a molecular perspective on procedures and...

A Molecular Perspective on Procedures andOutcomes with Assisted ReproductiveTechnologies

Monica A. Mainigi1, Carmen Sapienza2, Samantha Butts1, and Christos Coutifaris1

1Department of Obstetrics and Gynecologyand the Center for Research on Reproduction and Women’s Health,Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

2Fels Institute for Cancer Research and Molecular Biology and Department of Pathology and LaboratoryMedicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Correspondence: [email protected]

The emerging association of assisted reproductive technologies with adverse perinatal out-comes has prompted the in-depth examination of clinical and laboratory protocols andprocedures and their possible effects on epigenetic regulatory mechanism(s). The applicationof various approaches to study epigenetic regulation to problems in reproductive medicinehas the potential to identify relative risk indicators for particular conditions, diagnosticbiomarkers of disease state, and prognostic indicators of outcome. Moreover, whenapplied genome-wide, these techniques are likely to find novel pathways of disease patho-genesis and identify new targets for intervention. The analysis of DNA methylation, histonemodifications, transcription factors, enhancer binding and other chromatin proteins, DNase-hypersensitivity and, micro- and other noncoding RNAs all provide overlapping and oftencomplementary snapshots of chromatin structure and resultant “gene activity.” In terms ofclinical application, the predictive power and utility of epigenetic information will dependon the power of individual techniques to discriminate normal levels of interindividual var-iation from variation linked to a disease state. At present, quantitative analysis of DNAmethylation at multiple loci seems likely to hold the greatest promise for achieving thelevel of precision, reproducibility, and throughput demanded in a clinical setting.

Since the first live-birth resulting from in vi-tro fertilization (IVF) in 1978, the combina-

tion of improved success rates and increaseddemand for treatment has led to a dramaticrise in the number of infants born as a resultof this technology. In the United States alone,more than 130,000 assisted reproductive tech-nology (ART) cycles were initiated in 2013,

resulting in more than 42,000 live births and55,000 infants (www.sartcorsonline.com). World-wide, greater than three million children havebeen born with the assistance of in vitro repro-ductive technologies. (ESHRE 2006; Van Voo-rhis 2007).

In recent years, there have been increasingconcerns regarding the potential health impact

Editors: Diana W. Bianchi and Errol R. Norwitz

Additional Perspectives on Molecular Approaches to Reproductive and Newborn Medicine available

at www.perspectivesinmedicine.org

Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a023416

Cite this article as Cold Spring Harb Perspect Med 2016;6:a023416

1

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org

on June 20, 2020 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from

mailto:[email protected]



https://www.sartcorsonline.com



http://www.perspectivesinmedicine.org



http://perspectivesinmedicine.cshlp.org/

of infants conceived with the assistance of IVF.Although twin and higher order multiple preg-nancies are the main contributors to the mor-bidities associated with IVF conceptions, studieshave indicated that even singleton pregnanciesare at increased risk for a number of adverseperinatal outcomes such as preterm birth, lowbirth weight, congenital anomalies, preeclamp-sia, placental abruption, perinatal mortality, andseveral other pregnancy related complications(Wang et al. 2000; Ericson and Kallen 2001;Hansen et al. 2002; Schieve et al. 2002, 2004;Jackson et al. 2004; Shevell et al. 2005; Chunget al. 2006; Van Voorhis 2006; Kalra and Moli-naro 2008; Kalra et al. 2011, 2012; Henningsen2014). The contributing factors underlyingthese associations are not known, but the obser-vations, particularly in singleton pregnancies,suggest a relationship of adverse outcomes tosome aspects of IVF. There is ongoing debatewhether an underlying inherent increased riskin subfertile couples is central to the pathogen-esis of these adverse outcomes (Schieve 2004).Alternatively, these adverse outcomes may berelated to the protocols used to hyperstimulatepatients to maximize the number of oocytesretrieved (Sato et al. 2007; Market-Velker et al.

2010) and/or to the laboratory procedures usedfor oocyte retrieval, fertilization, and growth ofthe embryos (Fig. 1). Mechanical manipulationof gametes and embryos and exposure to tem-perature and oxygen variation (or even light)during a very critical time of developmentmay potentially have profound and lasting ef-fects.

Therefore, it is likely that multiple factorscontribute to the observed adverse outcomesand their pathophysiologic cellular mecha-nism(s) may differ. Given that most of the clin-ical observations can be attributed to a “placen-ta process” (Shevell et al. 2005; Sun et al. 2009),data from the mouse model and our own pub-lished and preliminary observations in the hu-man, lead us to hypothesize that some of theobserved adverse outcomes may be the resultof alterations in trophoblast function. This re-sults in perturbations in the processes of im-plantation and placentation (Fowden et al.2006). These processes depend on both em-bryonic factors regulating the adhesive and in-vasive properties of trophoblasts and maternaluterine and immunologic factors that play per-missive and regulatory roles modulating tro-phoblast invasion (Norwitz et al. 2001). Most

Ovarianstimulation

Egg retrieval

InseminationICSI

Incubation conditions/culture media

Blastomere Bx

Assisted hatching

Trophectoderm Bx

5/6tx

3tx

2tx

1 4

Other exposure:

Temperature-Light-Oxygen–Embryo manipulation/pipetting

Days postinsemination

Figure 1. Schematic representation of the different exposures during the process of clinical human in vitrofertilization (IVF). From gonadotropin exposure during controlled ovarian stimulation (depicted as an ultra-sound image) to culture and transfer (tx) at various stages of early development up to the blastocyst stage, thegametes and embryos are subjected to multiple chemical and physical exposures not encountered duringunassisted conception.

M.A. Mainigi et al.

2 Cite this article as Cold Spring Harb Perspect Med 2016;6:a023416

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



importantly, oxygen tension has emerged as acentral regulator of trophoblast differentiation(Genbacev et al. 1996, 1997; Maltepe and Simon1998; Red-Horse et al. 2004; Jiang and Mendel-son 2005; Robins et al. 2007).

Given the now well accepted associationof adult disease(s) to peri-conceptional andintrauterine environmental influences (Barkeret al. 1993, 2002, 2005; Gluckman et al. 2008;Woo and Patti 2008; Simmons 2009), it is crit-ical to methodically and comprehensibly eval-uate the cellular and molecular effects follow-ing the in vitro preimplantation growth ofhuman embryos. Elucidation of the specificcontributions of treatment to the perinatalmorbidity of children conceived with this tech-nology is of both scientific and public healthimportance. Moreover, a comparison of resultsobtained from placentas and newborns derivedafter in vitro versus in vivo conceptions can notonly provide information as to the pathogene-sis of perinatal adverse events associated withART, but can also suggest avenues for basicresearch that focus on understanding the regu-latory mechanisms involved in preimplantationhuman embryonic development and placenta-tion.

Here, we present background and summaryinformation on some of the evolving technolo-gies used to query epigenetic marks. We try toprovide an assessment of their suitability as di-agnostic or prognostic tools in a clinical setting.Although many of the technologies give deeplytextured information on the relationship be-tween individual epigenetic marks, or combina-tions of marks, and gene activity, not all arelikely to provide the robust level of reproduc-ibility and high-throughput amenable to rou-tine clinical laboratory testing. The demandsplaced on the technology are, in part, a byprod-uct of the overriding issue in many clinical tests:Given the great diversity of “normal” humanphenotypes, how does one distinguish an indi-vidual with an “abnormal” phenotype? Bothclinicians and researchers should be aware ofthe strengths, weaknesses, and pitfalls of suchtechnologies, as they are applied, in this case, tothe evaluation of epigenetic changes during theclinical application of ARTs.

EPIGENETIC REGULATION

Epigenetic regulation is thought to play a role inmediating the effects of many different expo-sures to long-term health and disease. However,linking an exposure and an outcome can be verydifficult. Reproduction is an excellent model tostudy the relationship between an exposure andan outcome, primarily because there is access totissue at birth (embryonic and extra-em-bryonic) and because there may be “early” conse-quences of exposure from as early as the periodof preimplantation development to later inpregnancy, such as is observed in preeclampsiaor intrauterine growth restriction (IUGR).Studies have shown that early exposure to highnutrient intake and rapid weight gain in infancyare associated with later metabolic risks inadolescence and adulthood (Fabricius-Bjerreet al. 2011). Exposures during pregnancy lead-ing to IUGR have been linked to long-term un-desirable outcomes in the offspring such as obe-sity, metabolic syndrome, and a predispositiontoward long-term morbidity from type 2 dia-betes and cardiovascular disease. Growing evi-dence suggests that the liver may represent oneof the candidate organs targeted by program-ming, undergoing structural, functional, andepigenetic changes following exposure to anunfavorable intrauterine environment (Cian-farani et al. 2012). Low-birth weight and babiesdelivered preterm may represent populationsat risk of long-term metabolic disorders andmay be valuable cohorts to study the role ofepigenetic programming.

Our own interest in exploiting emergingtechnologies to study epigenetic regulation toanswer questions in reproductive biology stemsdirectly from the possibility that epigeneticstructures can be altered by environmental fac-tors. Since the pioneering work of Barker etal. (2002) on the fetal origins of adult disease,we have understood that what occurs in uterocan have long-term consequences on healthand disease. Multiple studies have found anassociation of the peri-conceptional, peri-im-plantation, or in utero environments and DNAmethylation, and several are summarized in Ta-ble 1 (modified from data in Hogg et al. 2012).

Epigenetics and ART Outcomes

Cite this article as Cold Spring Harb Perspect Med 2016;6:a023416 3

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



This programming, at the cellular and molecu-lar levels, is still poorly understood, specificallyas it relates to the role of epigenetic alterations tothe development of adult disease (Fig. 2; mod-ified from data in Hogg et al. 2012).

Many different environmental effects, suchas exposure to ethanol or altered maternal diet,have been linked to adverse perinatal outcomes

that are believed to be regulated, in part, byepigenetic changes in the placenta. For example,ethanol exposure in an animal model led tochanges in DNA methylation in the placenta,with DNA methylation in the embryo unaffect-ed (Haycock 2009). Animal models have alsolinked maternal diabetes or a high-fat diet toepigenetic alterations in the placenta and off-

Table 1. Associations between DNA methylation and different human in utero exposures

Exposure type Exposure in utero Effect on DNA methylation Tissue Reference

Dietary/nutritional

Dutch famine Altered DNA methylation atseveral imprinted genes, somesex-specific effects

Peripheral blood Tobi et al. 2009

Folate andvitamin B12

Decreased global DNA methylation(LUMA) associated withmaternal B12 status

Cord blood McKay et al.2012

Endocrinedisruptingchemicals

Traffic airpollution

Lower global DNA methylation(methylamp)

Cord blood Herbstmanet al. 2012

Antiepileptic drugs(valproic acid)

Reduced global DNA methylation(Illumina HumanMethylation27array) and site-specific changes

Cord blood andplacenta

Smith et al.2012

Modified from data in Hogg et al. (2012).

Maternal/environmental exposureART, alcohol, tobacco, stress,

nutrition, endocrinedisrupters

Altered epigenetic profilesDNA methylation, histone modifications,

noncoding RNAs

Altered gene expression profiles

Adverse outcomes

Fetus Adult diseasePlacentaMother

- preeclampsia- gestational diabetes- recurrent miscarriage

- aberrant invasion- impaired vascularization- altered cell differentiation- poor growth

- growth restriction- metabolic changes- altered neurological- development- altered hormonal axes

- hypertension- cardiovascular disease- obesity- type II diabetes

Figure 2. Many of the problems of reproductive health can be linked to problems with placental morphology andfunction. (Created from modified data in Hogg et al. 2012.)

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



spring that can then lead to longer-term meta-bolic effects (Simmons 2009, 2013; Gatford andSimmons 2013). However, the majority of thesestudies focus on a small number of imprintedgenes (Table 1). It is likely that all of these ex-posures, as well as ART, have more broad epige-netic consequences and it is critical to developepigenetic technologies to broadly study theseconsequences.

Epigenomic Plasticity

Epigenetics is the study of differences in somati-cally heritable states of gene expression that arenot the result of differences in DNA sequence.Epigenetic alterations may also allow changesin gene expression to become transgenerational.It is largely through epigenetic means that or-ganisms achieve phenotypic plasticity, as wellas the capacity for a single genotype to resultin different cellular phenotypes that may alsobe tailored to the environment. The first studiesof epigenetic variation within populations sug-gest high levels of phenotypically relevant vari-ation, with the patterns of epigenetic regulationvarying between individuals and genome re-gions as well as with environment (Johnsonand Tricker 2010).

With respect to how one might measure thepotential effects of ART on the epigenome andthe effects of the epigenome on outcomes, thereare three classes of epigenetic molecules thatmight be able to make these distinctions: (1)DNA methylation, (2) modifications of his-tones and other chromosomal proteins, and(3) noncoding RNAs, including miRNAs andlong-noncoding RNAs. With special relevanceto environmental effects on the epigenome, allthree epigenetic regulatory mechanisms are notlikely to be equally capable of distinguishing theepigenetic differences between individuals thatmay be of clinical interest with regard to proce-dures used in assisted reproduction. The scopeof the problem lies in the observed level of in-terindividual variation, the expected effect-sizeof the laboratory exposure or clinical protocolor procedure and the precision and through-put with which the epigenetic measurementscan be made. These considerations make DNA

methylation the most likely candidate to be abiomarker of assisted reproduction exposures.DNA is a highly stable molecule. Levels of inter-individual variation in global or site-specificmethylation do vary but are constrained (i.e.,methylation at any one site can vary, as a fractionof molecules measured, between zero and one)and there are high-precision, highly reproduc-ible techniques available with the capacity forhigh-throughput (Eads et al. 2000; Weber et al.2005; Khulan et al. 2006; Irizarry et al. 2008; Zuoet al. 2009; Bibikova et al. 2011; Jelinek et al.2012). These techniques have the ability to dis-tinguish differences in population means of theexpected small magnitude in samples of mod-erate size. In other words, interindividual vari-ation is low enough and precision of the DNAmethylation measurement is high enough that itis likely that it can be used to distinguish theeffects of the intervention (i.e., ART proceduresor clinical protocols) on the epigenotype, even ifthose effects are expected to be small in magni-tude. With current technologies, the same can-not be said for histone modifications or even forgene expression arrays interrogating long non-coding or miRNAs.

DNA Methylation

Epigenetics play an important role in fetal de-velopment. Following fertilization, areas of thegenome that are not protected by imprintingundergo both active and passive demethyla-tion of DNA. DNA methylation patterns thenbegin to differentiate by developmental stageand by tissue, with �20% of CpG sites show-ing tissue-specific methylation patterns. Differ-ent cell types, individuals, and disease statesdevelop unique epigenomes. Because fetal de-velopment is a period of extensive cellular rep-lication and growth, environmentally inducedepigenetic changes may result in stable patternsof modified gene expression and phenotypicdifferences among exposed individuals (Menonet al. 2012). Such exposure-specific changescould represent epigenetic “signatures” of par-ticular exposures and be used for both forensicand diagnostic purposes.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



DNA methylation is a very important epi-genetic mark that has suspected regulatory rolesin a broad range of biological processes anddiseases. DNA methylation plays a crucial rolein embryonic development, maintenance ofpluripotency, X-chromosome inactivation andgenomic imprinting through regulation of tran-scription, chromatin structure, and chromo-somal stability (Robertson 2005).

In humans, DNA methylation occurs pre-dominantly at cytosine bases in the form of5-methylcytosines (mCs) at methyl cytosine-guanine dinucleotides (mCGs). hydroxyme-thylcytosines (hmCs), formylmethylcytosines(fmCs), carboxymethylctyosines (cmCs), andpossibly other, yet unknown, forms also occurat much lower frequencies. These bases com-prise the human DNA methylome and interin-dividual differences in the methylome mayidentify genomic regions involved in cell differ-entiation and disease. Experimental and bioin-formatic methods are now available for applyinggenome-wide DNA methylation mapping to abroad range of phenotypes, diseases, and bio-logical questions (Bock 2012). However, there isa growing need for establishing powerful andefficient tools for integrative data analysis to fil-ter biologically relevant findings from the grow-ing mass of epigenome data.

Gene-specific methods of measuring DNAmethylation include sodium bisulfite conver-sion of unmethylated cytosine to uracil, followedby methylation-specific (MS) polymerase chainreaction (PCR), pyrosequencing or mass spec-trometry-based analyses. Bisulfite conversion isneeded for many downstream applications todistinguish methylated DNA from nonmethy-lated DNA. Sodium bisulfite oxidatively deam-inates cytosine to uracil, however, 5-methylcy-tosine is resistant to this conversion. Althoughbisulfite treatment creates easily assayed, quali-tative differences in sequence from differencesin epigenotype, the method involves a harshchemical treatment resulting in high fragmen-tation and low-yield of DNA posttreatment. Ofnecessity, the technique also reduces sequencecomplexity (converting all unmethylated cyto-sines to uracil/thymine), which becomes a chal-lenge for primer design for individual genes, as

well as downstream mapping from “reads” backto the genome in sequencing-based whole-ge-nome approaches. Incomplete bisulfite conver-sion can also result in false positive methylationresults. There are several methylation-sensitiverestriction endonucleases that have the ability tocleave DNA at specific sequences, which is de-pendent upon the DNA methylation state of thesequence. Combined bisulfate restriction analy-sis (COBRA) combines bisulfite treatment withsequence-specific restriction endonucleases forlocus-specific analysis of DNA methylation.MS PCR allows for highly sensitive detectionof locus-specific DNA methylation using PCRamplification of bisulfite-converted DNA (Her-man et al. 1998). It is sensitive, quick, and inex-pensive. However, there are problems with spe-cificity (PCR design is difficult because thereduced complexity of converted DNA mini-mizes primer specificity); it is not quantitative,only one or two CpG sites are interrogated ata time and there is no bisulfite conversioncontrol. Pyrosequencing is site-specific, quanti-tative, and high-throughput, however, it onlycovers short reads (�100 bp) and the assay de-sign is difficult in CG dense areas. Mass spec-trometry-based methods allow for relativelyinexpensive long reads. However, site-specificeffects may be lost if CpG sites are close togetheror fall on small fragments.

Global restriction enzyme-based methodsof measuring DNA methylation focus on re-petitive elements such as long interspersedelement 1 (LINE1), an autonomous retrotrans-poson comprising �17% of the total genome,and Alu, a SINE family (short interspersed el-ement) which is a nonautonomous retrotrans-poson comprising �11% of the genome. Thereare quick and quantitative screens for these re-gions, collectively. However, the assay design forthese screens can be challenging (which region/CpGs to include, problems with variability),and sample quality and tissue of origin canaffect the success of the assay (Lander et al.2001). The restriction enzyme based lumino-metric methylation assay (LUMA) determinesmethylation at all CCGG sites (HpaII cleavesunmethylated CCGG; MspI cleaves both meth-ylated and unmethylated CCGG). Although

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



this method is advantageous because one doesnot need to know the exact target sequence, itdoes not give specific information on whichgenes are affected (Karimi et al. 2006).

DNA sequencing has become more af-fordable and capable of high-throughput ap-proaches. Whole-genome sequencing of bisul-fite-converted or MeDIP-precipitated DNA todetect genome-wide DNA methylation profilesis being more routinely used. With the rapiddevelopment of new methods for epigenomicanalysis, there is an acute need for a systemat-ic assessment of available technologies (Laird2010; Stolzenberg et al. 2011).

Genome-wide analysis of DNA methylationcan also be performed using the DREAM (dig-ital restriction enzyme analysis of methylation)assay. Genomic DNA is sequentially digestedusing SmaI and XmaI enzymes and the methyl-ation-specific signatures created at the endsof each cleavage product are analyzed usingnext generation sequencing. DREAM analyses�150K unique CpG sites, 39K of which are inCpG islands and 30K are at transcription startsites of 13K RefSeq genes. Compared with bi-sulfate pyrosequencing, DREAM is cost effec-tive, quantitative, and reproducible (Jelineket al. 2012).

HELP (HpaII tiny fragment enrichmentby ligation-mediated PCR) is another whole-genome methylation analysis technique. Thisassay is quantitative (compared methylation-sensitive HpaII sites with the methylation-in-sensitiveisoschizomer MspI sites) and support-ed by open source bioinformatic tools for anal-ysis. One version of the HELP assay generatedrepresentations of sizes 20022000 bp, allowingthe testing of roughly 800K loci throughoutthe human genome (Khulan et al. 2006; Odaet al. 2009). The MspI representation allows si-multaneous copy number variability testing(HELP-CNV) and as little as 10 ng of startingDNA can be used for the assay (nanoHELP).Another protocol uses HELP with massivelyparallel sequencing (MPS) rather than microar-rays (HELP-seq). This technique is similar tomethyl-seq and allows for more sensitive de-tection of hypomethylated loci. A large numberof polymorphisms in HpaII sites (.3%)

are also detected. These are a significant sourceof variability for microarray-based studies. AHELP-tagging assay (combining the power ofthe MspI normalization of HELP with the in-creased comprehensiveness of the methyl-sen-sitive cut counting, MSCC, assay) is based onIllumina sequencing and is highly quantitative(Suzuki et al. 2010).

Epigenome-wide analyses (arrays or parallelsequencing) of DNA methylation include plat-forms such as Illumina’s BeadChips for humanDNA (27K CpG island-heavy versus 450K withbetter whole-genome coverage), promoter ar-rays (NimbleGen, Agilent), which are biasedbut available for multiple species, and tiling ar-rays (Affymetrix, NimbleGen) for human DNA.These latter arrays are unbiased and have morethan 41 million probes. These protocols mayinclude antibody precipitation of methylatedDNAor MBP before array hybridization. Parallelsequencing or deep sequencing methods in-clude BS-Seq, MeDIP-Seq, and MethylPlex-Seq. Methylated DNA immunoprecipitation(MeDIP) uses an antibody that specifically rec-ognizes methylated DNA, and the immunopre-cipitated methylated DNA sequences can beidentified by PCR, Next-Gen sequencing or mi-croarray hybridization approaches. These meth-ods are global, with respect to genome coverage;however, a reference sequence is needed for theBS-Seq method, an antibody is required for theMeDIP method and in all cases and results mustbe validated (Pomraning et al. 2009).

For any epigenome-wide analyses, study de-sign considerations include sample require-ments (amount and quality; i.e., some restric-tion endonuclease digests require .2 mg DNA,Illumina Bead Arrays can still be effective withdegraded DNA, whereas affinity methods aremore tolerable of DNA impurity but requirelarger amounts), sample throughput (high-throughput 96 or 384 sample assays are low inlabor but high in reagent costs), genome cover-age and resolution (restriction endonucleasetechnologies are limited to the number/distri-bution of recognition sites and some technolo-gies work better for smaller genomes), accuracyand reproducibility (fragment lengths affect hy-bridization, sequencing, and reproducibility/



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



false positives and negatives, incomplete bi-sulfite conversion and validation), and finallybioinformatic and data storage requirements(Laird 2010). DNA methylation analyses, interms of cost, resolution, genome coverage,and accuracy were previously reviewed by Har-ris et al. (2010) and Bock (2012).

ASSISTED REPRODUCTIVETECHNOLOGIES (ART)

Major epigenetic events that occur during germcell development and preimplantation stagesof embryo development precisely coincidewith when clinical and laboratory proceduresrelated to ARTare being done. The combinationof epidemiological data indicating that childrenconceived through ART are at an increased riskfor a number of undesirable outcomes and thedemonstration that model organisms subjectedto similar manipulations exhibit epigenetic al-terations at specific loci (Mann et al. 2004; Ri-vera et al. 2008) suggest an association betweenthe two.

ART may affect proper establishment and/or maintenance of epigenetic marks. The mostprovocative observations have been the combi-nation of demonstrated effects of culture mediaon DNA methylation and imprinted gene ex-pression in model systems (Mann et al. 2004;Rivera et al. 2008) and the suggestion that twovery rare diseases resulting from alterations atimprinted genes (such as Beckwith–Wiede-mann and Angelman syndromes) (DeBaunet al. 2003; Maher et al. 2003; Odom and Segars2010; Lazaraviciute et al. 2014) have been ob-served at greater frequency among children con-ceived in vitro. These data are suggestive becausethe timing of epigenetic reprogramming and thetiming of clinical protocols used in assisted re-production overlap to a great degree.

Given that the timing of interventions usedin assisted reproduction coincide with the peri-od when genome-wide changes in DNA meth-ylation are thought to take place, it is only ashort logical extension to compare DNA meth-ylation levels between children conceived in vi-tro and children conceived in vivo to determinewhether differences observed in model or-

ganisms are also observed in human concep-tions. Such comparisons have been performedin multiple laboratories, initially targetingsmall numbers of imprinted genes (reviewedby Batcheller et al. 2011; systematic review andmeta-analysis by Lazaraviciute et al. 2014; Ne-lissen et al. 2014; Whitelaw et al. 2014; Melamedet al. 2015), under the assumption that imprint-ed genes might be more susceptible to effects ofepigenetic disruption (Table 2), and in a modestnumbers of patients.

However, it is also critical to expand ourknowledge beyond imprinted genes and CpGislands. DNA methylation on the edges of CpGislands, so-called “island shores,” may play a rolein affecting gene expression or affect chromatinstructure and secondarily gene expression (Iri-zarry et al. 2009). It is therefore increasingly nec-essary to examine global DNA methylation andnot focus solely on imprinted genes and theknown imprinting control regions. A recentstudy found that methylation of LINE1 was as-sociated with birth weight and methylation ofLINE1 and AluYb8 methylation also differedamong infants exposed to tobacco and alcohol(Wilhelm-Benartzi et al. 2012).

MOVING FROM BIOMARKERS TOPHYSIOLOGY AND FROM ASSOCIATIONSTO CAUSE/EFFECT: WHAT WILL IT TAKE?

In multiple studies, associations have been ob-served between DNA methylation levels at spe-cific sites and conception in vitro (Table 2);however, some inconsistencies have been ob-served. Many of the differences between studiescan be viewed as the most likely outcome ofreplication studies involving small numbers ofindividuals when the level of interindividualvariation is large and the expected differencebetween groups is small. As shown from thedata presented in Figure 3, there is significantinterindividual variation in both methyla-tion and transcript levels. Nevertheless, it isclear that many of the small differences inmean methylation level between groups arealso matched by differences in transcript level(Fig. 3), suggesting that the ART-associated dif-ferences in DNA methylation may play an

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



important role. Many such differences are dif-ficult to show because the level of interindivid-ual difference in transcript level is even largerthan the interindividual difference in methyla-tion levels (Katari et al. 2009). In total, the stud-

ies comparing DNA methylation between chil-dren conceived in vitro and children conceivedin vivo point to subtle differences at manygenes. In agreement with data derived from ex-periments in the mouse, extraembryonic tis-

Table 2. Studies comparing epigenetic differences between children conceived in vivo or using ART

Tissue Methodology Sample size Genes analyzed

Difference

observed Reference

Cord bloodandplacenta

GoldenGatearray and RT-PCR

10 ART, 13 in vivo 736 genes Yes Katari et al.2009

Placenta Bisulfite PCR 78 ART, 38 in vivo H19, GTL2, PEG1,KCNQ1OT1,ZAC, PEG3,SNRPN, XIST

Yes Kobayashiet al.2009

Cord blood,peripheralblood,amnion/chorion

Bisulfite PCR 77 ICSI, 35 IVF, 73 in vivo KvDMR1, H19,SNRPN, MEST,GRB10, MEG3,IG-DMR, GNAS,NESP55, GNAS,NESPas, GNASXL a-s, GNASExon1A

No Tierlinget al.2010

Cord bloodandplacenta

Methylationspecific-PCRand RT-PCR

45 ART, 56 in vivo IGF2/H19 andIGF2R DMR, X-inactivation

Yes Turan et al.2010

Placenta RT-PCR 65 ART, 924 in vivo IGF2, H19,KCNQ1OT1,CDKN1C

Yes Katagiriet al.2010

Placenta(CVS)

Bisulfite PCR 42 ART, 29 in vivo H19, MEG3, LIT1,MEST, NESP55,PEG3, SNRPN,NANOG, APC

Yes Zechneret al.2010

Cord Blood Bisulfite PCR,cloning andsequencing

29 IVF and 30 invivo conceived twin pairs

KvDMR1, PEG1,H19/IGF2 DMR

No Li et al.2011

Cord bloodandperipheralblood

Bisulfitesequencing

30 IVF, 30 ICSI and 60 invivo

PEG3, L3MBTL,PHILDA2

No Feng et al.2011

Placenta Pyrosequencing 35 IVF, 35 in vivo H19, MEST Yes Nelissenet al.2013

Chorionicvilli andmusclesamples

Pyrosequencing,bisulfitesequencingPCR

75 ART stillbirth, 73ART multifetalreduction, 90 naturalconception stillbirths,82 natural conceptioninduced abortion

H19, LIT1, SNRPN Yes Zheng et al.2013

Modified from data in Batcheller et al. (2011).



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



sues, in general, exhibit more and larger DNAmethylation differences than embryonic tissues(Mann et al. 2004; Rivera et al. 2008; de Waalet al. 2014).

CONCLUDING REMARKS

Infertile couples are increasingly turning toART to treat their infertility. Of growing con-cern is that ART-conceived pregnancies andchildren are at increased risk for specific loss-of-imprinting disorders, as well as congenitalmalformations, IUGR, and preeclampsia. Pub-lished reports from our own and other labora-tories show that following hormonal stimula-tion along with the in vitro manipulation of

human gametes and/or embryos, extra-embry-onic and fetal tissues exhibit particular suscept-ibility to both methylation and specific geneexpression differences.

Epigenetic changes occurring in response toART manipulations have been linked with ad-verse outcomes in the offspring. Although ini-tially ART was associated with diseases knownto be caused by epigenetic alterations, such asAngelman syndrome and Beckwith–Wiede-mann syndrome (Odom and Segars 2010), itis not these rare outcomes that are most con-cerning. The developmental origins of adultdisease hypothesis has gained strength as a pow-erful explanation for risk of some of the mostprevalent and morbid human conditions. It is

ADAM15 cg03379131

Bet

a va

lue

Bet

a va

lue

ControlsArt

ControlsArt ControlsArt

ControlsArt

0.7

0.65

0.55

0.45

0.35

0.3

0.6

0.5

0.4

0.7

0.65

0.55

0.45

0.35

0.25

0.2

0.3

0.6

0.5

0.4

2500

2000

1500

1000

500

0

2500

2000

1500

1000

500

0

PlacentaILMN_2384544

ADAM15

COL6A3 cg08950375Placenta

ILMN_1706643COL6A3

Ave

_sig

nal

Ave

_sig

nal

P = 0.009

P = 0.004

P = 0.0008

P < 10–4

Figure 3. Interindividual variation in gene-specific DNA methylation and transcription. Interindividual vari-ation in DNA methylation (left) at two CpG sites in genes that also show significant differences in transcript level(right) between ART and control placentas. Methylation was measured using Illumina’s Infinium 27K methyl-ation array and transcript level was measured using Illimina’s Human HT-12 Expression BeadChip in placentafrom 24 children conceived in vitro (ART) and 24 children conceived in vivo (controls).

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



likely that less severe epigenetic alterations areresponsible for other, more common, adverseoutcomes associated with IVF such as low birthweight, preeclampsia, and preterm labor. Thefull impact of impaired fetal growth may notbe completely evident until years after birthwhen later developmental and reproductivemilestones can be affected. Many of these out-comes are associated with long-term healthproblems such as hypertension, diabetes, andother adverse metabolic effects.

Studying the effects of ART on the epige-nome may help to predict long-term conse-quences of our early interventions. It is criticalto investigate our interventions to determinewhether they are the cause of these epigeneticdifferences. It is particularly important to inves-tigate the role of each individual interventionused in ART (i.e., superovulation, ICSI, extend-ed culture, etc.) on epigenetic outcome and toadapt current ART protocols to minimize riskto the pregnancy, the neonate and the long-termhealth of the offspring. Finally, it is also criticalto use this information to further our under-standing of epigenetic programming.

Advancement in the field of epigenetics mayhelp us understand many of the outcomes fol-lowing ART treatments and processes that de-crease IVF success rates, such as poor/abnormalembryo development, failed implantation, preg-nancy loss, and developmental anomalies. Weknow that chromosomally normal embryos donot implant 100% of the time and changes inDNA methylation may also play a role in lower“egg quality” associated with age (Yue et al.2012). Abnormal maintenance of methylationmay further play a role in early pregnancy loss(Yin et al. 2012; Zheng et al. 2013). Therefore,understanding epigenetic marks that predict ad-verse disease events may help us with developingbiomarkers that could aid in early diagnosis andtreatment of conditions that could lead to im-proved quality of life or even prevention of dis-ease. Furthermore, we know very little about theextended consequences of any ART-associatedepigenetic differences. Clinical ART has onlybeen in existence for less than four decades sothere may be yet undiscovered consequences inthe millions of children conceived by IVF.

Finally, epidemiologic data supporting a re-lationship between ARTs, low birth weight andrare conditions involving imprinted genes alsomakes it plausible that additional exposuresaround the time of conception may impact fetalgrowth. Growing evidence supports the conceptthat developmental programming may occurfrom a window that spans peri-conceptionthrough postnatal life. Nearly half of all preg-nancies conceived in the United States are un-planned (3.1 million). Such pregnancies are as-sociated with an increased risk of delayed or lackof prenatal care, fetal exposure to harmfulchemicals such as endocrine disrupters, smok-ing and alcohol, and low birth weight. Theseassociations highlight the fact that maternal ex-posures both preconception and in utero arecritical for programming fetal developmentand when these exposures are unfavorable peri-natal morbidity increases.

It should be noted that these issues go be-yond the effects of clinical and laboratory pro-tocols involved in assisted reproductive technol-ogies. Environmental and nutritional exposuresdiffer among populations of women accordingto socioeconomic status, race, and age. Epige-netic differences may not only be secondary toexposure (ART or other in utero exposure) butmay also be dependent on the maternal geno-type. A recent study found that polymorphismsin genes involved in folate absorption and me-tabolism may affect DNA methylation patterns(McKay et al. 2012). Abnormal methylation ofpaternal DNA is also important to study be-cause DNA methylation may be altered in thesperm of men with infertility. One study hasshown that in some infertile men perturbationsin methylation may contribute to abnormalembryo development (Aston et al. 2012). Thepossibility that some aspect(s) of infertility perse contributes to altered phenotype and/or epi-genotype is open for consideration and furtherinvestigation (Song et al. 2015).

Ultimately, we need additional epidem-iological research incorporating epigeneticmarkers of environmental exposure, taking alife-course approach with regards to assessingoutcomes. Compelling results from such studiescould help to broaden the scope of maternal care



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



to one that places more emphasis on preconcep-tion surveillance and treatment. Modificationsto maternal health, both before and at the ear-liest stages of gestation, could impact upon fetaldevelopment and reduce adult disease disk. Toaid us in achieving this, techniques for measur-ing DNA methylation are very useful in quanti-fying epigenetic effects of environment and nu-trition, correlating developmental epigeneticvariation with phenotypes, understanding epi-genetics of cancer and chronic diseases, measur-ing the effects of drugs on DNA methylation orderiving new biological insights into mamma-lian genomes (Jelinek et al. 2012). To conclude,it is necessary to study not only different tissues,but different time points during developmentand adulthood. Although there are conflictinghypotheses, epigenetic marks may be static andtissue-specific differences in DNA methylationmay change during different times of develop-ment (Novakovic et al. 2011; Yuen et al. 2011).

ACKNOWLEDGMENTS

The authors thank Dr. Nahid Turan for her helpin the preparation of this manuscript. This workis supported by the National Institutes ofHealth P50 HD068157 (C.C., C.S., S.B.), R01HD048730 (C.S., C.C.), and the K12 Reproduc-tive Scientists Development Program (RSDP) toM.M.

REFERENCES

Aston KI, Punj V, Liu L, Carrell DT. 2012. Genome-widesperm deoxyribonucleic acid methylation is altered insome men with abnormal chromatin packaging or poorin vitro fertilization embryogenesis. Fertil Steril 97(2):285–292.

Barker DJ, Hales CN, Fall CH, Osmond C, Phipps K, ClarkPM. 1993. Type 2 (non-insulin-dependent) diabetes mel-litus, hypertension and hyperlipidaemia (syndrome X):Relation to reduced fetal growth. Diabetologia 36: 62–67.

Barker DJ, Eriksson JG, Forsen T, Osmond C. 2002. Fetalorigins of adult disease: Strength of effects and biologicalbasis. Int J Epidemiol 31: 1235–1239.

Barker DJ, Osmond C, Forsen JJ, Kajantie E, Eriksson JG.2005. Trajectories of growth among children who havecoronary events as adults. N Engl J Med 353: 1802–1809.

Batcheller A, Cardozo E, Maguire M, DeCherney AH, SegarsJH. 2011. Are there subtle genome-wide epigenetic alter-

ations in normal offspring conceived by assisted repro-ductive technologies? Fertil Steril 96: 1306–1311.

Bibikova M, Barnes B, Tsan C, Ho V, Klotzle B, Le JM,Delano D, Zhang L, Schroth GP, Gunderson KL, et al.2011. High density DNA methylation array with singleCpG site resolution. Genomics 98: 288–295.

Bock C. 2012. Analysing and interpreting DNA methylationdata. Nat Rev Genet 13: 705–719.

Chung K, Coutifaris C, Chalian R, Lin K, Ratcliffe SJ, Cas-telbaum AJ, Freedman MF, Barnhart KT. 2006. Factorsinfluencing adverse perinatal outcomes in pregnanciesachieved through use of assisted reproductive technology.Fertil Steril 86: 1634–1641.

Cianfarani S, Agostoni C, Bedogni G, Berni Canani R,Brambilla P, Nobili V, Pietrobelli A. 2012. Effect of intra-uterine growth retardation on liver and long-term meta-bolic risk. Int J Obes (Lond) 36: 1270–1277.

DeBaun MR, Niemitz EL, Feinberg AP. 2003. Association ofin vitro fertilization with Beckwith-Wiedemann syn-drome and epigenetic alterations of LITI and H19. AmJ Hum Genet 72: 156–160.

de Waal E, Mak W, Calhoun S, Stein P, Ord T, Krapp C,Coutifaris C, Schultz RM, Bartolomei MS. 2014. In vitroculture increases the frequency of stochastic epigeneticerrors at imprinted genes in placental tissues from mouseconcepti produced through assisted reproductive tech-nologies. Biol Reprod 90: 22–28.

Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C,Shibata D, Dandenberg PV, Laird PW. 2000. MethyLight:A high-throughput assay to measure DNA methylation.Nucleic Acids Res 28: E32.

Ericson A, Kallen B. 2001. Congenital malformations ininfants born after IVF: A population-based study. HumReprod 16: 504–509.

ESHRE (European Society of Human Reproduction andEmbryology). 2006. World report on assisted reproduc-tion technology. www.eshre.eu

Fabricius-Bjerre S, Jensen RB, Færch K, Larsen T, MølgaardC, Michaelsen KF, Vaag A, Greisen G. 2011. Impact ofbirth weight and early infant weight gain on insulin re-sistance and associated cardiovascular risk factors in ad-olescence. PLoS ONE 6: e20595.

Feng C, Tian S, Zhang Y, He J, Zhu XM, Zhang D, Sheng JZ,Huang HF. 2011. General imprinting status is stable inassisted reproduction conceived offspring. Fertil Steril 96:1417–1423.

Fowden AL, Sibley C, Reik W, Constancia M. 2006. Imprint-ed genes, placental development and fetal growth. HormRes 65: 50–58.

Gatford KL, Simmons RA. 2013. Prenatal programming ofinsulin secretion in intrauterine growth restriction. ClinObstet Gynecol 56: 520–528.

Genbacev O, Joslin R, Damsky CH, Pllliotti BM, Fisher SJ.1996. Hypoxia alters early early gestational human cyto-trophoblast differentiation/invasion in vitro and modelsthe placental defects that occur in preeclampsia. J ClinInvest 97: 540–550.

Genbacev O, Zhou U, Ludlow JW, Fisher SJ. 1997. Regula-tion of human placental development by oxygen tension.Science 277: 1669–1672.

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Gluckman PD, Hanson MA, Cooper C, Thornburg KL.2008. Effect in in utero and early-life conditions on adulthealth and disease. N Engl J Med 359: 61–73.

Hansen M, Kurinczuk JJ, Bower C, Webb S. 2002. The risk ofmajor birth defects after intracytoplasmic sperm injec-tion and in-vitro fertilization. N Engl J Med 346: 725–730.

Harris RA, Wang T, Coarfa C, Nagarajan RP, Hong C, Dow-ney SL, Johnson BE, Fouse SD, Delaney A, Zhao Y. 2010.Comparison of sequencing-based methods to profileDNA methylation and identification of monoallelic epi-genetic odifications. Nat Biotechnol 28: 1097–1105.

Haycock PC, Ramsay M. 2009. Exposure of mouse embryosto ethanol during preimplantation development: Effecton DNA methylation in the H19 imprinting control re-gion. Biol Reprod 81(4): 618–627.

Henningsen AK, Pinborg A. 2014. Birth and perinatal out-comes and complications for babies conceived followingART. Semin Fetal Neonatal Med 19: 234–238.

Herbstman JB, Tang D, Zhu D, Qu L, Sjodin A, Li Z, Ca-mann D, Perera FP. 2012. Prenatal exposure to polycyclicaromatic hydrocarbons, benzo[a]pyrene-DNA adducts,and genomic DNA methylation in cord blood. EnvironHealth Perspect 120: 733–738.

Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JPJ,Markowitz S, Willson JKV, Hamilton SR, Kinzler KW.1998. Incidence and functional consequences ofhMLH1 promoter hypermethylation in colorectal carci-noma. Proc Natl Acad Sci 95: 6870–6875.

Hogg K, Price EM, Hanna CW, Robinson WP. 2012. Prenataland perinatal environmental influences on the humanfetal and placental epigenome. Clin Pharmacol Ther 92:716–726.

Irizarry RA, Ladd-Acosta C, Carvalho B, Wu H, Branden-burg SA, Jeddeloh JA, Wen B, Feinberg AP. 2008. Com-prehensive high-throughput arrays for relative methyla-tion (CHARM). Genome Res 18: 780–790.

Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C,Onyango P, Cui H, Gabo K, Rongione M, Webster M.2009. Genome-wide methylation analysis of human co-lon cancer reveals similar hypo- and hypermethylation atconserved tissue-specific CpG island shores. Nat Genet41: 178–186.

Jackson R, Gibson KA, Wu YW, Croughan MS. 2004. Peri-natal outcomes in singletons following in vitro fertiliza-tion: A meta-analysis. Obstet Gynecol 130: 551–563.

Jelinek J, Liang S, Lu Y, He R, Ramagli LS, Shpall EJ, EstecioMR, Issa JP. 2012. Conserved DNA methylation patternsin healthy blood cells and extensive changes in leukemiameasured by a new quantitative technique. Epigenetics 7:1368–1378.

Jiang B, Mendelson CR. 2005. O2 enhancement of humantrophoblast differentiation and hCYP19 (Aromatase)gene expression are mediated by proteasomal degrada-tion of USF1 and USF2. Molec Cell Biol 25: 8824–8833.

Johnson LJ, Tricker PJ. 2010. Epigenomic plasticity withinpopulations: Its evolutionary significance and potential.Heredity 105: 113–121.

Kalra SK, Molinaro T. 2008. The association of in vitro fer-tilization and perinatal morbidity. Semin Reprod Med 26:423–435.

Kalra SK, Ratcliffe SJ, Coutifaris C, Molinaro T, Barnhart KT.2011. Ovarian stimulation and low birth weight in new-borns conceived through in vitro fertilization. Obstet Gy-necol 118: 863–871.

Kalra SK, Ratcliffe SJ, Barnhart KT, Coutifaris C. 2012. Ex-tended embryo culture and an increased risk of pretermdelivery. Obstet Gynecol 120: 69–75.

Karimi M, Johansson S, Ekstrom TJ. 2006. Using LUMA: Aluminometric-based assay for global DNA-methylation.Epigenetics 1: 45–48.

Katagiri Y, Aoki C, Tamaki-Ishihara Y, Fukuda Y, KitamuraM, Matsue Y, So A, Morita M. 2010. Effects of assistedreproduction technology on placental imprinted geneexpression. Obstet Gynecol Int 2010: 437528.

Katari S, Turan N, Bibikova M, Erinle O, Chalian R, FosterM, Gaughan JP, Coutifaris C, Sapienza C. 2009. DNAmethylation and gene expression differences in childrenconceived in vitro or in vivo. Hum Mol Genet 18: 3769–3778.

Khulan B, Thompson RF, Ye K, Fazzari MJ, Suzuki M, Sta-siek E, Figueroa ME, Glass JL, Chen Q, Montagna C.2006. Comparative isoschizomer profiling of cytosinemethylation: The HELP assay. Genome Res 16: 1046–1055.

Kobayashi H, Hiura H, John RM, Sato A, Otsu E, KobayashiN, Suzuki R, Suzuki F, Hayashi C, Utsunomiya T. 2009.DNA methylation errors at imprinted loci after assistedconception originate in the parental sperm. Eur J HumGenet 17: 1582–1591.

Laird PW. 2010. Principles and challenges of genome-wideDNA methylation analysis. Nat Rev Genet 11: 191–203.

Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC,Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W.2001. Initial sequencing and analysis of the human ge-nome. Nature 409: 860–921. Erratum: Nature 412: 565and Nature 411: 720.

Lazaraviciute G, Kauser M, Bhattacharya S, Haggarty P,Bhattacharya S. 2014. A systematic review and meta-anal-ysis of DNA methylation levels and imprinting disordersin children conceived by IVF/ICSI compared with chil-dren conceived spontaneously. Hum Reprod Update 20:840–852.

Li L, Wang L, Le F, Liu X, Yu P, Sheng J, Huang H, Jin F. 2011.Evaluation of DNA methylation status at differentiallymethylated regions in IVF- conceived newborn twins.Fertil Steril 95: 1975–1979.

Maher ER, Brueton LA, Bowdin SC. 2003. Beckwith-Wie-demann syndrome and assisted reproduction technology(ART). J Med Genetics 40: 62–64.

Maltepe E, Simon MC. 1998. Oxygen, genes, and develop-ment: An analysis of the role of hypoxic gene regulationduring murine vascular development. J Molec Med 76:391–401.

Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD,Schultz RM, Bartolomei MS. 2004. Selective loss of im-printing in the placenta following preimplantation devel-opment in culture. Development 131: 3727–3735.

Market-Velker BA, Zhang L, Magri LS, Bonvissuto AC,Mann MRW. 2010. Dual effects of superovulation: Lossof maternal and paternal imprinted methylation in adose-dependent manner. Hum Molec Genet 19: 36–51.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



McKay JA, Groom A, Potter C, Coneyworth LJ, Ford D,Mathers JC, Relton CL. 2012. Genetic and non-geneticinfluences during pregnancy on infant global and sitespecific DNA methylation: Role for folate gene variantsand vitamin B12. PLoS ONE 7: e33290.

Melamed N, Choufani S, Wilkins-Haug LE, Koren G, Weks-berg R. 2015. Comparison of genome-wide and gene-specific DNA methylation between ART and naturallyconceived pregnancies. Epigenetics doi: 10.4161/15592294.2014.988041.

Menon R, Conneely KN, Smith AK. 2012. DNA methyla-tion: An epigenetic risk factor in preterm birth. ReprodSci 19: 6–13.

Mercer TR, Dinger ME, Mattick JS. 2009. Long non-codingRNAs: Insights into functions. Nat Rev Genet 10: 155–159.

Nelissen EC, Dumoulin JC, Daunay A, Evers JL, Tost J, VanMontfoort AP. 2013. Placentas from pregnancies con-ceived by IVF/ICSI have a reduced DNA methylationlevel at the H19 and MEST differentially methylated re-gions. Hum Reprod 28: 1117–1126.

Nelissen EC, Dumoulin JC, Busato F, Ponger L, Eijssen LM,Evers JL, Tost J, van Montfoort AP. 2014. Altered geneexpression in human placentas after IVF/ICSI. Hum Re-prod 29: 2821–2831.

Norwitz ER, Schust DJ, Fisher SJ. 2001. Implantation andthe survival of early pregnancy. N Engl J Med 345: 1400–1408.

Novakovic B, Yuen RK, Gordon L, Penaherrera MS, SharkeyA, Moffett A, Craig JM, Robinson WP, Saffery R. 2011.Evidence for widespread changes in promoter methyla-tion profile in human placenta in response to increasinggestational age and environmental/stochastic factors.BMC Genomics 12: 529.

Oda M, Glass JL, Thompson RF, Mo Y, Olivier EN, FigueroaME, Selzer RR, Richmond TA, Zhang X, Dannenberg L.2009. High-resolution genome-wide cytosine methyla-tion profiling with simultaneous copy number analysisand optimization for limited cell numbers. Nucleic AcidsRes 37: 3829–3839.

Odom LN, Segars J. 2010. Imprinting disorders and assistedreproductive technology. Curr Opin Endocrinol DiabetesObes 17: 517–522.

Pellegrini M, Ferrari R. 2012. Epigenetic analysis: ChIP-chipand ChIP-seq. Methods Mol Biol 802: 377–387.

Petronis A. 2010. Epigenetics as a unifying principle in theaetiology of complex traits and diseases. Nature 465:721–727.

Pomraning KR, Smith KM, Freitag M. 2009. Genome-widehigh throughput analysis of DNA methylation in eukary-otes. Methods 47: 142–150.

Red-Horse K, Zhou Y, Genbacev O, Prakobphol A, Foulk R,McMaster M, Fisher SJ. 2004. Trophoblast differentiationduring embryo implantation and formation of the ma-ternal-fetal interface. J Clin Invest 114: 744–754

Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Barto-lomei MS. 2008. Manipulations of mouse embryos priorto implantation result in aberrant expression of imprint-ed genes on day 9.5 of development. Hum Mol Genet 17:1–14.

Robertson KD. 2005. DNA methylation and human disease.Nat Rev Genet 6: 597–610.

Robins JC, Heizer A, Hardiman A, Hubert M, HandwergerS. 2007. Oxygen tension directs the differentiation path-way of human cytotrophoblast cells. Placenta 28: 1141–1146.

Ross JP, Shaw JM, Molloy PL. 2013. Identification of differ-entially methylated regions using streptavidin bisulfiteligand methylation enrichment (SuBLiME), a new meth-od to enrich for methylated DNA prior to deep bisulfitegenomic sequencing. Epigenetics 8: 113–127.

Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. 2007.Aberrant DNA methylation of imprinted loci in super-ovulated oocytes. Hum Reprod 22: 26–35.

Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng GT, Wil-cox LS. 2002. Low and very low birth weight in infantsconceived with use of assisted reproductive technology. NEngl J Med 346: 725–730.

Schieve LA, Ferre C, Peterson HB, Macaluso M, ReynoldsMA, Wright VC. 2004. Perinatal outcome among single-ton infants conceived through assisted reproductive tech-nology in the United States. Obstet Gynecol 103: 1144–1153.

Shevell T, Malone FC, Vicaver J, Porter JF, Luthy DA, Corn-stock CH, Hankins GD, Eddleman K, Dolan S, Dugoff L,et al. 2005. Assisted reproductive technology and preg-nancy outcome. Obstet Gynecol 106: 1039–1045.

Simmons RA. 2009. Developmental origins of adult disease.Pediatr Clin North Am 56: 449–454.

Simmons RA. 2013. Programming of DNA methylation intype 2 diabetes. Diabetologia 56: 947–948.

Smith AK, Conneely KN, Newport DJ, Kilaru V, SchroederJW, Pennell PB, Knight BT, Cubells JC, Stowe ZN, Bren-nan PA. 2012. Prenatal antiepileptic exposure associateswith neonatal DNA methylation differences. Epigenetics7: 458–463.

Song S, Ghosh J, Mainigi M, Turan N, Weinerman R,Truongcao M, Coutifaris C, Sapienza C. 2015. DNAmethylation differences between in vitro- and in vivoconceived children are associated with ART proceduresrather than infertility. Clin Epigenetics doi: 10.1186/s13148-015-0071-7.

Stolzenberg DS, Grant PA, Bekiranov S. 2011. Epigeneticmethodologies for behavioral scientists. Horm Behav59: 407–416.

Sun LM, Walker M, Cao HL, Yang Q, Duan T, Kingdom J.2009. Assisted reproductive technology and placenta-me-diated adverse pregnancy outcomes. Obstet Gynecol 114:818–824.

Suzuki M, Jing Q, Lia D, Pascual M, McLellan A, Greally JM.2010. Optimized design and data analysis of tag-basedcytosine methylation assays. Genome Biol 11: R36.

Tierling S, Souren NY, Gries J, Loporto C, Groth M, Lutsik P,Neitzel H, Utz-Billing I, Gillessen-Kaesbach G, KentenichH. 2010. Assisted reproductive technologies do not en-hance the variability of DNA methylation imprints inhuman. J Med Genet 47: 371–376.

Tobi EW, Lumey LH, Talens RP, Kremer D, Putter H, SteinAD, Slagboom PE, Heijmans BT. 2009. DNA methylationdifferences after exposure to prenatal famine are common

M.A. Mainigi et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



and timing- and sex-specific. Hum Mol Genet 18: 4046–4053.

Turan N, Katari S, Gerson LF, Chalian R, Foster MW,Gaughan JP, Coutifaris C, Sapienza C. 2010. Inter- andintra-individual variation in allele-specific DNA methyl-ation and gene expression in children conceived usingassisted reproductive technology. PLoS Genet 6:e1001033.

Van Voorhis BJ. 2006. Outcomes from assisted reproductivetechnology. Obstet Gynecol 107: 183.

Van Voorhis BJ. 2007. In vitro fertilization. N Engl J Med356: 379–386.

Wang YA, Sullivan EA, Black D, Dean J, Bryant J, ChapmanM. 2000. Preterm birth and low birth weight after assistedreproductive technology-related pregnancy in Australiabetween 1996 and 2000. Fertil Steril 83: 1650–1658.

Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, LamWL, Schubeler D. 2005. Chromosome-wide and promot-er-specific analyses identify sites of differential DNAmethylation in normal and transformed human cells.Nat Genet 37: 853–862.

Whitelaw N, Bhattacharya S, Hoad G, Horgan GW, Hamil-ton M, Haggarty P. 2014. Epigenetic status in the off-spring of spontaneous and assisted conception. Hum Re-prod 29: 1452–1458.

Wilhelm-Benartzi CS, Andres Houseman E, Maccani MA,Poage GM, Koestler DC, Langevin SM, Gagne LA, Ban-ister CE, Padbury JF, Marsit CJ. 2012. In utero exposures,infant growth, DNA methylation of repetitive elements

and developmentally related genes in human placenta.Environ Health Perspect 120: 296–302.

Woo M, Patti ME. 2008. Diabetes risks begins in utero. CellMetab 8: 5.

Yin LJ, Zhang Y, Lv P, He W, Wu Y, Liu A, Ding G, Dong M,Qu F, Xu C, et al. 2012. Insufficient maintenance DNAmethylation is associated with abnormal embryonic de-velopment. BMC Med 10: 26.

Yue MX, Fu XW, Zhou GB, Hou YP, DU M, Wang L, Zhu SE.2012. Abnormal DNA methylation in oocytes could beassociated with a decrease in reproductive potential inold mice. J Assist Reprod Genet 29: 643–650.

Yuen RK, Neumann SM, Fok AK, Penaherrera MS, McFad-den DE, Robinson WP, Kobor MS. 2011. Extensive epi-genetic reprogramming in human somatic tissues be-tween fetus and adult. Epigenetics Chromatin 4: 7.

Zechner U, Pliushch G, Schneider E, El Hajj N, Tresch A,Shufaro Y, Seidmann L, Coerdt W, Muller AM, Haaf T.2010. Quantitative methylation analysis of developmen-tally important genes in human pregnancy losses afterART and spontaneous conception. Mol Hum Reprod 16:704–713.

Zheng HY, Tang Y, Niu J, Li P, Ye DS, Chen X, Shi XY, Li L,Chen SL. 2013. Aberrant DNA methylation of imprintedloci in human spontaneous abortions after assisted re-production techniques and natural conception. Hum Re-prod 28: 265–273.

Zuo T, Tycko B, Liu T, Lin H, Huang T. 2009. Methods inDNA methylation profiling. Epigenomics 1: 331–345.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



January 8, 20162016; doi: 10.1101/cshperspect.a023416 originally published onlineCold Spring Harb Perspect Med

Monica A. Mainigi, Carmen Sapienza, Samantha Butts and Christos Coutifaris Reproductive TechnologiesA Molecular Perspective on Procedures and Outcomes with Assisted

Subject Collection Molecular Approaches to Reproductive and Newborn Medicine

Information in SpermIntergenerational Transfer of Epigenetic

Oliver J. Rando TechnologiesOutcomes with Assisted Reproductive A Molecular Perspective on Procedures and

Butts, et al.Monica A. Mainigi, Carmen Sapienza, Samantha

Programming: Molecular ApproachesEffects of Maternal Obesity on Fetal

Caterina Neri and Andrea G. Edlow Have Single-Gene DisorderstoSequencing in Critically Ill Neonates Suspected

Whole-Exome Sequencing and Whole-Genome

KingsmoreLaurie D. Smith, Laurel K. Willig and Stephen F.

The Neonatal Salivary TranscriptomeJill L. Maron Blood Group Using Next-Generation Sequencing

Noninvasive Antenatal Determination of Fetal

Morten Hanefeld DziegielKlaus Rieneck, Frederik Banch Clausen and

Tract Development, Adult Function, and Fertility Genes in Female ReproductiveHoxThe Role of

Hongling Du and Hugh S. Taylor Newborn ScreeningGenome-Scale Sequencing to Augment Current Potential Uses and Inherent Challenges of Using

Jonathan S. Berg and Cynthia M. Powell

InterfaceMaternal−Molecular Cross-Talk at the Feto

Gendie E. Lashthe Decidual ClockMolecular Regulation of Parturition: The Role of

Snegovskikh, et al.Errol R. Norwitz, Elizabeth A. Bonney, Victoria V.

PerspectiveMolecular Regulation of Parturition: A Myometrial

Montalbano, et al.Nora E. Renthal, Koriand'r C. Williams, Alina P.

Molecular Mechanisms of Preeclampsia

KarumanchiTammy Hod, Ana Sofia Cerdeira and S. Ananth

of Fetal Single-Gene DisordersGenome-Wide Sequencing for Prenatal Detection

Ignatia B. Van den Veyver and Christine M. Eng Maternal Plasma DNADiseases Using Massively Parallel Sequencing of Noninvasive Prenatal Screening for Genetic

Lyn S. Chitty and Y. M. Dennis LoMicroRNA in Ovarian Biology and Disease

ChristensonLynda K. McGinnis, Lacey J. Luense and Lane K. The Oocyte's Perspective on the Incoming Sperm

Confrontation, Consolidation, and Recognition:

David Miller

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved


http://perspectivesinmedicine.cshlp.org/cgi/collection/

http://perspectivesinmedicine.cshlp.org/cgi/collection/


a molecular perspective on procedures and...

Documents