ovarian stimulation only 10 of the 16 germinal vesicle (GV)/metaphase I (MI) oocytes were methylated at MEST ( Sato et al. , 2007 ). The cause of subfertility in the couples donating the affected GV/MI primary oocytes was male factor or tuba obstruction, suggesting a genuine ovulation induction effect. The methylation status of two other maternally imprinted DMRs [ SNRPN ( Geuns et al. , 2003 ) and KCNQ1OT1 ( Geuns et al. , 2007b )] was in most cases undisturbed. However, in one of the 12 GV/MI oocytes KCNQ1OT1 DMR was completely unmethylated (Table  III ). In the study of GV and MI oocytes of Khoueiry et al. , around 60–70% of the alleles was methylated at KCNQ1OT1 DMR, while in metaphase II (MII) oocytes, which are mostly used for IVF treatment, the methylation level was around 90% ( Khoueiry et al. , 2008 ). This would suggest that around 10% of the MII oocytes can lead to BWS, which is not in agreement with the true incidence of BWS in ART. In this study of ( Khoueiry et al. , 2008 ), that included 52 ICSI cycles, some women suffered from polycystic ovary syndrome (PCOS, n = 11), endometriosis ( n = 4) or dysovulation ( n = 3). The methylation level of oocytes from women with or without PCOS was similar. Concerning SNRPN , El-Maarri et al. (2001) found a completely unmethylated DMR in a pool of 20–30 human MII oocytes obtained after ovarian stimulation. This is in agreement with neither the data from Geuns et al. (2003) where at the GV and MI stage SNRPN is already completely methylated, nor with the SNRPN methylation pattern in mouse MII oocytes ( Lucifero et al. , 2002 ). Also, the relative low incidence of AS and PWS after ART is not what would be expected with a completely unmethylated DMR. Regarding the expected paternal DMR demethylation in oocytes, two out of six GV/MI oocytes were erroneously methylated at H19 after ovarian stimulation ( Sato et al. , 2007 ), a finding not substantiated by Borghol et al. (2006) where the paternal methylation imprint at H19 DMR was absent, as expected. Also, the paternally imprinted intergenic DMR of DLK1 and MEG3 (IG-DMR) was mainly unmethylated in superovulated oocytes at the GV and MI stages ( Geuns et al. , 2007a ). IVM of oocytes has been introduced to retrieve several oocytes for IVF treatment avoiding exogenous gonadotrophins, especially for patients at risk for the ovarian hyperstimulation syndrome and/or PCOS ( Jurema and Nogueira, 2006 ). In general, small and medium-sized antral follicles are aspirated and the associated oocytes are cultured for 24–48 h before fertilization, depending on procedure at the clinic. At the antral follicle stage, most DMR CpG methylation has been established although not completely so (see text above and Table  III ). Hence, in theory, IVM could interfere with imprint establishment or maintenance. To prove this point, GV and MI oocytes from women undergoing IVF treatment with hormonal stimulation were cultured in maturation medium. The normal maternal imprints of SNRPN and KCNQ1OT1 in the GV and MI oocytes were stably maintained in vitro up to the secondary oocyte stage ( Geuns et al. , 2003 , 2007b ; Table  III ). However, in a study comparing in vivo derived and IVM derived MII oocytes, the methylation level of KCNQ1OT1 DMR in the second group was statistically lower ( Khoueiry et al. , 2008 ). These authors point out that the maturation time (28 h when compared with ∼36 h in vivo ) might be too short to finish the methylation process but this is disputed by the correct methylation pattern in oocytes cultured overnight as reported in Geuns et al . ( 2003 , 2007b ). The paternal imprint at the IG-DMR was correctly absent after IVM of GV and MI oocytes ( Geuns et al. , 2007a ). Borghol et al. (2006) obtained evidence that the H19 DMR is more vulnerable to the environment. After GV maturation for 24 h, two of the six pools with a maximum of three MII oocytes showed complete methylation in at least one allele, while in MI-derived MII oocytes methylation was completely absent. In the human male germline (Table  IV and Supplementary data, Table ), the imprints of both maternal ( MEST ) and paternal ( H19 ) DMRs are completely erased in fetal prospermatogonia ( Kerjean et al. , 2000 ). The maternally methylated MEST DMR remains unmethylated during spermatogenesis. For H19 , the imprint is established during the adult spermatogonial stage or at least before the spermatocytes enter meiosis I, and is maintained thereafter ( Kerjean et al. , 2000 ), resembling the reprogramming in the mouse ( Ueda et al. , 2000 ). Hence in mature spermatozoa the paternally imprinted DMRs are completely methylated, while the maternally imprinted ones are unmethylated (see Table  IV for references). Table IV DNA CpG methylation studies on human spermatozoa from normal probands and subfertile patients. Reference Conditions analysed DMR Methylation (M/P) Technique ART outcome analysed Normal Concentration Motility Morphology Other Geuns et al. (2007a ) + IG-DMR P Bisulphite sequencing Geuns et al. (2003 ) + SNRPN M Bisulphite sequencing Geuns et al. (2007b ) + KCNQ1OT1 M Bisulphite sequencing Kerjean et al. (2000 ) + Spermatids, Testicular (fetal) spermatogonia and spermatocytes H19 MEST P M Bisulphite sequencing Marques et al. (2004 ) + + H19 MEST P M Bisulphite sequencing Marques et al. (2008 ) + + H19 MEST LINE1 P M Global Bisulphite sequencing Kobayashi et al. (2007 ) + + H19 IG-DMR MEST KCNQ1OT1 PLAGL1 PEG3 SNRPN LINE1 Alu P P M M M M M Global Global Bisulphite sequencing + COBRA + (1 child) Sato et al. (2011 ) + + ZDBF2 H19 IG-DMR MEST KCNQ1OT1 PEG3 SNRPN PLAGL1 P P P M M M M M Bisulphite polymerase chain reaction Luminex, bisulphite sequencing and COBRA Hammoud et al. (2010 ) + + Protamine replacement defect H19 KCNQ1OT1 MEST SNRPN PLAGL1 IGF2 PEG3 P M M M M M M Bisulphite sequencing Poplinski et al. (2010 ) + + + + H19 MEST P M Bisulphite sequencing Benchaib et al. (2003 ) + + + + Overall global Immunostaining Houshdaran et al. (2007 ) + + + + 37 regions 1421 regions – – MethyLight Illumina array Boissonnas et al. (2010 ) + + OAT H19-CTCF3 H19 CTCF6 IGF2-DMR0 IGF2-DMR2 LINE1 P P P P Global Pyrosequencing + (fertilization rate, cleavage and fragmentation) Navarro-Costa et al. (2010 ) + OAT DAZL DAZ M+P M+P Bisulphite sequencing El Hajj et al. (2011 ) + OAT H19 IG-DMR MEST KCNQ1OT1 PEG3 SNRPN NESPAS LINE1 P P M M M M M Global Pyrosequencing + (Fertilization rate, pregnancy rate, live birth rate, abortion rate) Manning et al. (2001 ) + Testicular round spermatids, testicular elongated spermatids SNRPN M Methylation sensitive PCR with fragment length analysis Hartmann et al. (2006 ) + Arrested spermatogonia Arrested spermatocytes H19 P Bisulphite PCR with single-strand conformation polymorphism (SSCP) analysis Marques et al. (2010 ) Testicular spermatozoa form patients azoospermic due to ANJ, OAZI, CBAVD, HP H19 MEST P M Bisulphite sequencing + (embryonic developmental arrest in HP patient) COBRA = combined bisulphite-PCR restriction analysis, OAT = patients patients presenting with combined  oligozoospermia,  asthenozoospermia and teratozoospermia, ANJ = Anejaculation, OAZI = secondary inflammatory obstructive azoospermia, CBAVD = obstructive azoospermia due to congenital bilateral absence of the vas deferens, HP = secretory azoospermia due to hypospermatogenesis. DNA CpG methylation studies on human spermatozoa from normal probands and subfertile patients. H19 MEST P M H19 MEST P M H19 MEST LINE1 P M Global H19 IG-DMR MEST KCNQ1OT1 PLAGL1 PEG3 SNRPN LINE1 Alu P P M M M M M Global Global ZDBF2 H19 IG-DMR MEST KCNQ1OT1 PEG3 SNRPN PLAGL1 P P P M M M M M H19 KCNQ1OT1 MEST SNRPN PLAGL1 IGF2 PEG3 P M M M M M M H19 MEST P M 37 regions 1421 regions – – MethyLight Illumina array H19-CTCF3 H19 CTCF6 IGF2-DMR0 IGF2-DMR2 LINE1 P P P P Global DAZL DAZ M+P M+P H19 IG-DMR MEST KCNQ1OT1 PEG3 SNRPN NESPAS LINE1 P P M M M M M Global Arrested spermatogonia Arrested spermatocytes H19 MEST P M COBRA = combined bisulphite-PCR restriction analysis, OAT = patients patients presenting with combined  oligozoospermia,  asthenozoospermia and teratozoospermia, ANJ = Anejaculation, OAZI = secondary inflammatory obstructive azoospermia, CBAVD = obstructive azoospermia due to congenital bilateral absence of the vas deferens, HP = secretory azoospermia due to hypospermatogenesis. ART as such is unlikely to affect methylation in spermatozoa since these patterns, including the paternal imprints, are established before any manipulation occurs, the normal sperm nucleus being metabolically inert. Several studies show that a disturbed spermatogenesis itself is associated with incorrect imprinting (Table  IV , Supplementary data, Table ). In spermatozoa from oligozoospermic men, the occurrence of hypermethylation of several maternally imprinted DMRs or hypomethylation of the H19 and IG-DMR is increased, especially in ejaculates of <10 × 10 6 /ml ( Supplementary data, Table ). The number of affected CpG sites ranges from only a few in normozoospermia to the whole DMR in severe azoospermia, only occurring in a minority of alleles sampled ( Supplementary data, Table ). Further evidence for an association between methylation and sperm concentration comes from a study by Boissonnas et al. (2010) who analysed the H19 DMR (CTCF6 region) in teratozoospermic (TZ) and oligo-astheno-teratozoospermic (OAT) patients. In spermatozoa from TZ patients, only 2out of 16 CpGs were significantly hypomethylated. In OAT spermatozoa, methylation was drastically reduced for all CpGs, reaching significance in subgroups with a sperm concentration of <10 × 10 6 /ml. Sperm concentration is positively correlated with H19 methylation and negatively correlated with MEST methylation that is normally absent ( Boissonnas et al. , 2010 , Poplinski et al. , 2010 ). OAT spermatozoa also show reduced IG-DMR methylation ( El Hajj et al. , 2011 ). Alteration of the protamine 1 to protamine 2 ratio, which should be around 1, generally denotes affected spermatogenesis [either as cause or as consequence ( Nanassy et al. , 2011 )] and led to hypermethylation of several normally maternally methylated loci DMRs ( KCNQ1OT1 , SNRPN , MEST , PEG3 , PLAGL1 , IGF2 ) and to hypomethylation of the H19 DMR ( Hammoud et al. , 2010 ). Together, these data clearly indicate that DMR methylation defects are associated with poor spermatogenesis. Besides oligozoospermia, also other aetiologies of male subfertility are associated with epigenetic defects (Table  IV , Supplementary data, Table ). Azoospermia caused by anejaculation and secondary inflammatory obstruction was related to an increase in MEST methylation ( Marques et al. , 2010 ), as was a sperm motility <40, or <5% of sperm with normal morphology ( Poplinski et al. , 2010 ). Global DNA methylation of non-imprinted repetitive sequences, such as long and short interspersed nucleotide elements (LINE1 and SINE (Alu)), did not show a decrease in spermatozoa from oligozoospermic or OAT patients ( Kobayashi et al. , 2007 ; Marques et al. , 2008 ; Boissonnas et al. , 2010 ; El Hajj et al. , 2011 ), except for Alu element methylation in the study by El Hajj et al. ( Supplementary data, Table ). The methylation of non-imprinted genes and a repetitive sequence was also affected ( Houshdaran et al. , 2007 ), typically for sequences showing large intra- and interindividual methylation variation in spermatozoa from normozoospermic males ( Flanagan et al. , 2006 ). Recently, it has been shown that patients with OAT had an increased level of methylation in the promotor of the normally unmethylated germline regulator gene DAZL, the autosomal substitute of Y-linked DAZ that correctly remained unmethylated ( Navarro-Costa et al. , 2010 ; Supplementary data, Table ). It is not known to what extent DMR CpG methylation in both degree and prevalence can be ablated before germline transmission of this mark suffers. Kobayashi et al. (2009) compared the methylation defect that was found in trophoblastic villi from ART-miscarriages between 6-9 weeks of gestation with the imprints in the semen from the father. In 7 out of the 17 ART pregnancies with a placental H19 methylation defect, this was also found in the spermatozoa, suggesting transfer from the father. In a patient with hypospermatogenesis and with almost complete hypomethylation of the H19 DMR, the embryos obtained after ICSI all showed developmental arrest (stage unknown) ( Marques et al. , 2010 ). As in the human H19 is not expressed up to the blastocyst stage ( Salpekar et al. , 2001 ), a common paternal factor might be at stake. As no analysis of the embryos was undertaken, there is no formal proof of paternal inheritance of H19 DMR hypomethylation . In patients with OAT with a partial hypomethylation of H19, the fertilization rate after ICSI was reduced ( Boissonnas et al. , 2010 ). Developmental parameters, such as embryo quality, implantation rate, gestational age and birthweight, were similar to normally methylated paternal controls ( Boissonnas et al. , 2010 ). In another case, with spermatozoa showing both a maternal and a paternal methylation imprinting error ( MEST DMR was methylated in 60% of the alleles and PLAGL1 in 20%, and H19 DMR was unmethylated in 90% of the alleles), a normal pregnancy was achieved with normal methylation ( Kobayashi et al. , 2007 ). Methylation analysis in 19 ISCI children born small for gestational age revealed that one of them had hypermethylation in KCNQ1OT1 and MEST . As both parents had a normal methylation profile (and hypermethylation can only refer to the normally hypomethylated paternal allele), the methylation must have appeared de novo , maybe in the male germline because of the oligozoospermia of the father ( Kanber et al. , 2009 ; Table  V ). Table V DNA CpG methylation studies of mainly DMRs in human offspring following ART. Reference ART n Control Sample DMR M/P Results Kanber et al. (2009 ) ICSI + SGA 19 Normal weight children after spontaneous conception Buccal smear KCNQ1OT1 MEST PEG3 H19 GTL2 PLAGL1 M M M P P M 1/19 children had hypermethylation of KCNQ1OT1 and MEST Manning et al. (2000 ) ICSI 92 – Blood SNRPN M In all 92 children the expected methylation pattern was seen Gomes et al. (2009 ) IVF and ICSI 18 Naturally conceived children and BWS patients Peripheral blood, UCB or placenta KCNQ1OT1 M 3 of 18 IVF children showed hypomethylation at KCNQ1OT1, without BWS phenotype Tierling et al. (2010 ) IVF and ICSI 112 Naturally conceived children UCB, amnion membrane KCNQ1OT1 H19 SNRPN MEST GRB10 GTL2 GNAS locus M P M M M PM+P Only MEST was slightly hypermethylated in IVF compared with ICSI and control samples Katari et al. (2009 ) IVF 10 Naturally conceived children UCB, placenta 1536 CpG sites – 23% CpG sites differed in UCB and 16% in placenta. Imprinted genes are not extra vulnerable for deregulation. 4/11 tested genes with differential methylation also showed differential expression Zechner et al. (2010 ) IVF and ICSI 42 Abortions/stillbirths after spontaneous conception Chorion villi H19 MEG3 KCNQ1OT1 MEST NESP55 PEG3 SNRPN NANOG APC P P M M P M M M+P M+P IVF villi showed a hypomethylation (3% less) of KCNQ1OT1 Turan et al. (2010 ) IVF (ICSI unknown) 45–98 Naturally conceived children UCB, cord, placenta H19 P After IVF the intra- and inter-individual variation in methylation is higher. The expression of H19 and IGF2 in placenta and UCB was reduced in the IVF group M/P, maternally or paternally methylated; SGA, small for gestational age; UCB, umbilical cord blood. DNA CpG methylation studies of mainly DMRs in human offspring following ART. KCNQ1OT1 MEST PEG3 H19 GTL2 PLAGL1 M M M P P M KCNQ1OT1 H19 SNRPN MEST GRB10 GTL2 GNAS locus M P M M M PM+P H19 MEG3 KCNQ1OT1 MEST NESP55 PEG3 SNRPN NANOG APC P P M M P M M M+P M+P M/P, maternally or paternally methylated; SGA, small for gestational age; UCB, umbilical cord blood. There is only one case report in which part of the methylation defect of the child was also detected in leucocytes from the father. In a child with SRS conceived by IVF, eight CpGs were hypermethylated in PEG1/MEST DMR, four of which were also hypermethylated in the father ( Kagami et al. , 2007 ). Loss of 5methyl CpG immunostaining in human embryos after fertilization resembles that reported in mammalian embryos ( Santos et al. , 2002 , 2010 ). Active loss of immuno-recognition of paternal 5methylC also takes place in the human zygote indicating active demethylation ( Beaujean et al. , 2004 ; Fulka et al. , 2004 ). After gamete fusion, a global passive maternal demethylation takes place, clearly visible at the 4-cell stage. At the end of the morula stage, remethylation starts ( Fulka et al. , 2004 ; Santos et al. , 2010 ). In blastocysts, the methylation level of the trophectoderm and inner cell mass diverge, with more methylation in the inner cell mass ( Santos et al. , 2010 ). Abnormal chromatin organization, as observed via DNA-specific YOYO staining and aberrant mCpG staining, seemed to be correlated in arrested IVF embryos, suggesting proper chromatin organization for early development ( Santos et al. , 2010 ). Transcripts of several imprinted genes like SNRPN , MEST , UBE3A and IGF2 (but not H19 ) are already present at the preimplantation embryonic stages ( Lighten et al. , 1997 ; Huntriss et al. , 1998 ; Salpekar et al. , 2001 ). The monoallelic expression starts from the 4 ( SNRPN ) and 8 ( IGF2 ) cell stage, meaning that the primary imprints laid down during oogenesis and spermatogenesis are resistant to active and passive demethylation during the cleavage divisions. Effects of IVF or subsequent development in culture medium alone are difficult to investigate in the human, since both are inevitably connected with each other. Moreover, the in vivo comparison cannot be made. In vitro conditions could affect maintenance of imprinting: at Day 3, 19% of human surplus embryos of low-quality (not suitable for transfer or for cryopreservation) showed hypomethylation of H19 ( Chen et al. , 2010 ). Paternal transmission was unlikely as none of the sperm samples showed hypomethylation. Similar results were obtained in a study where 8 of the 21 arrested surplus embryos showed loss of paternal methylation at H19 DMR, while the corresponding sperm samples were normal ( Ibala-Romdhane et al. , 2011 ) It is not known whether the hypomethylation (likely as a correlated response) leads to growth arrest or whether the growth arrest (induced by in vitro conditions) leads to loss of methylation. In the same study, eight arrested embryos showed methylation of the maternal allele (three of which also had hypomethylation at the paternal allele). After analysis of the unfertilized oocytes of two patients by using a distinguishing single nucleotide polymorphism, it was hypothesized that a defect in the erasure of the paternal imprint in the maternal germline led to this hypermethylation. Interestingly, five cryopreserved blastocysts that were donated for research after several years, all showed normal methylation ( Ibala-Romdhane et al. , 2011 ). As to the mode of IVF, there is no convincing evidence that ICSI elevates the risk for epigenetic abnormalities when compared with IVF or vice versa. Nucleus structure and methylation levels (immunofluorescence) seen in arrested embryos and fully grown blastocysts did not differ between IVF and ICSI ( Santos et al. , 2010 ). An increased risk of imprinting disorders applies to pregnancies originating from both IVF and ICSI (Table  II ). Recently, proof has been obtained of an effect of culture medium on offspring. IVF children derived from embryos that were cultured in two different media showed a significant difference in birthweight of almost 250 g ( Dumoulin et al. , 2010 ). This resembles the animal studies where the addition of serum to the culture medium affects the growth of the fetus ( Khosla et al. , 2001 ; Young et al. , 2001 ; Fernandez-Gonzalez et al. , 2004 ), although the effect in the human (not involving serum) seems less severe and a causative epigenetic variable has not been found yet. Except for the described imprinting disorders, induced epigenetic variants (Table  V ) that do not have clear phenotypical effects might be transmitted to the offspring. Gomes et al. (2009) analysed KCNQ1OT1 , that was hypomethylated in 3 out of 18 IVF children. These were all part of a dizygotic twin, with the co-twin showing normal methylation. The methylation level was reduced from 41.5% in naturally conceived children to around 14% in these three probands without clinical symptoms. BWS patients show 1% methylation. Another group did not find a difference in KCNQ1OT1 methylation in amnion/chorion membranes, umbilical cord blood and maternal peripheral blood of IVF and control conceptions ( Tierling et al. , 2010 ). Eight other DMRs also showed a normal methylation pattern ( H19 , SNRPN , GRB10 , IG-DMR and 4 DMRs in the GNAS region). Only MEST was slightly hypermethylated in IVF compared with ICSI and control samples. No correlation between the methylation level of any of these genes and birthweight was found. In chorion villus samples from spontaneous miscarriages and stillbirths, a hypomethylation of KCNQ1OT1 (significant) as well as H19 (trend) was seen in samples derived after IVF ( n = 42) ( Zechner et al. , 2010 ). Five other DMRs did not show a difference compared with a control group. The intra- and interindividual variation in methylation as assessed for H19 makes comparison in humans harder ( Turan et al. , 2010 ). The variation is higher in placental tissue compared with umbilical cord blood but also increased after IVF compared with in vivo fertilization, probably because IVF offspring result from embryos with fewer trophoblast stem cells ( Turan et al. , 2010 ). The expression of both IGF2 and H19 was reduced in placental tissue of ART pregnancies. The expression of IGF2 was not correlated with birthweight. An extended DNA methylation analysis of more than 1500 genes, including DMRs, in placental tissue and umbilical cord blood from IVF and control pregnancies indicated that imprinted genes are not more vulnerable to methylation differences than non-imprinted genes ( Katari et al. , 2009 ). Around 16% of the analysed CpG sites showed a difference (hypo- or hypermethylation) in placental tissue and 23% in umbilical cord blood. Four out of 11 tested genes that showed a difference in methylation between the two groups also showed a difference in level of transcription. Zhang et al. (2010) analysed global gene expression patterns in placentae from three IVF and three control pregnancies. Twenty-six genes were differentially expressed, none of them imprinted. In all the above mentioned studies that found a methylation effect in ART children, the methylation of the single investigated CpG within the analysed tissue was never completely (100%) methylated or demethylated (0%). This suggests that the methylation defects are not transmitted from the oocyte or sperm cell. Unfortunately, the type of analyses and the presentation of the results do not allow us to accurately specify mosaicism. Three meta-analyses with similar results on perinatal outcome have been published ( Helmerhorst et al. , 2004 ; Jackson et al. , 2004 ; McDonald et al. , 2009 ). The studies included in the analyses were selected either on the use of an appropriate control group ( Helmerhorst et al. , 2004 ), or whether they controlled for maternal age ( McDonald et al. , 2009 ) or maternal age and parity ( Jackson et al. , 2004 ). The substantial number of included singletons ranged from 5361 ( Helmerhorst et al. , 2004 ) to more than 31 000 ( McDonald et al. , 2009 ) and all three meta-analyses showed an increased risk in the IVF group for very preterm birth [relative risk (RR) ranged from 3.0 to 3.3], preterm birth (RR 1.9–2.0), very low-birthweight (RR 2.7–3.8), low-birthweight (RR 1.4–1.8), small for gestational age (RR 1.4–1.6), Caesarean section (RR 1.5–2.1), admittance to neonatal intensive care unit (RR 1.3–1.6) and mortality (RR 1.7–2.4). For congenital malformations, more controversy exists, also mainly because of the relative small sample sizes in comparison with the frequency of the malformation. Recently, a Swedish group analysed two consecutive cohorts, each consisting of more than 15 000 singleton IVF children ( Kallen et al. , 2005 , 2010b ). Besides esophageal atresia (OR = 5.2) and urogenital defects (OR = 2.3) ( Kallen et al. , 2005 ), in both cohorts an increased risk for limb reduction (OR = 1.7–2.0), neural tube defects (OR = 2.9–4.2) cardiovascular malformations (OR = 1.3–1.7) and syndromes associated with imprinting defects like Prader–Willi (RR 4.0) was reported. These malformations were also found in smaller cohorts [e.g. ( Ericson and Kallen, 2001 ; Hansen et al. , 2002 ; Klemetti et al. , 2005 )]. Regarding cancer, Kallen et al. found a relative risk of 1.4 in a large cohort of almost 27 000 IVF children. Pertinent to this review is the aetiology of these malformations, i.e. the balance between genetic and epigenetic aberrations. Ceelen et al. dedicated themselves to the physical development of IVF children aged 8–18 years. They investigated systolic and diastolic blood pressure, skinfold thickness, fasting glucose/insulin levels, fat, growth velocity, bone development and endocrine status during puberty. Systolic and diastolic blood pressure, peripheral skinfold thickness, fasting glucose level, weight and height gain between 3 months and 1 year and dehydroepiandrosterone sulphate (DHEAS) and LH level in pubertal girls were all higher in the IVF group compared with a control group (consisting of naturally conceived children from subfertile couples) even after adjustment for potential confounders, such as maternal BMI ( Ceelen et al. , 2007 , 2008a , c , 2009 ). The higher blood pressure was confirmed in a cohort of 4–14-year-old IVF children, together with a higher triglyceride level. DHEAS levels did not differ from the control group ( Sakka et al. , 2010 ). In a younger IVF group of around 6 years old, the IVF children were taller and had a slightly more favourable lipid profile when compared with naturally conceived children ( Miles et al. , 2007 ). For all these parameters, under the assumption of a random participation in ART among the genotypic variance present in our population, no genetic component is indicated. It may therefore well be the result of an epigenetic adaptive response to the (preimplantation) environment.

It is clear that a number of questions regarding possible epigenetic effects of ART can be answered (see Fig.  6 ). Figure 6 Overview of the results of studies on the effect of ART on methylation and expression of imprinted genes. ( A ) Overview of mouse data. ( B ) Overview of human data. A/C = Amnion/Chorion, Bl = Blastocyst, E = embryo with the age, Ex = Expression which indicates either the level of expression or the allelic expression, ET = embryo transfer, GV = germinal vesicle oocyte, M = methylation, MI = oocyte in meiosis I, MII = oocyte in meiosis II, P = placenta with the age, UCB = umbilical cord blood. The numbers refer to the studies: 1. Sato et al. (2007 ), 2. Market-Velker et al. (2010a ), 3. Fauque et al. (2007 ), 4. Fortier et al. (2008 ), 5. Rivera et al. (2008 ), 6. Doherty et al. (2000 ), 7. Mann et al. (2004 ), 8. Li et al. (2005 ), 9. Market-Velker et al. (2010b ), 10. Khosla et al. (2001 ), 11. Fauque et al. (2010a ), 12. Geuns et al. (2003 ), 13. Geuns et al. (2007b ), 14. Khoueiry et al. (2008 ), 15. El-Maarri et al. (2001 ), 16. Borghol et al. (2006 ), 17. Geuns et al. (2007a ) 18. Chen et al. (2010 ), 19. Ibala-Romdhane et al. (2011 ), 20. Gomes et al. (2009 ), 21. Tierling et al. (2010 ), 22. Turan et al. (2010 ), 23. Zechner et al. (2010 ), 24. Zhang et al. (2010 ), 25. Katari et al. (2009 ). Overview of the results of studies on the effect of ART on methylation and expression of imprinted genes. ( A ) Overview of mouse data. ( B ) Overview of human data. A/C = Amnion/Chorion, Bl = Blastocyst, E = embryo with the age, Ex = Expression which indicates either the level of expression or the allelic expression, ET = embryo transfer, GV = germinal vesicle oocyte, M = methylation, MI = oocyte in meiosis I, MII = oocyte in meiosis II, P = placenta with the age, UCB = umbilical cord blood. The numbers refer to the studies: 1. Sato et al. (2007 ), 2. Market-Velker et al. (2010a ), 3. Fauque et al. (2007 ), 4. Fortier et al. (2008 ), 5. Rivera et al. (2008 ), 6. Doherty et al. (2000 ), 7. Mann et al. (2004 ), 8. Li et al. (2005 ), 9. Market-Velker et al. (2010b ), 10. Khosla et al. (2001 ), 11. Fauque et al. (2010a ), 12. Geuns et al. (2003 ), 13. Geuns et al. (2007b ), 14. Khoueiry et al. (2008 ), 15. El-Maarri et al. (2001 ), 16. Borghol et al. (2006 ), 17. Geuns et al. (2007a ) 18. Chen et al. (2010 ), 19. Ibala-Romdhane et al. (2011 ), 20. Gomes et al. (2009 ), 21. Tierling et al. (2010 ), 22. Turan et al. (2010 ), 23. Zechner et al. (2010 ), 24. Zhang et al. (2010 ), 25. Katari et al. (2009 ). In the mouse, an effect of ART, from imprinting maintenance to physiological homeostasis to behaviour, has generally been found. The placenta stands out as much more vulnerable to the influences of ART on imprinting compared with the embryo proper. This is likely related to the underlying theoretical basis of imprinting, the male–female conflict hypothesis in mammalian reproduction ( Moore and Haig, 1991 ). With respect to the information that is lacking in the mouse, the clearest omissions are the absence of an OAT model and the effects of ART at increased maternal age. Also, the effect of in vitro culture without ovulation induction has not yet been studied. The effect of ovulation induction on maintenance of imprinting can be expressed at three levels. (i) That on maintenance of imprinting at recruitment of an antral follicle, (ii) that expressed as a maternal early embryonic (‘zygotic’) cellular effect on maintenance of the imprint after gamete fusion and (iii) that expressed via the maternal tractus. Not much work has been done on the first effect, for which some indication is found in the human but none in the mouse. It should be noted that in mice maturation of multiple oocytes in one cycle is natural, while in humans it is not. That might explain why in humans ovulation induction interferes with the maintenance of imprinting in the antral follicle while in the mouse the effect is only after in vitro preimplantation development and/or through the maternal tractus. In both species, the unusual phenomenon of methylation of the H19 DMR has been observed in oocytes, the etiology of which could be different as in the human germline, erasure is indicated to be later (during first meiotic prophase) compared with the mouse (before first meiotic prophase). Since in the mouse no ovulation induction effect in oocytes was reported (except H19), and a mosaic pattern for DMR methylation status was observed in blastocysts and the placenta, a ovulation induction effect on imprinting must be based on the maternal cellular effect in combination with the maternal environment. In the mouse, a negative effect of ovulation induction on the maternal tractus is generally accepted ( de Boer et al. , 1991 ; Van der Auwera et al. , 1999 ; Van der Auwera and D'Hooghe, 2001 ). After changing this environment to normal by transferring the embryo to a non-stimulated uterus, the ovulation induction effect on imprinting lessened ( Fortier et al. , 2008 ). At mid-gestation, in vitro culture aggravated the impact of ovulation induction on imprinting maintenance ( Rivera et al. , 2008 ). In all, enough evidence has been obtained in the mouse as to an effect of hormonal intervention and the in vitro steps (fertilization and subsequent culture) on maintenance of imprinting, especially for the placenta. In the only study that analysed superovulated in vivo matured human MII oocytes, the number of the oocytes not methylated at KCNQ1OT1 was much higher than the prevalence of BWS after ART. Most likely this suggests that the great majority of embryos derived from these oocytes are not viable. This non-viability of methylation disturbed embryos is substantiated by the hypo- and hypermethylation of H19 DMR in arrested embryos, while non-arrested embryos showed normal methylation ( Ibala-Romdhane et al. , 2011 ). It would be of interest to know whether a milder ovarian stimulation would lead to a reduction of BWS cases (single embryo transfer is increasingly applied and therefore fewer embryos are needed). In the human, the influence of poor spermatogenesis on maintenance of imprinting methylation and methylation of other sequences is undisputed, making sperm a potential vehicle for transmitting paternal methylation abnormalities. However, the chance of transmission to the offspring appears rather small. The type of IVF (conventional IVF or ICSI) does not seem to make a difference regarding methylation defects. In vitro culture conditions and methylation defects might be associated since in human arrested embryos, H19 hypomethylation is reported, without methylation defects in the spermatozoa ( Chen et al. , 2010 ; Ibala-Romdhane et al. , 2011 ). An in vivo comparison can, however, not be made. The first array-based analysis on CpG methylation at birth ( Katari et al. , 2009 ) gives the impression of an ART-induced vulnerability for CpG methylation disturbances for a considerable frequency of sites, with more CpGs differentially methylated in umbilical cord blood than in placental tissue. When a difference was observed, in cord blood most CpGs were hypermethylated in the in vitro group while in placental tissue most CpGs were hypomethylated. An affected 5methyl CpG maintenance can be without any effect but might bring IVF progeny closer to a threshold, making them more vulnerable to physiological reported effects at adolescence ( Ceelen et al. , 2007 , 2008a , c , 2009 ) or late-onset diseases, such as cardiovascular disease, cancer ( Kallen et al. , 2010a ) or other minor effects that have not yet been observed. Differences in gene expression of metabolism-related genes set by embryonic and/or fetal programming are assumed to underlie the relation between low-birthweight [also reported in IVF neonates ( Helmerhorst et al. , 2004 ; Jackson et al. , 2004 ; McDonald et al. , 2009 )] and chronic diseases in later life, like cardiovascular diseases and diabetes mellitus ( Barker, 2006 ). This finding could also be one explanation of the parallel observation of an effect of ART on both systolic blood pressure and glucose tolerance in mice and men ( Watkins et al. , 2007 ; Ceelen et al. , 2008a ; Scott et al. , 2010 ). The recently isolated maternally imprinted gene KLF14 ( Small et al. , 2011 ) could well be the master regulator, at least for diabetes II and adipocyte-related metabolic disease risk. In general, more evidence for an epigenetic effect of ART has been obtained in the mouse than in man. However, the definite answer to many questions in both mouse and human will await genome-wide epigenetic profiling in the different variants of ART. From the patient's perspective, those that contribute sperm from poor spermatogenesis stand out as very interesting for such an analysis as do the oocytes from older women.

In 2009, Katari et al. have reported that some genes from babies conceived by means of IVF show a gene expression pattern that is different from naturally conceived children ( Katari et al. , 2009 ). The observed changes were associated with the mechanism that switches genes on and off, which is heritable to forthcoming cell generations, hence under epigenetic control. In their study, Katari et al. have stated that this mechanism could put children conceived by means of assisted reproduction treatment (ART) at a greater risk of diseases, such as diabetes and obesity, later in life. Epigenetic deregulation already received increasing attention as a possible common cause of adverse ART outcomes, since the incidence of disorders that involve imprinted genes, especially the Beckwith–Wiedemann syndrome (BWS), is often reported to be increased in the offspring ( Amor and Halliday, 2008 , Ceelen et al. , 2008b , Manipalviratn et al. , 2009 ). This led to an expansion of ART literature on epigenetic effects, also including mouse model studies. The subfertility of one or both parents as a causative factor has to be taken into account, which often is difficult to achieve. Moreover, mouse models in this area are almost absent. Another question is if the assumed epigenetic effects of ART can be transmitted to the next generation. Besides classical Mendelian inheritance of information stored in the DNA sequence, other mechanisms are active in the transmission of phenotypic traits across generations too. No insight into underlying molecular mechanisms was available when the theory of non-genetic transmission was first put forward by Waddington (1953) to describe the acquired characteristics in the offspring of Drosophila exposed to heat ( McLaren, 1999 ). In the broad sense coined by Waddington, these observations are termed transgenerational epigenetic effects ( Youngson and Whitelaw, 2008 ). When these phenotypic alterations are caused by transfer of chromosome/chromatin modifications through the gametes, the term transgenerational epigenetic ‘inheritance’ is used ( Youngson and Whitelaw, 2008 ; Jablonka and Raz, 2009 ). In this definition, the word epigenetic refers to the mechanisms involved in the mitotic and meiotic transfer of non-genetic (i.e. not DNA sequence based) information. Transgenerational epigenetic inheritance has been proved in organisms ranging from bacteria and plants to the mouse and humans ( Jablonka and Raz, 2009 ). Epigenetic mechanisms exist as an interplay between DNA methylation, RNA-mediated chromatin modifications, histone modifications and histone variants, but likely also less well-studied mechanisms, such as the organization of nuclear structure including chromosome replication behaviour ( Jablonka and Raz, 2009 ; Margueron and Reinberg, 2010 ). In the context of this review, inheritance via DNA methylation will be mainly discussed. The molecular epigenetic mechanisms are instrumental in the specification of cell identity and potency within generations. Epigenetic mechanisms are thought to concertedly orchestrate the spatial and temporal regulation of cell differentiation throughout development ( Goldberg et al. , 2007 ; Margueron and Reinberg, 2010 ; Zaidi et al. , 2010 ). Since all cells of an organism have the same genotype, epigenetic marks are deposited to alter transcription and achieve cell-type specific gene expression patterns in different tissues. In fact, the definition of epigenetics given by Jabonka and Raz (2009) as ‘the study of the processes that underly developmental plasticity and canalization and that bring about persistent developmental effects in both prokaryotes and eukaryotes’ underlines its role in embryogenesis and cell differentiation. Sex-specific genomic imprinting and stable female X-inactivation are also under epigenetic control. Between generations, the germ line is subjected to two distinct reprogramming events [one in the primordial germ cells (PGCs) and one in the preimplantation embryo], in order to prepare the cells for pluri- and totipotency and down-regulate the inheritance of epigenetic information between generations [reviewed in ( Reik et al. , 2001 ; Morgan et al. , 2005 ; Feng et al. , 2010 )]. Both phases are pertinent to ART and especially the second one (in the preimplantation embryo) owing to the in vitro circumstances at this epigenetically crucial phase of development. The discovery that some loci, notably imprinted ones, escape reprogramming in the early embryo, provided a first hint regarding the mechanism behind epigenetic inheritance as this brought into question the rigidity of epigenetic erasure between generations as a principle. Epigenetic marks are generally thought to be stable through rounds of somatic mitosis after initial deposition in development ( Margueron and Reinberg, 2010 ). This careful balance between somatic maintenance of epigenetic marks and dynamic reprogramming in the germline has led epigenetic mechanisms to be put forward as a vehicle for ‘soft inheritance’ ( Youngson and Whitelaw, 2008 ), a term first introduced to describe a more pliable system of inheritance, which would allow organisms to quickly adapt to fluctuations in nutrition, predation or disease ( Mayr and Provine, 1980 ; Mayr, 1982 ). The question we will pursue here is whether the conditions during gametogenesis and the in vitro phases intrinsic to ART could elicit epigenetic effects and, if so, whether these could be transmitted to the next generation. We will first present the processes of mitotic epigenetic inheritance. Next, we will describe the known molecular mechanisms involved in the escape of germline reprogramming and present environmental and hormonal cues that induce alterations in the epigenome to be passed on to the next generation. In the second part of this review, observations of a molecular epigenetic nature made in mouse and human ART will be presented. Finally, we will attempt to integrate these observations, including the likelihood of transgenerational epigenetic inheritance, and designate the areas of human reproduction in the context of ART where insight is lacking most.

Supplementary data are available at http://humupd.oxfordjournals.org/ .