Preimplantation genetic diagnosis at 20 years

PRENATAL DIAGNOSISPrenat Diagn 2010; 30: 682–695.Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/pd.2552

30th Anniversary Issue of Prenatal Diagnosis

REVIEW OF CURRENT PRACTICE

Preimplantation genetic diagnosis at 20 years

Joe Leigh Simpson*Wertheim College of Medicine, Florida International University, Miami, FL, USA

First reported in 1990, PGD has evolved into a complementary form of prenatal diagnosis offering novelindications. DNA for PGD can be recovered with equal safety and facility from polar bodies I and II, blastomere(8 cell embryo) and trophectoderm (5–6 day blastocyst). Diagnostic accuracy is very high (>99%) for bothchromosomal abnormalities and single gene disorders. Traditional application of FISH with chromosomespecific probes for detecting aneuploidy and translocations may be replaced or complemented by arraycomparative genome hybridization (array CGH); biopsied embryos can now be cryopreserved (vitrification)while analysis proceeds in orderly fashion. PGD has been accomplished for over 200 different single genedisorders. Novel indications for PGD not readily applicable by traditional prenatal genetic diagnosis includeavoiding clinical pregnancy termination, performing preconceptional diagnosis (polar body I), obtainingprenatal diagnosis without disclosure of prenatal genotype (nondisclosure), diagnosing adult-onset disordersparticularly cancer, and identifying HLA compatible embryos suitable for recovering umbilical cord bloodstem cells. Copyright 2010 John Wiley & Sons, Ltd.

KEY WORDS: PGD; PGS; embryo biopsy; aneuploidy; single gene; birth defects

INTRODUCTION

Twenty years ago preimplantation genetic diagnosis(PGD) was first accomplished in humans. Since thenperhaps 50 000 cycles have been performed world-wide (www.pgdis.org). The first 1000 PGD births werereached by 2004 (Verlinsky et al., 2004a), and per-haps close to 10 000 additional PGD babies have sincebeen born. PGD has evolved into not simply an earlierextension of traditional prenatal genetic diagnosis, but amodality offering novel indications. In this update, weshall consider evolving indications, current approachesto obtaining cell(s) for PGD, diagnostic accuracy andmethods to maximize safety.

HISTORY

Although often considered a recent entry in prenatalgenetic diagnosis, PGD has actually long been envi-sioned; see Harper (2009) for detailed history. Gardnerand Edwards (1968) biopsied a rabbit blastocyst andperformed X-chromatin analysis, suggesting applicationfor X-linked recessive traits. Over the next decade,a number of mouse geneticists (Andrzej Tarkowski,Andrei Pavlovich Dyban, Alfred Gropp and others)demonstrated the ability to manipulate cells and obtainmetaphase chromosomes from mouse blastomeres

*Correspondence to: Joe Leigh Simpson, Wertheim College ofMedicine, Florida International University, 11200 SW 8th Street,HLS 693 Miami, FL 33199, USA. E-mail: [email protected]

(Dyban, 1991). However, the progress in human PGDwas delayed until in vitro fertilization (IVF), the main-stay of the assisted reproductive technologies (ART),was achieved in 1978 (Steptoe and Edwards, 1978).During the next decade, animal studies paved the wayfor human PGD. Marilyn Monk and various colleaguesbiopsied mouse blastomeres and showed feasibility ofdetecting a single gene disorder (Monk and Handy-side, 1988). Pioneering work was done in Melbourneby Leanda Wilton and Alan Trounson and in Brusselsby Andre Van Steirteghem and colleagues.

The dawning of the molecular diagnostic era in1986 with polymerase chain reaction (PCR) openedthe field for PGD. In Europe, emphasis in the late1980s focused on blastomere biopsy, whereas in theUnited States mostly on polar body biopsy. In theUnited Kingdom, Alan Handyside, Peter Braude andRobert Winston pursued blastomere biopsy and analysis,succeeding in 1990 with determining sex in a pregnancyat risk for ornithine transcarbamylase deficiency (OTC)(Handyside et al., 1990). This was soon followed bydetection of cystic fibrosis, using nested primer PCR(Handyside et al., 1992). In the United States, YuryVerlinsky and colleagues pursued polar body biopsy.Clinical application was first reported in 1987 at aninternational IVF Congress (Verlinsky et al., 1987), butthe first peer publication of a PGD (α1-antitrypsiondeficiency) was not until 1990 (Verlinsky et al., 1990).(The delay was due to Nature initially rejecting thesubmission.) In 1990, PGD for cystic fibrosis wasreported by this group (Strom et al., 1990) using polarbody biopsy.

Copyright 2010 John Wiley & Sons, Ltd. Received: 28 December 2009Revised: 25 March 2010Accepted: 13 April 2010

PREIMPLANTATION GENETIC DIAGNOSIS 683

Progress in detecting chromosomal abnormalitiesawaited development of fluorescence in situ hybridiza-tion (FISH). In the United Kingdom, Darren Griffinsuccessfully performed FISH on blastomeres (Griffinet al., 1991). In the United States, Jamie Grifo did so(Grifo et al., 1990, 1992a). Working with Jacques Cohenand a highly productive team at Cornell Medical Col-lege (New York), Grifo reported a pregnancy followingembryo biopsy subjected to X and Y FISH (Grifo et al.,1992b). Santiago Munne joined the group and soonshowed practicality of multicolor FISH in blastomeresusing chromosome-specific probes (Munne et al., 1993).This set the stage for modern PGD aneuploidy testing.Munne also first applied FISH for PGD of chromosomaltranslocations (Munne et al., 1998a, 2000). In the UnitedKingdom, Joy Delhanty and Joyce Harper performedrapid FISH (Harper et al., 1994) and became concernedabout mosaicism; Munne has by contrast long believedthis problem was manageable with optimal diagnostics.Verlinsky et al. (1995) independently applied FISH topolar bodies. Verlinsky and Anver Kuliev have longfavored polar body biopsy reasoning greater predictivevalue with respect to embryonic chromosomal comple-ment. This approach has belatedly become vindicated(Geraedts et al., 2010).

The field of PGD has now become more formallyorganized. Pivotal to progress in early years wereopen international meetings arranged by Yury Verlin-sky (Edwards et al., 2009). One, especially, importantearly meeting in the 1990s was solely dedicated topreimplantation genetics and PGD, yielding a well-citedvolume (Verlinsky and Kuliev, 1991). Yearly Interna-tional Working Group on PGD meetings followed, heldaround the world often in conjunction with other inter-national meetings. These meetings kept the field pro-gressing until technology caught up with vision. By themid-1990s, PGD had evolved from boutique medicineto mainstream prenatal diagnosis with its own distinctindications (Simpson et al., 1994), setting the stage forprogress that rapidly followed (Simpson, 2001; Kulievet al., 2004b, Harper, 2009; Verlinsky and Kuliev,2010).

The erstwhile International Working Group evolved in2003 into the Preimplantation Genetic Diagnosis Inter-national Society (PGDIS), incorporated by this authorand others to preserve continuity of the invaluableInternational Working Groups. At its first meeting inLondon in 2005, PGDIS elected Yury Verlinsky aspresident and Luca Gianaroli as president elect. Thenext President was Joe Leigh Simpson, followed byRobert G. Edwards (2009–2011) and next Renee Mar-tin (2011–2013). Anver Kuliev serves as ExecutiveDirector (www.pgdis.org). In Europe, PGD is organizedthrough the European Society for Human Reproductionand Embryology (ESHRE). In 1997 in Europe, the Spe-cial Interest Group (SIG) in Genetics formed an ESHREPGD Consortium; guiding lights were Karen Sermon,Joep Geraedts and Joyce Harper. The PGD Consor-tium Registry has been invaluable (Harper et al., 2008;Goossens et al., 2008b, 2009). The American Societyfor Reproductive Medicine (ASRM) formed its own SIGon PGD in 2005. Presidents have been Yury Verlinsky,

Jacques Cohen, Marcus Hughes (2009–2010) and nextSantiago Munne (2011–2013). Precongress, postgrad-uate courses and workshops on PGD topics are nowconducted around the world by PGDIS, ESHRE andASRM.

OBTAINING CELLS FOR PREIMPLANTATIONGENETIC DIAGNOSIS

PGD requires access to DNA from gametes or embryosbefore 6 days of conception, when implantation occurs.There are three potential approaches: (1) polar bodybiopsy, (2) blastomere biopsy (aspiration) from the3 day six- to eight-cell cleaving embryos and (3)trophectoderm biopsy from the 5- to 6-day blastocyst;see Verlinsky and Kuliev (2005a) for details on biopsytechnique.

Blastomere (6–8 cell) biopsy

In blastomere biopsy, the zona pellucida, a glycoproteinlayer surrounding the embryo, is traversed by mechani-cal, laser or chemical means in order to extract a cell(s).The first two methods are used most commonly. Themajority of centers remove one cell. Even one cell lessis believed to reduce embryo survival, manifested by a10% reduction in pregnancy rate; removal of two cellsreduces the pregnancy rate even more (Cohen et al.,2007). These figures are based on data-derived numbersof blastomeres remaining following thawing of cryopre-served embryos. Extrapolating these pregnancy rates tothose of biopsied embryos not subjected to cryopreser-vation may or may not be completely applicable. Irre-spective of the above, the 40% reduction in pregnancyrate associated with loss of two blastomeres would castaspersions on protocols that call for removal of two blas-tomeres. DeVos et al. (2009), whose experienced center(Brussels) once routinely removed two cells, recentlyreached similar conclusions; live birth rates were 37.4and 22.4% after removal of one versus two cells, respec-tively.

More than one biopsied embryos are usually trans-ferred in the United States. ‘Take-home’ baby ratesapproximate 30% per cycle in the United States, compa-rable to non-biopsied embryos. In parts of Europe, singleembryo transfer is practiced more often. In the mostrecent ESHRE PGD Consortium data collection (2005cycles), delivery rates were 16% per oocyte retrieval and22% per embryo transfer; clinical pregnancy rates were19 and 26%, respectively (Goossens et al., 2008a,b).

Polar body biopsy

Oocyte genotype can be deduced by analysis of the firstand second polar biopsy (Verlinsky and Kuliev, 2005c).The underlying principle is that the first polar body froma heterozygous individual showing a mutant maternalallele should be complemented by a primary oocyte hav-ing the normal allele. Oocytes deduced to be genetically

Copyright 2010 John Wiley & Sons, Ltd. Prenat Diagn 2010; 30: 682–695.DOI: 10.1002/pd

684 J. L. SIMPSON

normal can be allowed to fertilize in vitro and be trans-ferred for potential implantation. Conversely, a normalpolar body indicates an abnormal oocyte; thus, fertil-ization would not proceed. Similarly, if the first polarbody failed to show chromosome 21, the oocyte wouldbe presumed to have two 21 chromosomes and, hence,generate a trisomic zygote.

The first polar body is present before fertilization;thus, its analysis offers the unique possibility of pre-conceptional diagnosis. The second polar body is notextruded until the mature oocyte is exposed to sperm.Another advantage of polar body biopsy is that no reduc-tion occurs in cell number. The obvious disadvantage isinability to assess paternal genotype, precluding appli-cation if the father has an autosomal dominant disorderand making analysis less efficient in managing couplesat risk for autosomal recessive traits.

One must take into account recombination, whichobligatorily occurs between homologous chromosomesin order to assure orderly disjunction. If recombinationwere not to occur in the first polar body in the regionin question, the second polar body would be identical tothe oocyte. If crossing over were to involve the regioncontaining the gene in question, the single chromosome(two chromatids) in the first polar body would showboth alleles (heterozygosity). Genotype of the oocytecould not be predicted without either biopsy of thesecond polar body or biopsy of the embryo per se(blastomere). If analysis of the second polar body werenot informative, the 8-cell embryo or blastocyst wouldneed to be biopsied. In practice, both first and secondpolar bodies are biopsied in almost all centers.

Polar body biopsy has recently gained popularity fortwo very different reasons. (1) The first polar bodybiopsy can, as stated, uniquely provide preconceptionalinformation. This becomes the only option useful if onemust limit the number of oocytes that can be fertil-ized or embryos transferred; biopsy of the first polarbody allows, in the absence of recombination, normaloocytes to be identified. The majority of euploid oocytesmay be fertilized and reasonable pregnancy rates main-tained, despite restrictive legislation (Gianaroli et al.,2009), even without examining the second polar body.(2) Chromosomal status of the oocyte, as deduced fromits complementary first and second polar bodies, para-doxically appears to be a more reliable indication ofembryo status itself, but sequential testing of blastomereor blastocyst is still necessary to exclude rarer paternallyderived abnormalities (Kuliev et al., 2005; Kuliev andVerlinsky, 2007). A single blastomere could be the prod-uct of a single, unique mitotic non-disjunctional eventand, hence, unrepresentative of the embryo. A random-ized clinical trial (RCT) planned by ESHRE to assesswhether PGD aneuploidy testing increases ART suc-cess rates will utilize polar body biopsy (Geraedts et al.,2010).

Centers technically experienced in polar body biopsyappear to have pregnancy rates comparable to thoseachieved using blastomere biopsy. No attempts havebeen made to compare relative safety of blastomere ver-sus polar body biopsy, reflecting until recently the realitythat few centers performed polar body biopsy. RCTs

would be difficult and potentially misleading unless tech-nical expertise in a given center was comparable for bothtechniques.

Surprisingly, biopsy of polar bodies followed laterby biopsy of the embryo does not seem to decreasepregnancy rates compared to either alone (Cieslak et al.,2006).

Blastocyst biopsy

Biopsy of the trophectoderm in the 5- to 6-day, 120-cell blastocyst is the third approach. More cells can beremoved at this stage, potentially facilitating diagnosis.The trophectoderm forms the placenta; thus, embryoniccells per se are not removed. In fact, before developmentof PGD, Buster et al. (1985) recovered human blasto-cysts by uterine lavage. Thereafter, lavage to recoverblastocysts was widely envisioned to be the approachby which cells removed by trophectoderm biopsy couldallow genetic diagnosis (Carson et al., 1991). In fact,prior to PCR no other approach seemed feasible. How-ever, after PCR lavage for PGD was not pursued becauseof fear of retained, undiagnosed embryos. Analysis ofin vitro embryos cultured to blastocysts was envisionedfor PGD by Dorkas et al. (1990), who showed that tro-phectoderm biopsy could be accomplished in humans.Blastocyst biopsy began to receive less attention, how-ever, as ART routinely generated salutary pregnancyrates transferring 3- to 4-day morulae.

Practitioners of ART have now returned to the blas-tocyst as a preferred stage, after showing value oftransfer after 5 days in vitro culture. The additional 2to 3 days in culture, beyond that required for an 8-cell embryo, allows self-selection against non-thrivingembryos. Approximately one third of embryos withchromosomal abnormalities are selected against betweendays 3 and 5. PGD is still necessary to exclude remaininganeuploidies, whose frequency depends upon maternalage. McArthur, Jansen and their Australian team demon-strated feasibility of blastocyst biopsy with FISH forPGD (McArthur et al., 2005, 2008), as has Schoolcraftet al. (2009).

Embryo biopsy and cryopreservation

In the years immediately following the first success-ful ART (Steptoe and Edwards, 1978), cryopreservationof embryos was not considered possible; thus, ‘spare’embryos were often donated for research. Cryopreser-vation is now routine in ART, although pregnancy ratesusing cryopreserved, non-biopsied embryos remain per-haps 10% lower than with fresh non-biopsied embryos.

Embryo survival following polar body or blastomerebiopsy was likewise once considered almost impossible.Biopsied embryos thus had to be transferred by day 6,and all diagnostic results had to be available by then.Cryopreservation of biopsied embryos is now quite fea-sible (Zheng et al., 2005; Kuwayama, 2007; Schlenkeret al., 2009). As a result of this progress, one verypromising approach has evolved: blastocyst biopsy fol-lowed by vitrification and thawing for transfer 1 month



Table 1—Total number of PGD cycles in two non-lapping data sets

ESHRE PGD Consortium: pooleddata from 57 centers and labs

of varying size (Goossens et al.,2008b) (1997–2006 cycles)

Reproductive Genetics Institute: single U.S.lab performing in-house and transport PGD from

multiple centers (Rechitsky et al., 2009; Kuliev andRechitsky, 2009, personal communication) (1990–2009 cycles)

Single gene and HLA 3523 1737Chromosomal 11 795 4792

Translocations (2260) (475)Sex chromosomes (239) a

Aneuploidy testing (9153) (4017)Other (203)

Social sexing 497 a

15 815 6229

a Not tabulated separately from aneuploidy testing.

later. In the intervening month, new diagnostic appli-cations like array comparative genome hybridization(CGH) for single nucleotide polymorphisms (SNPs) orcopy number variants (CNVs) can be applied to preim-plantation embryos (Handyside et al., 2009; Schoolcraftet al., 2009; Vanneste et al., 2009; Johnson et al., 2010).

DIAGNOSTIC ACCURACY

Cytogenetics

The most common indication for PGD is detection ofchromosomal abnormalities (Table 1). Preimplantationcytogenetic analysis (numerical or structural) generallydepends on FISH using chromosome-specific probes(Griffin et al., 1991; Grifo et al., 1992a,b; Munne et al.,1993).

Pivotal to obtaining accurate results is the presence ofan intact nucleus. A high percentage of informative casescan be achieved only if removal of a blastomere or polarbody can be accomplished in 3 to 5 min, preferably less.Otherwise, the embryo (and blastomere or polar body)may be damaged as a result of dessication, temperaturechanges and alterations in osmolarity. The high errorrates and high numbers of non-informative embryos incertain RCTs probably reflect embryo damage (Munneet al., 2007a,b; Simpson, 2008).

Sensitivity should increase as the number of chro-mosomes tested increases. Approximately 70 to 80% ofaneuploidy can be detected using 8 to 12 chromosomes(e.g. X, Y, 13, 18, 21, 16, 17, 18, 15, 22). These canbe interrogated over 2 to 3 hybridization cycles. Whena given probe fails to yield results for a given chro-mosome, re-testing with a different probe on the samechromosome can lower the ‘no result’ rate (Colls et al.,2007).

Two trends are being pursued to derive information onall 24 chromosomes. (1) Perform additional hybridiza-tion cycles to cover all chromosomes. Using single bloodcells, Aurich-Costa et al. (2009) enumerated 24 chro-mosomes on a single slide, performing 6 successiveoligo-FISH cycles each with 4 chromosomes. Griffinet al. (2009) developed a 24-color FISH assay, usingfour rounds of hybridization completed within 24 h.

Munne et al. (2010) noted that 90% aneuploidy can bedetected by properly selecting 10 to 12 chromosomes;those selected might differ depending on maternal age(e.g. chromosomes 2, 4, 7 in younger women, 11 in olderwomen). Munne et al. (2010) noted that rarer trisomiesare often found only concomitant with another trisomy(double trisomy). Thus, screening for only 10 chromo-somes predicts 89% (382/427) embryos to be eithernormal or abnormal; testing 12 predicts 91% (389/427).

Alternatively, (2) apply genome-wide molecularapproaches using CNVs or SNPs. These approaches uti-lize either metaphase or array CGH, the latter usingSNPs. Given DNA from male and female ART part-ners and other family member(s), one can deduce thenumber of chromosomes in a single cell on the basisof number of transmission of SNPs to the embryo. Oneexpects either two or three SNPs, or altered allele ratios.The presence or absence of trisomy can then be deduced.Metaphase CGH was reported a decade ago (Voullaireet al., 2000; Wells and Delhanty, 2000, Wilton et al.,2001), but only recently has single cell CGH becomesufficiently reliable for clinical application. Wells et al.(2008, 2009a) applied metaphase (CGH) to test for allchromosomes. In a small series, over 90% of blasto-cysts gave informative results, and 36 of 42 cycles(86%) resulted in clinical pregnancy; non-PGD blasto-cysts showed a 60% pregnancy rate. This same grouphad estimated earlier that 9 chromosome FISH wouldhave failed to detect 18% of abnormal embryos (Fragouliet al., 2009). Single cell array CHG testing for genome-wide SNPs can be accomplished reliably on a sin-gle blastomere (Handyside et al., 2009; Vanneste et al.,2009; Johnson et al., 2010). Results can be providedquickly enough for normal embryos to be transferred inthe same cycle. Using SNPs, Treff et al. (2009) accom-plished this in 4 h.

Thought to minimize formation of intracellular icecrystals, vitrification is the cryopreservation method nowapplied following trophectoderm biopsy. Vitrificationallows array CGH by SNPs or CNV, with transfer at alater date. Schoolcraft et al. (2009) showed 95% survivalwhen blastocyst biopsy was followed by vitrification.Wells (2009b) performed array CGH (Illumina platform)and found the probability of an individual embryoforming a pregnancy was 66.7 versus 27.9% withoutPGD; however, sample size was small. Sher et al. (2009)


686 J. L. SIMPSON

reported a remarkable increase in birth rates transferringblastocysts, which as 3-day embryos underwent biopsyand CGH followed by vitrification and then thawingfor transfer at a later time: 48% birth rate (45/94)per transferred CGH-tested blastocyst versus only 15%(57/382) in non-CGH-tested blastocysts.

Single gene disorders

Approximately one fourth of PGD cases are performedfor disorders caused by a single mutant gene (Verlin-sky and Kuliev, 2010) (Table 1). Accuracy is very high,although diagnostic errors are recorded. One persistentfear is contamination, now largely obviated by oblig-atory use of intracytoplasmic sperm injection (ICSE).The other problem is allele dropout (ADO). One expla-nation for ADO is simply preferential amplificationof the other allele. Another is double-stranded break-age involving DNA strands of only one allele, therebyresulting in amplification of only the complementaryallele. This might especially be prone to occur in poorquality embryos or nuclei. The amplification rates rarelyexceed 90 to 95% per allele even in experienced hands(PGDIS, 2008). Less than 100% amplification could alsomerely reflect stochastic phenomena (failure of probes tolocate patient DNA) or embryo damage that has resultedin loss of embryonic DNA. The Reproductive Genet-ics Institute (RGI, Chicago) has observed three errors(β-thalassemia, cystic fibrosis, fragile X) in more than1700 PGD cycles resulting in more than 500 babies(0.3%) (Rechitsky et al., 2009; Verlinsky, Rechitsky andKuliev, 2009, personal communication). Liebaers et al.(2010) reported 0.6% misdiagnosis in 581 PGD preg-nancies, excluding one case in which linkage had beendeduced incorrectly (Goossens et al., 2008a,b; Wiltonet al., 2009). In the ESHRE PGD Consortium, therehave been 10 errors in 2599 single gene cases over9 years, 1997 to 2007 (Goossens et al., 2009). That therate of diagnostic accuracy is so high, despite unavoid-able ADO, probably reflects the policy in PGD centersof transferring only the most diagnostically robust andmorphologically normal embryos, which presumably areless likely to have ADO.

Linkage data is now becoming obligatory. Thisapproach confirms results of mutation analysis per seand allows transfer of embryos even if ADO hasoccurred at the locus being interrogated. Establishingparental haplotype phase usually involves identifyingpolymorphic short tandem repeats (STRs) linked oneither side of the mutant allele; SNPs can accomplishthe same purpose. Although typically established by ana-lyzing affected and unaffected family members, phaseis not always achievable in this manner. An examplearises when a male has a de novo germinal autosomaldominant disorder. By analysis of single sperm, how-ever, phase can be established. Half the sperm havethe mutant allele, accompanied by polymorphic allelesto form a haplotype; the other set shows the wild-typeallele and its different haplotype. In a female with a denovo mutation, deductions can be made from polar bodyanalysis (I and II). A final approach is to deduce phase

during the actual cycle, on the basis of alleles found inan unequivocally affected or normal embryo.

CURRENT INDICATIONS

Avoiding clinical termination

Many couples may wish to avoid an abnormal fetus,yet are opposed to pregnancy termination for reli-gious or other reasons. PGD allows information to beobtained prior to implantation, thus obviating neces-sity for pregnancy termination. In one ESHRE PGDConsortium report, 36% of 1561 couples stated thattheir reason for undergoing PGD was avoiding clini-cal pregnancy termination (ESHRE et al., 2002). In fact,considerable disquiet occurs in any couple undergoingpregnancy termination. This becomes exacerbated whenrepeated pregnancy terminations are necessary becauseof consecutively affected offspring. In the report citedabove, 21% of women undergoing PGD had a previ-ous termination and wished to avoid the possibility ofanother.

Non-disclosure of parental genotype

PGD is the only practical approach if a person at riskfor an adult-onset disorder wishes to remain unawareof his/her genotype, but still desires not to transmitthe mutation to his/her offspring. Prototypic indicationsinvolve Huntington disease and autosomal dominantAlzheimer disease. Traditional prenatal genetic diagno-sis using chorionic villus sampling (CVS) or amnio-centesis theoretically could accomplish the same goal,but would be unwieldy. Using PGD, many embryos canbe screened to identify unaffected embryos suitable fortransfer.

A caveat is that the scenario must be repeated insubsequent cycles, even if the (undisclosed) patientproves unaffected. Otherwise, an at-risk patient couldreadily deduce his/her genotype. The number of PGDcases potentially performed for non-disclosure consti-tutes about 5 to 10% of single gene disorders in theESHRE data collection and in two large U.S. centers(Table 2).

Cancer and other adult-onset disorders

In addition to Huntington disease and other neurodegen-erative disorders, other adult-onset Mendelian disordershave been interrogated by PGD. Performing prenatalgenetic diagnosis for adult-onset Mendelian conditionshad been considered arguable before application throughPGD (Simpson, 2002). In the United States, little contro-versy now exists, but there is still reticence in much ofEurope. The first case of PGD performed for adult-onsetcancer involved Li-Fraumeni syndrome, which is causedby a p53 perturbation (Verlinsky et al., 2001). Detec-tion of other disorders soon followed (Rechitsky et al.,2002). BRCA1, multiple endocrine neoplasia, familial



Tabl

e2

—PG

Dcy

cles

perf

orm

edfo

rsi

ngle

gene

indi

catio

nsin

thre

epr

esum

ptiv

ely

non-

over

lapp

ing

data

sets

a

ESH

RE

PGD

Con

sort

ium

(mos

tlyE

urop

ean

dM

iddl

eE

ast)

Dat

ase

tV

III

(200

5cy

cles

)

Dat

ase

tI–

VII

(199

7–

2004

cycl

es)

Pool

edfr

om39

cent

ers

and

mul

tiple

labs

(Har

per

etal

.,20

08)

(200

5)

Pool

edfr

omup

to45

cent

ers

and

mul

tipl

ela

bs(G

ooss

ens

etal

.,20

08b)

(199

7–

2004

)

Rep

rodu

ctiv

eG

enet

ics

Inst

itute

(Chi

cago

):in

-hou

sean

dtr

ansp

ort

PGD

case

sto

asi

ngle

lab

(Rec

hits

kyet

al.,

2009

;K

ulie

van

dR

echi

tsky

,20

09,

pers

onal

com

mun

icat

ion)

(199

0–

2004

,19

90–

2009

)

Rep

roge

netic

s(N

ewJe

rsey

):tr

ansp

ort

PGD

case

sfr

om59

cent

ers

toa

sing

lela

b(G

utie

rrez

-Mat

eoet

al.,

2009

)(2

004

–20

08)

Cys

ticfib

rosi

s55

403

106

See

belo

wb

73β

-Tha

lass

emia

and

sick

lece

llan

emia

61(i

nclu

des

HL

Aal

so)

N/A

N/A

N/A

β-T

hala

ssem

iaN

/A22

5(i

nclu

des

HL

Aal

so)

108

(exc

lude

sca

ses

conc

urre

ntly

unde

rgoi

ngH

LA

typi

ng)

9

Sick

lece

llan

emia

N/A

54(i

nclu

des

HL

Aal

so)

177

Spin

alm

uscu

lar

atro

phy,

I27

151

71

Orn

ithi

netr

ansc

arba

myl

ase

defic

ienc

y(O

TC

)

——

5—

Hun

ting

ton

dise

ase

5627

010

14M

yoto

nic

dyst

roph

y76

317

333

Tub

erou

ssc

lero

sis

15—

3—

Mar

fan

synd

rom

e13

—8

6

Duc

henn

e/B

ecke

rm

uscu

lar

dyst

roph

y7

7914

6

Li-

Fram

eni

synd

rom

e(p

53)

——

5—


688 J. L. SIMPSON

Tabl

e2

—(C

onti

nued

)

ESH

RE

PGD

Con

sort

ium

(mos

tlyE

urop

ean

dM

iddl

eE

ast)

Dat

ase

tV

III

(200

5cy

cles

)

Dat

ase

tI–

VII

(199

7–

2004

cycl

es)

Pool

edfr

om39

cent

ers

and

mul

tiple

(Har

per

etal

.,20

08)

(200

5)

Pool

edfr

omup

to45

cent

ers

and

mul

tipl

e(G

ooss

ens

etal

.,20

08b)

(199

7–

2004

)

Rep

rodu

ctiv

eG

enet

ics

Inst

itute

(Chi

cago

):in

-hou

sean

dtr

ansp

ort

PGD

case

sto

asi

ngle

(Rec

hits

kyet

al.,

2009

;K

ulie

van

dR

echi

tsky

,20

09,

pers

onal

com

mun

icat

ion)

(199

0–

2004

,19

90–

2009

)

Rep

roge

netic

s(N

ewJe

rsey

):tr

ansp

ort

PGD

case

sfr

om59

cent

ers

toa

sing

le(G

utie

rrez

-Mat

eoet

al.,

2009

)(2

004

–20

08)

Hem

ophi

lia

Aan

dB

1138

50

Von H

ippe

l–L

inda

usy

ndro

me

——

5—

Frag

ileX

(FM

R1)

5110

843

13N

euro

fibro

mat

osis

,I

and

II—

—5

17

Spin

ocer

ebel

lar

atax

ia—

—7

—

Fam

ilial

dysa

uton

omia

——

—9

Gau

cher

dise

ase

——

58

Tors

ion

dyst

onia

——

6—

HL

Aty

ping

+ge

netic

diso

rder

sSe

eβ

-th

alas

sem

ia/s

ickl

ece

llan

emia

See

abov

e47

172

1

HL

Aty

ping

alon

e18

—16

92—

Oth

erM

ende

lian

diso

rder

s11

045

413

959

All

exce

ptH

LA

N/A

N/A

N/A

1473

bN

/ATo

tal

cycl

es50

020

9956

217

37

N/A

,no

tap

plic

able

;—

,no

tsp

ecifi

ed.

aD

ata

from

Har

per

etal

.(2

008)

,G

ooss

ens

etal

.(2

008b

),R

echi

tsky

etal

.(2

009)

,K

ulie

van

dR

echi

tsky

(200

9,pe

rson

alco

mm

unic

atio

n)an

dG

utie

rrez

-Mat

eoet

al.

(200

9).

bIn

divi

dual

indi

catio

nsno

tta

bula

ted.



adenomatous polyposis (FAP), Li-Fraumeni syndrome,retinoblastoma and Von Hippel–Lindau (VHL) syn-drome are the most common indications. Given knowndiagnosis in a prospective parent under active clini-cal surveillance, non-disclosure PGD is not typicallyapplicable. However, PGD to exclude transmission ofan autosomal dominant trait seems far more acceptableto many at-risk families than the prospect of repetitivepregnancy termination using CVS or amniocentesis.

Selecting HLA-compatible embryos

Among sibs, one in four is HLA-compatible (identical).Having an HLA-compatible sibling is invaluable if anolder, moribund sibling with a lethal disease couldpotentially benefit from stem cell transplantation torepopulate his/her bone marrow. The ideal source ofstem cells is from a sibling, including its umbilical cordblood. Stem cell transplantation using cord blood is verysuccessful if the cord blood is HLA-compatible, butmuch less so if there is not 100% HLA-compatibility.Given risk for a single gene disorder usually co-existing,the couple can not only avoid another geneticallyabnormal child but also take advantage of resultingumbilical cord blood to generate stem cells, whichif transplanted allow their older offspring to survive.Assuming the pregnancy is also at risk for an autosomalrecessive disorder, the likelihood of a genetically normalHLA-compatible embryo is 3 in 16 (1 in 4 HLA-compatible embryos multiplied by the 3 in 4 likelihoodof also being unaffected = 3/16).

PGD for the purpose of transferring HLA-compatibleembryos was first performed by Verlinsky and col-leagues in a couple at risk for Fanconi anemia (Ver-linsky et al., 2001). By 2004, 45 cycles for humanleukocyte antigen (HLA) typing had been performed(Kuliev and Verlinsky, 2004a; Verlinsky et al., 2004b);17.5% embryos were genetically suitable for transfer,very near the expected 18.7% (3/16). The most com-mon genetic indication is β-thalassemia (Kuliev andVerlinsky, 2006). A caveat that should be mentionedin counseling is that recombination can occur within theHLA locus in gametes, thus producing an affected childin which a 100% HLA match is not possible. Someembryos will similarly have undergone recombinationand, hence, not be useful.

In the United States and Turkey, testing for HLA-compatible embryos without risk of genetic disease iswidely accepted. The prime indication is an older sibwith leukemia (Kuliev and Verlinsky, 2004a). PGD forHLA typing alone is an increasing indication in theUnited States, accounting for approximately one thirdof HLA PGD cases (Table 2). This indication is uncom-mon in the ESHRE PGD Consortium (Goossens et al.,2008a,b). A 2004 compilation by RGI encompassingits first 500 single gene cycles (1990–2004) is shownin Table 2; 11% involved HLA testing. By 2009, 15%of their cumulative total of 1666 single gene PGDcases had been performed for HLA typing (Rechit-sky et al., 2009). In Istanbul, 261 HLA cases havebeen performed, 200 for β-thalassemia (Ekmecki et al.,

2009). Data specific to the outcome of umbilical cordblood stem cell transplants in PGD sibs have not beenreported, although success should logically approximatethose in non-PGD, HLA-matched umbilical cord bloodtransplants.

Aneuploidy testing should be useful in couples desir-ing HLA-compatible embryos because most are ofadvanced reproductive age. At RGI, aneuploidy test-ing was performed together with HLA typing in 57cycles, yielding a 48.5% pregnancy rate; this preg-nancy rate was twice that in age-matched HLA cases(>35 years) not undergoing aneuploidy testing (Rechit-sky et al., 2009).

Other Mendelian disorders

PGD can be performed for any single gene disor-der whose chromosomal location is known, even ifthe causative mutation is not known (linkage analysiscan still be performed). In the 2005 ESHRE PGDConsortium cycles (Goosens et al., 2008a,b), the mostcommon single gene indications were myotonic dys-trophy (N = 76 of 500), Huntington disease (N =56), cystic fibrosis (N = 55), fragile X syndrome(N = 51), spinal muscular atrophy (N = 27), tuberoussclerosis (N = 15) and Marfan syndrome (N = 13);β-thalassemia and sickle cell anemia combined for 61(Table 2). A total of 110 other conditions were interro-gated. In this data set, 18 cases undergoing HLA typingwere not at risk for a genetic condition.

In the United States, RGI (Chicago) has tested over202 different conditions(Rechitsky et al., 2009), themost frequent being hemoglobinopathies and cysticfibrosis; see Verlinsky and Kuliev (2010) for a com-plete list of disorders tested. Reprogenetics (New Jersey)recently reported their 51-month experience with trans-port PGD, ending March 2008. A total of 162 couples in224 cycles were at risk for 46 different single gene dis-orders (Gutierrez-Mateo et al., 2009). Biopsy was per-formed in 59 different ART centers, blastomeres beingsent to New Jersey for laboratory analysis. By far themost common indication was cystic fibrosis (N = 73cycles). A few disorders were more common in thiscohort than in the ESHRE PGD Consortium and RGI,examples being neurofibromatosis and familial dysau-tonomia; however, the spectrum of disorders tested wassimilar. The implantation rate per embryo was 27.1%(gestational sacs/embryos transferred). Of interest, preg-nancy rates varied widely by referring centers, probablyreflecting underlying differences in embryo biopsy tech-niques and, hence, damage.

Chromosomal rearrangements

Chromosomal rearrangements (translocation or inver-sion) may result in unbalanced gametes and, hence,an unbalanced zygote. Many couples with rearrange-ments are diagnosed only after repeated spontaneousabortions, reflecting lethality conferred by unbalancedgametes. Using PGD, reproductive efficiency can be


690 J. L. SIMPSON

improved by transferring only cytogenetically normalembryos (Munne et al., 1998b, 2000). Such transferswould not only preclude another loss but also an abnor-mal liveborn. Relatively few embryos are normal orbalanced, for which reason success requires a signifi-cant number of embryos from which to choose the fewthat are normal.

Using commercially available chromosome-specificprobes that include telomeric probes, one cannot distin-guish an embryo with a balanced translocation from agenetically normal embryo. Breakpoint-specific probescould accomplish this, and were indeed employed inthe early years of PGD translocation analysis (Munneet al., 1998a); however, costs are prohibitive. An alter-native that can distinguish genetically normal from bal-anced gametes is conversion. The second polar bodyor blastomeres can be fused to a mouse cell to pro-duce a karyotype (Verlinsky et al., 1994). Other methodsinclude subjecting blastomeres to caffeine or electri-cal stimulation (Verlinsky and Evsikov, 1999; Verlin-sky et al., 2002; Shkumatov et al., 2007; Kuliev et al.,2010). These approaches have not always been efficient,but recent experience is more promising. Haplotypingcan also be applied (Traversa and Leigh, 2009).

Ideally, RCTs would have demonstrated efficacy ofPGD increasing pregnancy rates in couples having abalanced translocation who present with repeated preg-nancy losses. None have been conducted, but data arenonetheless compelling. Otani et al. (2006) observedonly 5.3% abortions after PGD for translocations, farfewer than expected. The lifetime cumulative pregnancyrate using PGD was 57.6%, involving an average of only1.24 cycles. The short time-frame during which translo-cation couples undergoing PGD achieve pregnancy con-trasts with the much longer interval (mean 4–6 years)necessary for couples who do not use PGD (Goddijnet al., 2004; Sugiura-Ogasawara et al., 2004; Stephen-son and Sierra, 2006). For that reason, the Societyfor Assisted Reproductive Technology (SART) guide-lines (Fritz and Schattmen, 2008) support this indicationfor PGD.

Repeated pregnancy loss (recurrentaneuploidy)

At least 50% of first-trimester spontaneous abortionshave numerical chromosomal abnormalities(aneuploidy). Non-random distribution occurs in suc-cessive miscarriages; abortuses tend to be either suc-cessively aneuploid or successively euploid (Warburtonet al., 2004). Non-random distribution also occurs inpreimplantation embryos in successive cycles (Rubioet al., 2003). Of relevance also is that 50% of morpho-logically normal embryos in women >35 years old arechromosomally abnormal (Munne et al., 1995). Thus,selecting an embryo optimal for transfer cannot be basedsolely upon morphology.

Given all the above, the rationale for performingPGD aneuploidy testing and transferring only euploidembryos would seem unassailable. Because the ratio-nale is mostly applicable for couples experiencing recur-rent aneuploidy, ideally at least one loss should have

documented aneuploidy. If no information is available,one can perform FISH or array CGH on archivedspecimens embedded in paraffin. If this is not possi-ble, one should acknowledge pitfalls, specifically thathalf of pregnancy losses will not have been due toaneuploidy.

RCTs have not been performed for this indication, butPGD seems beneficial (Gianaroli et al., 1999; Munneet al., 1999, 2005; Verlinsky et al., 2005; Verlinsky andKuliev, 2005). A good surrogate involves comparisonto objective criteria like the Brigham formula (Brighamet al., 1999), which takes into account maternal age andthe number of prior abortions to derive the likelihood ofa pregnancy loss. Munne et al. (2005) observed lossesin only 13% of couples who used PGD, compared to anexpected rate (Brigham) of 33%. Benefit was greatest forwomen older than 35 (39 vs expected 13%; P < 0.001).

Aneuploidy testing improving pregnancyrates

A controversial indication for PGD aneuploidy testing(often termed preimplantation genetic screening or PGS)is improving pregnancy rates in women who requireART for reasons other than for genetic indications. Preg-nancy rates in ART decline precipitously beginning latein the fourth decade. The primary reason is high embry-onic loss due to aneuploidy. Endometrial factors are notparamount, as witnessed by successful pregnancies fol-lowing transfer of donor embryos or use of a donoroocyte for women in their fifth decade or beyond. Theoverall ‘take-home baby rate’ for ART in the UnitedStates (deliveries per retrieval) was 31% in cycles begunin 2001; rates were 38.9% for women <35 years old,32.9% for age 35 to 37, 24.3% for age 38 to 40 and11.1% for age >40 years. (Society for ReproductiveTechnology and the American Society for Reproduc-tive Medicine, 2007). Not only does aneuploidy increasewith increasing maternal age, but miscarriage rates doso as well. Thus, an obvious strategy is to performPGD, transfer euploid embryos and increase the pro-portion of viable pregnancies. Based on success ratesprior to and after PGD, favorable results were reportedfrom experienced centers worldwide beginning in thelate 1990s (Gianaroli et al.,1999, 2005; Munne et al.,1999, 2003; Verlinsky and Kuliev, 2005b,c; Verlinskyet al., 2005). The same held when compared to histori-cal expectations for age-matched women not undergoingPGD. Two RCTs conducted in the United States (Werlinet al., 2003; Mersereau et al., 2008) showed improvedpregnancy rates, although neither was sufficiently pow-ered to show statistical benefit.

By 2000, larger PGD and ART centers in the UnitedStates and Europe increasingly offered PGD to improvepregnancy rates in older women. However, the largestcenters in the United States were not able to complete anRCT. This was due partly to ‘embryo research’ not beingfunded federally (National Institutes of Health); the 2008change in administration in the United States has stillnot resulted in a federally funded trial. Even if funded,only embryo biopsy and FISH components would likely



be covered in the United States; traditional ART costsas incurred during a non-PGD cycle would not havebeen expected to be covered in a research setting.Despite recent adverse publicity for PGD, difficulty inperforming an RCT for aneuploidy testing has thuspersisted in the United States. In the United States, self-funded patients do not appear willing to participate inRCTs if a procedure perceived to be beneficial will notbe available as one (control) arm. This was explicitlyshown by a recently registered RCT that is not provingsuccessful (Munne, 2008, Study NCT 006646893).

Given inability to conduct RCTs in larger centers,other centers, mostly European, have conducted RCTs.None have shown significant improvement in pregnancyrates (Staessen et al., 2004, 2008, Mastenbroek et al.,2007; Hardarson et al., 2008; Mersereau et al., 2008;Schoolcraft et al., 2009; Debrock et al., 2010). At leastone (Mastenbroek et al., 2007) showed harmful effects;others were inconclusive or showed non-significantlyhigher rates, all confounded by limited power. Conclu-sions of some of these studies have been widely criti-cized (Cohen and Grifo, 2007; Munne et al., 2007a,b;Simpson, 2008) on grounds of questionable technicalprowess, diagnostic uncertainty and arguable indications.

The charge of inexperience did not apply, however,to the first RCT, conducted in one of the premierEuropean ART centers (Staessen et al., 2004). Womenaged 36 to 39 assigned to PGD had a clinical pregnancyrate (>12 weeks) of 16.5% per embryo versus 10.4%in controls (P = 0.06); however, the take-home babyrate was not significantly different. A major pitfallwas removal of two blastomeres because, as discussedearlier, loss of even one single blastomere diminishesembryo viability by approximately 10%, with loss of twocells diminishing viability by 40% (Cohen et al., 2007).Indeed, the same Brussels group that conducted the RCTdescribed earlier (Staessen et al., 2004) later confirmeddeleterious effects of removing two cells (DeVos et al.,2009). Given this, they acknowledged that the criticismof Cohen et al. (2007) and others concerning removal oftwo cells ‘seems justified’.

The second RCT concluding harmful effects of PGDaneuploidy testing was conducted by Mastenbroek et al.(2007). A major problem here was method of analysis,at least when generalized beyond their center. WhenPGD was successful (defined as seven chromosomestested and at least one euploid embryo transferred), thepregnancy rate was 16.8% per embryo. When no biopsywas performed (true control), the pregnancy rate was14.7% per embryo, or 13% lower than with PGD. In20% of blastomeres, however, there were no diagnosticresults, twice the expected rate (PGDIS, 2008; Gutierrez-Mateo et al., 2009). This problem was exacerbated bythe low mean number of embryos (4.8/cycle), less thanthe minimum number of 6 recommended for proceedingwith PGD (Simpson, 2008; PGDIS, 2008). Given lackof results, a third (unintended) group existed in whichbiopsy was performed but there was no diagnostic result.The pregnancy rate was only 6% in this group. Theauthors then applied intent-to-treat statistical analysis,a method dictating that all cases remain assigned totheir original group even if they do not complete

their assigned technical protocol (i.e. PGD). Using thismethod, the true PGD group (16.8% pregnancy perembryo) was pooled with the de facto sham group (6%)to yield a blended ‘PGD’ live birth rate of 24% percycle, compared to the statistically higher 35% in thenon-biopsied, non-PGD group.

Considering existing RCTs, the ESHRE PGD Con-sortium generated a position statement recommendingthat PGD aneuploidy testing (termed PGS) should inter-rogate embryonic cells other than blastomeres and usemethods other than FISH (Harper et al., 2010). Polarbodies biopsy using array CGH was specifically recom-mended for an RCT. There is much to commend thisrecommendation. Awaiting these results, the followingseem appropriate clinical counsel if PGD aneuploidytesting were offered, irrespective of stage of biopsy:(1) interrogate embryos only from women of advancedmaternal age, perhaps >37 years old; (2) proceed only ifthere are six to eight morphologically normal embryos;two to three chromosomally normal embryos can thus bereasonably expected. If fewer embryos exist, PGD prob-ably should not be pursued. (3) Use only highly skilledembryologists. (4) Interrogate at least 8 and preferably10 to 12 chromosomes by FISH, or preferably all 24chromosomes either by FISH or array/metaphase CGH.

Congenital anomalies in PGD

Removal of one or more blastomeres, or presumablyany embryonic cell, might logically decrease survivalor implantation and, hence, reduce pregnancy rates.However, the totipotential nature of embryonic cellsshould confer safety against organ-specific anomalies inresulting liveborns. Loss of one or more cells prior toirrevocable commitment toward a specific embryologi-cally developmental pathway is obviated by another cellhaving capacity to accomplish that same purpose. Themalformation rate should thus be that of the general pop-ulation. Actually, comparison in PGD should be to abirth defect rate of 30% over background because that isthe rate observed in non-PGD ART pregnancies (Hansenet al., 2005). It is unclear whether this increase reflectsART per se or, more likely, the underlying infertilitythat necessitated ART (Zhu et al., 2006). Irrespective ofthis, available data indicate no increased rate in livebornspreviously subjected to PGD.

Liebaers et al. (2010) recently reported a thorough,systematic study of PGD offspring judged on the basisof a physical examination 2 months after birth. Anoma-lies in 563 PGD liveborns, 18 stillborns and 9 neonataldeaths were compared to these in a previously reportedcohort study of Intracytoplasmic Sperm Injection (ICSI)offspring not undergoing PGD (Bonduelle et al., 2003).Approximately half the PGD cases of Liebaers et al.(2010) were at risk for a single gene disorder, whereasthe others underwent aneuploidy testing. Structural mal-formations were found in 2.13% for PGD alone and3.38% for ICSI. There were no differences betweenoffspring resulting from single gene PGD and fromPGD aneuploidy testing. A smaller matched pair study(N = 102 in each arm) involved more tests and also


692 J. L. SIMPSON

showed no statistical difference between PGD and ICSI(Desmyttere et al., 2009)

The anomaly rate observed in PGD cases in Brusselsis similar to that observed and regularly updated by RGI(Chicago). The latest reported anomaly rate in RGI caseswas 1.9% of 1230 babies (Ginsberg et al., 2009), poolingall indications. It is also reassuring that neither Brusselsnor Chicago cohorts showed anomalies disproportionallyclustered in any given organ system. As more dataare accumulated, stratification can be performed byindication, procedure and use of cryopreservation. Inconclusion, it can be confidentially concluded that PGDis safe for liveborns (Simpson, 2010).

CONCLUSION

PGD can be applied for any Mendelian disorder whoselocation is known, using linkage analysis even whenthe causative mutation is not known. More than 200different conditions have been tested, with high accuracyand safety. An increasing indication is HLA testingto identify a compatible embryo whose umbilical cordblood can be used for stem cell transplantation for anolder, moribund sib. Other indications applicable only byPGD are non-disclosure testing and avoidance of clinicalpregnancy terminations.

PGD for translocations is accepted, and PGD to avoidrepeated pregnancy losses in couples having recurrentaneuploidy is efficacious. Controversy exists concern-ing whether PGD aneuploidy testing (PGS) improvespregnancy rates, for which reason adherence to spe-cific indications is recommended while the issue is beingadjudicated.

REFERENCES

Aurich-Costa J, Ng C, Selvaggio S, Colls P, Bradley S. 2009.Oligonucleotide (ODN) fluorescence in situ hybridization (Oligo-FISH) and conventional FISH allow enumeration of 24chromosomes in 6 successive hybridizations performed in asingle day. 65th Annual Meeting of the American Society forReproductive Medicine, Atlanta, Georgia, October 17–21. FertilSteril 92(3): Suppl. O-170): S50.

Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P,Liebaers I. 2003. Developmental outcome at 2 years of age forchildren born after ICSI compared with children born after IVF.Hum Reprod 18: 342–350.

Brigham SA, Conlon C, Farquharson RG. 1999. A longitudinal studyof pregnancy outcome following idiopathic recurrent miscarriage.Hum Reprod 14: 2868–2871.

Buster JE, Bustillo M, Rodi IA, et al. 1985. Biologic and morphologicdevelopment of donated human ova recovered by nonsurgicaluterine lavage. Am J Obstet Gynecol 53: 211–217.

Carson S. 1991. Biopsy of blastocysts. In Preimplantation Genetics.Verlinsky Y, Kuliev A (eds). Plenum Press: New York; 85–88.

Cieslak J, Tur-Kaspa I, Ilkevitch Y, Bernal A, Morris R, Verlinsky Y.2006. Multiple micromanipulations for preimplantation geneticdiagnosis do not affect embryo development to the blastocyst stage.Fertil Steril 85: 1826–1829.

Cohen J, Grifo J. 2007. Multicentre trial of preimplantation geneticscreening reported in the New England Journal of Medicine: anin-depth look at the findings. Reprod Biomed Online 15: 305–366.

Cohen J, Wells D, Munne S. 2007. Removal of 2 cells from cleavagestage embryos is likely to reduce the efficacy of chromosomal

tests that are used to enhance implantation rates. Fertil Steril 87:496–503.

Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munne S.2007. Increased efficiency of preimplantation genetic diagnosis forinfertility using “no result rescue”. Fertil Steril 88: 53–61.

Debrock S, Melotte C, Spiessens C, et al. 2010. Preimplantationgenetic screening for aneuploidy of embryos after in vitrofertilization in women aged at least 35 years: a prospectiverandomized trial. Fertil Steril 93: 364–373.

Desmyttere S, Bonduelle M, Nekkebroeck J, Roelants M, Liebaers I,De Schepper J. 2009. Growth and health outcome of 102 2-year-old children conceived after preimplantation genetic diagnosis orscreening. Early Hum Develop; 85: 788–759.

DeVos A, Staessen C, De Rycke M, et al. 2009. Impact of cleavage-stage embryo biopsy in view of PGD on human blastocystimplantation: a prospective cohort of single embryo transfers. HumReprod 24: 2988–2996.

Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. 1990.Trophectoderm biopsy in human blastocysts. Hum Reprod 5:821–825.

Dyban AP. 1991. Experimental cytogenetics of preimplantationdevelopment. In Preimplantation Genetics. Verlinsky Y, Kuliev A(eds). Plenum Press: New York; 15–23.

Edwards RG, Cohen J, Gianaroli L, et al. 2009. Obituary: YuryVerlinsky (01/09/1943–16/07/2009: pioneer in CVS, PGD andhESC. Reprod Biomed Online 19: 298–299.

Ekmecki CG, Beyazyurek C, Tac HA, et al. 2009. Seven yearsof experience on preimplantation HLA typing. 9th AnnualInternational Conference on Preimplantation Genetics, Miami,Florida, April 23–25. Reprod Biomed Online (Suppl) 18: S11.

ESHRE Preimplantation Genetic Diagnosis Consortium. 2002. Datacollection III (May 2001). Hum Reprod 17: 233–246.

Fragouli E, Katz-Jaffe M, Schoolcraft WB, Munne S, Wells D. 2009.Comprehensive cytogenetic analysis of human blastocysts. 9thInternational Conference on Preimplantation Genetics, Miami,Florida, April 23–25. Reprod Biomed Online (Suppl) 18: S9.

Fritz MA, Schattman G. 2008. Reply of the Committee: Parentaltranslocations and need for preimplantation genetic diagnosis?Distorting effects of ascertainment bias and the need forinformation-rich families. Fertil Steril 90: 892–893.

Gardner RL, Edwards RG. 1968. Control of the sex ratio at full termin the rabbit by transferring sexed blastocysts. Nature 218(5139):346–349.

Geraedts J, Collins J, Gianroli L, et al. 2010. What next forpreimplantation genetic screening? A polar body approach! HumReprod 25: 575–577.

Gianaroli L, Magli MC, Ferraretti AP, Munne S. 1999. Preimplan-tation diagnosis for aneuploidies in patients undergoing in vitrofertilization with poor prognosis: identification of the categoriesto which it should be proposed. Fertil Steril 72: 837–844.

Gianaroli L, Magli C, Ferraretti AP, et al. 2005. The beneficial effectsof PGD for aneuploidy support extensive clinical application.Reprod BioMed Online 10: 633–640.

Gianaroli L, Magli MC, Lappi M, Capoti A, Robles F, Ferraretti AP.2009. Preconception diagnosis. 9th International Conference onPreimplantation Genetics, Miami, Florida, April 23–25. ReprodBiomed Online (Suppl) 18: S5.

Ginsberg N, Rechitsky S, Kuliev A, Verlinsky Y. 2009. Clinicaloutcomes of over thousand deliveries after preimplantation geneticdiagnosis (PGD) for genetic and chromosomal disorders. 9thInternational Conference on Preimplantation Genetics, Miami,Florida, April 23–25. Reprod Biomed Online (Suppl) 18: S35.

Goddijn M, Joosten JHK, Knegt AC, can der, et al. 2004. Clinicalrelevance of diagnosing structural chromosome abnormalities incouples with repeated miscarriage. Hum Reprod 19: 1013–1017.

Goossens V, De Rycke M, De Vos A, et al. 2008a. Diagnosticefficiency, embryonic development and clinical outcome after thebiopsy of one or two blastomeres for preimplantation geneticdiagnosis. Hum Reprod 23: 481–492.

Goossens V, Harton G, Moutou C, et al. 2008b. ESHRE PGDConsortium data collection VIII: cycles from January to December2005 with pregnancy follow-up to October 2006. Hum Reprod 12:2629–2645.

Goossens V, Harton G, Moutou C, Traeger-Synodinos J, Van Rij M,Harper JC. 2009. ESHRE PGD Consortium: cycles from January to



December 2006 with pregnancy follow-up to October 2007. HumRepord 24: 1786–1810.

Griffin DK, Handyside AH, Penketh RJ, Winston RM, Delhanty JD.1991. Fluorescent in-situ hybridization to interphase nuclei ofhuman preimplantation embryos with X and Y chromosome specificprobes. Hum Reprod 6: 101–105.

Griffin DK, Ioannou D, Ganriel AS, et al. 2009. Novel perspectiveson 24 chromosome diagnosis in human preimplantation embryos.Chromosome Res 17: 554.

Grifo JA, Boyle A, Fischer E, et al. 1990. Preembryo biopsy andanalysis of blastomere by in situ hybridization. Am J Obstet Gynecol163(6 Pt 1): 2013–2019.

Grifo JA, Boyle A, Tang YX, Ward DC. 1992a. Preimplantationgenetic diagnosis. In situ hybridization as a tool for analysis. ArchPathol Lab Med 116: 393–397.

Grifo JA, Tang YX, Cohen J, Gilbert F, Sanyal M, Rosenwaks Z.1992b. Pregnancy after embryo biopsy and coamplification of DNAfrom X and Y chromosomes. JAMA 268: 727–729.

Gutierrez-Mateo C, Sanchez Garcia J, Fischer J, et al. 2009.Preimplantation genetic diagnosis of single gene disorders:experience with more than 200 cycles conducted by a referencelaboratory in the United States. Feril Steril 92: 1544–1556.

Handyside AH, Kontogianni EH, Hardy K, Winston RML. 1990.Pregnancies from biopsied human preimplantation embryos sexedby Y-specific DNA amplification. Nature 244: 768–770.

Handyside AH, Lesko JG, Tarin JJ, Winston RM, Hughes MR. 1992.Birth of a normal girl after in vitro fertilization and preimplantationdiagnostic testing for cystic fibrosis. N Engl J Med 327: 905–909.

Handyside AH, Harton GL, Mariani B, et al. 2009. Karyomapping: auniversal method for genome wide analysis of genetic disease basedon mapping crossovers between parental haplotypes. J Med GenetOctober 25 [Epub ahead of print].

Hansen M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. 2005.Assisted reproductive technologies and the risk of birth defects—asystematic review. Hum Reprod 20: 328–338.

Hardarson T, Hanson C, Lundin K, et al. 2008. Preimplantationgenetic screening in women of advanced maternal age caused adecrease in clinical pregnancy rate: a randomized controlled trial.Hum Reprod 23: 2617–2621.

Harper J. 2009. Preimplantation Genetic Diagnosis. (2nd edn),Cambridge University Press: Cambridge.

Harper J, Coonen E, De Rycke M, et al. 2010. What next forpreimplantation genetic screening (PGS)? A position statement fromthe ESHRE PGD Consortium steering committee. Hum Reprod Feb2 [Epub ahead of print].

Harper JC, Coonen E, Ramaekers FCS, et al. 1994. Identification ofthe sex of human preimplantation embryos in two hours using animproved spreading method and fluorescent in situ hybridisationusing directly labelled probes. Hum Reprod 9: 721–724.

Harper JC, de Die-Smulders C, Goossens V, et al.. 2008. ESHREPGD consortium data collection VII: cycles from January toDecember 2004 with pregnancy follow-up to October 2005. HumReprod 23: 741–755.

Johnson DS, Genelos G, Baner J, et al. Preclinical validation of amicroarray method for full molecular karyotyping of blastomeresin a 24-h protocol. Hum Reprod January 24 [Epub ahead of print].

Kuliev A, Verlinsky Y. 2004a. Thirteen years’ experience ofpreimplantation diagnosis: report of the Fifth InternationalSymposium on Preimplantation Genetics. Reprod Biomed Online8: 28–235.

Kuliev A, Verlinsky Y. 2004b. Preimplantation HLA typing and stemcell transplantation: report of International Meeting, Cyprus, 27–28March 2004. Reprod Biomed Online 9: 205–209.

Kuliev A, Verlinsky Y. 2006. The future of preimplantation diagnosis.Expert Rev Obstet Gynecol 1: 95–102.

Kuliev A, Verlinsky Y. 2007. Preimplantation genetic diagnosis:technological advances to improve accuracy and range. ReprodBiomed Online 16: 532–538.

Kuliev A, Cieslak J, Verlinsky Y. 2005. Frequency and distribution ofchromosomal abnormalities in human oocytes. Cytogenet GenomeRes 111: 193–198.

Kuliev A, Cieslak J, Jansen J, et al. 2010. Conversion and non-conversion approach to preimplantation diagnosis for chromosomalrearrangements in 475 cycles. Reprod Biomed Online (in press).

Kuwayama M. 2007. Highly efficient vitrification for cryopreservationof human oocytes and embryos: the cryotop method. Theriogenol-ogy 67: 73–80.

Liebaers I, Desmyttere S, Verpoest W, et al. 2010. Report on aconsecutive series of 581 children born after blastomere biopsyfor preimplantation genetic diagnosis. Hum Reprod 25: 275–282.

Mastenbroek S, Twisk M, van Echten-Arends J, et al. 2007. In vitrofertilization with preimplantation genetic screening. N Engl J Med357: 9–17.

McArthur SJ, Leigh D, Marshall JT, De Boer KA, Jansen RPS. 2005.Pregnancies and live births following biopsy and PGD analysis ofhuman embryos at the blastocyst stage. Fertil Steril 84: 1628–1636.

McArthur SJ, Leigh D, Marshall JT, Gee AJ, De Boer KA, Jansen RP.2008. Blastocyst trophectoderm biopsy and preimplantation geneticdiagnosis for familial monogenic disorders and chromosomaltranslocations. Prenat Diagn 28: 434–442.

Mersereau J, Pergament E, Zhang X, et al. 2008. Preimplantationgenetic screening to improve in vitro fertilization pregnancyrates: a prospective randomized controlled trial. Fertil Steril 90:1287–1289.

Monk M, Handyside A. 1988. Sexing of preimplantation embryosby measurement of X-linked gene dosage in a single blastomere.J Reprod Fert 82: 365–368.

Munne S. 2008. Comparison of Double Embryo Transfer (D.E.T.)with D.E.T. combined with PGS for the indication of advancedproductive age (37–42) in patients undergoing ART StudyNCT00646893. www.clinicaltrials.gov.

Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. 1993. Diagnosisof major chromosome aneuploidies in human preimplantationembryos. Hum Reprod 8: 2185–2192.

Munne S, Alikani M, Tomkin F, Grifo J, Cohen J. 1995. Embryomorphology, developmental rates, and maternal age are correlatedwith chromosome abnormalities. Fertil Steril 64: 382–391.

Munne S, Fung J, Cassel C, Marquez C, Weier HUG. 1998a.Preimplantation genetic analysis of translocations case-specificprobes for interphase cell analysis. Hum Genet 102: 663–674.

Munne S, Morrison L, Fung J, et al. 1998b. Spontaneous abortionsare reduced after pre-conception diagnosis of translocations. J AssistReprod Genet 15: 290–296.

Munne S, Magli C, Cohen J, et al. 1999. Positive outcome afterpreimplantation diagnosis of aneuploidy in human embryos. HumReprod 14: 2191–2199.

Munne S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J.2000. Outcome of preimplantation genetic diagnosis of transloca-tion. Fertil Steril 73: 1209–1218.

Munne S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sad-owy S, Cohen J, Sable D. 2003. Improved implantation after preim-plantation genetic diagnosis of aneuploidy. Reprod Biomed Online7: 91–97.

Munne S, Chen S, Fischer J, et al. 2005. Preimplantation geneticdiagnosis reduces pregnancy loss in women aged 35 years and olderwith a history of recurrent miscarriages. Fertil Steril 84: 331–335.

Munne S, Cohen J, Simpson JL. 2007a. In vitro fertilization withpreimplantation genetic screening. N Engl J Med 25(357):1769–1770.

Munne S, Gianaroli L, Tur-Kaspa I, et al. 2007b. Substandardapplication of preimplantation genetic screening may interfere withits clinical success. Fertil Steril 88: 781–784.

Munne S, Howles CM, Wells D. 2009. The role of preimplantationgenetic diagnosis in diagnosing embryo aneuploidy. Curr OpinObstet Gynecol 21: 442–449.

Munne S, Fragouli E, Colls P, Katz-Jaffe M, Schoolcraft W, Wells D.2010. Improved detection of aneuploid blastocysts using a new 12chromosome FISH test. Reprod Biomed Online 20: 92–97.

Otani T, Roche M, Mizuike M, Colls P, Escudero T, Munne S.2006. Preimplantation genetic diagnosis significantly improves thepregnancy outcome of translocation carriers with a history ofrecurrent miscarriage and unsuccessful pregnancies. Reprod BiomedOnline 13: 879–894.

Preimplantation Genetic Diagnosis International Society (PGDIS).2008. Guidelines for good practice in PGD: programmerequirements and laboratory quality assurance. Reprod BiomedOnline 16: 134–147.

Rechitsky S, Verlinsky O, Chistokhina A, et al. 2002. Preimplantationgenetic diagnosis for cancer predisposition. Reprod Biomed Online5: 148–155.


694 J. L. SIMPSON

Rechitsky S, Kuliev A, Sharapova T, et al. 2009. PGD Impact onstem cell transplantation. 9th Annual International Conference onPreimplantation Genetics, Miami, Florida, April 23–25. ReprodBiomed Online (Suppl) 18: S2.

Rubio C, Simon C, Vidal F, et al. 2003. Chromosomal abnormalitiesand embryo development in recurrent miscarriage couples. HumReprod 18: 182–188.

Schlenker Y, Stevens J, Rawlins M, McCormick S, Katz-Jaffe MG,Schoolcraft WB. 2009. Clinical success with vitrification followingtrophectoderm biopsy for comprehensive chromosomal screening.65th Annual Meeting of the American Society for ReproductiveMedicine, Atlanta, Georgia, October 17–21. Fertil Steril 92(3):Suppl. O-243): S71.

Schoolcraft WB, Fragouli E, Stevens J, Munne S, Katz-Jaffe MG,Wells D. 2009. Clinical application of comprehensive chromosomalscreening at the blastocyst stage. Fertil Steril Nov 23. [Epub aheadof print].

Sher G, Keskintepe L, Keskintepe M, Maassarani G, Tortoriello D,Brody S. 2009. Genetic analysis of human embryos by metaphasecomparative genomic hybridization (mCGH) improves efficiencyof IVF by increasing embryo implantation rate and reducingmultiple pregnancies and spontaneous miscarriages. Fertil Steril 92:1886–1894.

Shkumatov A, Kuznyetsov V, Cieslak J, Ilkevitch Verlinsky Y. 2007.Obtaining metaphase spreads from single blastomeres for PGD ofchromosomal rearrangements. Reprod Biomed Online 14: 498–503.

Simpson JL. 2001. Changing indications for preimplantation geneticdiagnosis (PGD). Mol Cell Endocrinol 183(Suppl 1): S69–S75.

Simpson JL. 2002. Celebrating preimplantation genetic diagnosis ofp53 mutations in Li-Fraumeni syndrome. Reprod Biomed Online 3:2–3.

Simpson JL. 2008. What next for preimplantation genetic screening?Randomized clinical trial in assessing PGS: necessary but notsufficient. Hum Reprod 23: 2179–2181.

Simpson JL. 2010. Children born after preimplantation geneticdiagnosis show no increase in congenital anomalies. Hum Reprod25: 6–8.

Simpson JL, Carson SA, Buster J, Bishop CE, Elias S. 1994.Preimplantation genetic diagnosis: Indications and pitfalls. InPerspectives on Assisted Reproduction. Mori T, Tominaga T,Aono T, Hiroi M (eds). Ares-Serono Symposia Publications: Rome;689–696.

Society for Assisted Reproductive Technology and the AmericanSociety for Reproductive Medicine. 2007. Assisted reproductivetechnology in the United States: 2001 results generated from theAmerican Society for Reproductive Medicine/Society for AssistedReproductive Technology registry. Fertil Steril 87: 1253–1266.

Staessen C, Platteau P, Van Assche E, et al. 2004. Comparison ofblastocyst transfer with or without preimplantation genetic diagnosisfor aneuploidy screening in couples with advanced maternalage: a prospective randomized controlled trial. Hum Reprod 19:2849–2858.

Staessen C, Verpoest W, Donoso P, et al. 2008. Preimplantationgenetic diagnosis does not improve delivery rate in women underthe age of 36 following single-embryo transfer. Hum Reprod 23:2818–2825.

Stephenson MD, Sierra S. 2006. Reproductive outcomes in recurrentpregnancy loss associated with a parental carrier of a structuralchromosome rearrangement. Hum Reprod 21: 1076–1082.

Steptoe PC, Edwards RG. 1978. Birth after the reimplantation of ahuman embryo. Lancet 2(8085): 366.

Strom CM, Verlinsky Y, Milayeva S, et al. 1990. Preconceptiongenetic diagnosis of cystic fibrosis. Lancet 336: 306–307.

Sugiaura-Ogasawara M, Ozaki Y, Sata T, Suzumori N, Suzumori K.2004. Poor prognosis of recurrent aborters with either maternal orpaternal reciprocal translocation. Fertil Steril 81: 367–373.

Traversa M, Leigh D. 2009. The application of a molecularstrategy using STR for routine PGD in both reciprocal andRobertsonian translocation carriers. 9th International Conferenceon Preimplantation Genetics, Miami, Florida, April 23–25. ReprodBiomed Online (Suppl) 18: S10.

Treff N, Tao X, Su J, Taylor D, Miller K, Scott R. 2009. Four hour 24chromosome aneuploidy screening using high throughput PCR SNPallele ratio analysis. 65th Annual Meeting of the American Societyfor Reproductive Medicine, Atlanta, Georgia, October 17–21. FertilSteril 92(3): Suppl O-169): S49.

Vanneste E, Voet T, Le Caignec C, et al. 2009. Chromosomeinstability is common in human cleavage-stage embryos. Nat Med15: 577–583.

Verlinsky Y, Evsikov S. 1999. A simplified and efficient methodfor obtaining metaphase chromosomes from individual humanblastomeres. Fertil Steril 72: 1127–1133.

Verlinsky Y, Kuliev A. 1991. Preimplantation Genetics. PlenumPress: New York.

Verlinsky Y, Kuliev A. 2005a. Atlas of Preimplantation GeneticDiagnosis. (2nd edn), Taylor and Francis: Boca Raton.

Verlinsky Y, Kuliev A. 2005b. PGD and its role in ART. In Textbookof IVF and Assisted Reproduction. (Third Edition), Brinsden P (ed).Parthenon Publishing Group: London.

Verlinsky Y, Kuliev A. 2005c. Practical Preimplantation GeneticDiagnosis. Springer-Verlag: London.

Verlinsky Y, Kuliev A. 2010. Preimplantation genetic diagnosis. InGenetic Disorders and the Fetus. (6th ed), Milunsky A, andMulinsky JM (eds). Blackwell Publishing: Sussex; 950–977.

Verlinsky Y, Pergament E, Binor Z, Rawlins R. 1987. Geneticanalysis of human embryos prior to implantation: Futureapplications of in-vitro fertilization in treatment and preventionof human genetic diseases. In Future Aspects in HumanIn-Vitro Fertilization. Ferchlinger and (eds).Kemeter). Springer-Verlag: Berlin; 262–266.

Verlinsky Y, Ginsberg N, Lifchez A, et al. 1990. Analysis of thefirst polar body: preconception genetic diagnosis. Hum Reprod 5:826–829.

Verlinsky Y, Dozortsev D, Evsikov S. 1994. Visualization andcytogenetic analysis of second polar body chromosomes followingits fusion with one-cell mouse embryo. J Assist Reprod Genet 11:123–131.

Verlinsky Y, Cieslak J, Freidine M, Ivaknenko V, Strom C, Kuliev A.1995. Pregnancies following preconception diagnosis of commonaneuploidies by FISH. Hum Reprod 10: 1923–1927.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. 2001a. Preimplantationdiagnosis for p53 tumour suppressor gene mutations. ReprodBiomed Online 2: 102–105.

Verlinsky Y, Rechitsky S, Schoolcraft W, et al. 2001b. Preimplanta-tion diagnosis for Fanconi anemia combined with HLA matching.JAMA 285: 1–4.

Verlinsky Y, Cieslak J, Evsikov S, Galat V, Kuliev A. 2002. Nucleartransfer for full karyotyping and preimplantation diagnosis fortranslocations. Reprod BioMed Online 5: 300–305.

Verlinsky Y, Cohen J, Munne S, et al. 2004a. Over a decade ofexperience with preimplantation genetic diagnosis: a multicenterreport. Fertil Steril 82: 292–294.

Verlinsky Y, Rechitsky S, Sharapova T, Morris R, Taranissi M,Kuliev A. 2004b. Preimplantation HLA testing. JAMA 291:2079–2085.

Verlinsky Y, Tur-Kaspa I, Cieslak J, et al. 2005. Preimplantationtesting for chromosomal disorders improves reproductive outcomeof poor-prognosis patients. Reprod Biomed Online 11: 219–225.

Voullaire L, Slater H, Williamson R, Wilton L. 2000. Chromosomeanalysis of blastomeres from human embryos by using comparativegenomic hybridization. Hum Genet 106: 210–217.

Warburton D, Dallaire L, Thangavelu M, Ross L, Levin B, Kline J.2004. Trisomy recurrence: a reconsideration based on NorthAmerican data. Am J Hum Genet 75: 376–385.

Wells D, Delhanty JD. 2000. Comprehensive chromosomal analysis ofhuman preimplantation embryos using whole genome amplificationand single cell comparative genomic hybridization. Mol HumReprod 6: 1055–1062.

Wells D, Alfarawati S, Fragouli E. 2008. Use of comprehensivechromosomal screening for embryo assessment microarrays andCGH. Mol Hum Reprod 14: 703–710.

Wells D, Fragouli E, Alfarawaty S, Munne S, Schoolcraft WB, Katz-Jaffe MG. 2009a. Highly significant improvement in embryoimplantation and increased live birth rate achieved aftercomprehensive chromosomal screening: Implications for singleembryo transfer. 65th Annual Meeting of the American Society forReproductive Medicine, Atlanta, Georgia, October 17–21. FertilSteril 92(3): Suppl. O-268): S79.

Wells D, Fragouli E, Alfarawaty S, Schoolcraft WB, Munne S, Katz-Jaffe MG. 2009b. Increased embryo implantation and high birthrates following comprehensive chromosomal screening of in vitrofertilized embryos. 9th Annual International Conference on



Preimplantation Genetics, Miami, Florida, April 23–25. ReprodBiomed Online (Suppl) 18: S10.

Werlin L, Rodi I, DeCherney A, Marello E, Hill D, Munne S. 2003.Preimplantation genetic diagnosis as both a therapeutic anddiagnostic tool in assisted reproductive technology. Fertil Steril 80:467–468.

Wilton L, Williamson R, McBain J, et al.. 2001. Birth of ahealthy infant after preimplantation confirmation of euploidy bycomparative genomic hybridisation. N Engl J Med 345: 1537–1541.

Wilton L, Thornhill A, Traeger-Synodinos J, Sermon KD, Harper JC.2009. The causes of misdiagnosis and adverse outcomes in PGD.Hum Reprod 5: 1221–1228.

Zheng WT, Zhuang GL, Zhou CQ, et al. 2005. Comparison of thesurvival of human biopsied embryos after cryopreservation withfour different methods using non-transferable embryos. Hum Reprod20: 1615–1618.

Zhu JL, Basso O, Obel C, Billie C, Olsen J. 2006. Infertility,infertility treatment and congenital malformations: Danish nationalbirth cohort. BMJ 333: 679–683.


Documents

Preimplantation genetic diagnosis at 20 years