PGD for aneuploidy screening: an expensive hoax?

Best Practice & Research Clinical Obstetrics and GynaecologyVol. 21, No. 1, pp. 157e168, 2007

doi:10.1016/j.bpobgyn.2006.09.006available online at http://www.sciencedirect.com

11

PGD for aneuploidy screening: an

expensive hoax?

Patricio Donoso* MD

Fellow

Paul Devroey MD, PhD

Professor

Centre for Reproductive Medicine, University Hospital, Vrije Universiteit Brussel,

Laarbeeklaan 101, B-1090 Brussels, Belgium

Pre-implantation genetic diagnosis for aneuploidy screening (PGD-AS) constitutes a techniquedeveloped to improve embryo selection in patients with a poor outcome after in-vitro fertiliza-tion treatment due to an increased frequency of numerical chromosome abnormalities in theembryos. Although multiple studies have evaluated the performance of PGD-AS in differentgroups of patients, inconsistencies in the evidence available have not enabled definitive conclu-sions to be drawn. According to randomized trials, PGD-AS does not improve the outcome ofwomen of advanced age when there is no limitation on the number of embryos to be trans-ferred. In patients who have experienced recurrent implantation failure or recurrent miscar-riage, AS only seems to provide diagnostic information, especially when aneuploid embryosalone are found. Additional evidence is needed before AS is implemented as part of routine clin-ical practice.

Key words: pre-implantation genetic diagnosis; aneuploidy screening.

Pre-implantation genetic diagnosis (PGD) was introduced over a decade ago as a strat-egy to prevent the inheritance of sex-linked diseases, by performing DNA amplifica-tion and exclusion of male embryos.1 The further development of new techniquessuch as fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR)enabled the detection of structural and numerical chromosomal abnormalities aswell as the detection of most single gene disorders.2

* Corresponding author. Tel.: þ5622101120; Fax: þ5622101037.

E-mail address: [email protected] (P. Donoso).

1521-6934/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.

mailto:[email protected]

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158 P. Donoso and P. Devroey

PGD for aneuploidy screening (PGD-AS) evaluates the numerical chromosomalconstitution of cleavage-stage embryos through FISH with the aim of selecting euploidembryos for transfer. Although the evaluation of embryo morphology on day 5 enablesthe selection of embryos with a high chance of implantation3e5, this strategy does notensure an euploid chromosomal status as it has been shown that 37% of trisomicembryos develop to blastocyst stage.6 Hence, it has been suggested that in patientswith a high rate of numerical chromosomal abnormalities in embryos, such as womenof advanced maternal age7,8, and women who have experienced unexplained recurrentmiscarriage9,10 or recurrent implantation failure11,12, embryo selection based on PGD-AS could enhance the chance of success. In addition, a reduction in the number ofmultiple pregnancies could be gained by reducing the number of embryos transferred.

The initial optimism generated by observational studies has weakened following theperformance of randomized trials13 that have not shown an improved outcome in womenof advanced age. Furthermore, the recent finding of a similar proportion of abnormalembryos (64%) in young women (under 38 years)14 adds further uncertainty regardingthe value of PGD-AS. The inclusion of patients of all ages is therefore mandatory in thedesign of future randomized studies. Regarding unexplained recurrent miscarriage andrecurrent implantation failure, the lack of consistent evidence supporting the effective-ness of AS raises the need for randomized trials to be performed in order to improvethe counselling of these patients. To date, therefore, the effectiveness of PGD-AS remainsunproven. This article will discuss the available evidence concerning the added value of ASfor embryo selection, and its advantages and limitations.

EMBRYO BIOPSY AND FISH TECHNIQUE

The removal of cells from a cleavage-stage embryo constitutes a delicate procedure; ifit is not performed at the appropriate moment, it can jeopardize embryo developmentas well as its implantation potential. It has been established that the most appropriatestage at which to perform the biopsy is when the embryo has developed to eight cells,as blastomeres are still totipotent and the embryo has not yet undergone compacta-tion.15 Indeed, when biopsies have been carried out on an embryo at an early stage ofdevelopment (two to four cells), a significant reduction in the inner cell mass has beendemonstrated.16 The opening of the zona pellucida can be made with acid Tyrode0smedium or laser. Laser offers the advantages of being less time consuming and ofachieving a higher rate of intact blastomeres than the chemical approach.17 The re-moval of one or two cells is a matter of current debate, as it has been suggestedthat the extraction of two blastomeres can disturb the polarization process towardstrophectoderm and inner cell mass, thus possibly impairing the implantation potentialof these embryos.18 Nevertheless, data derived from both retrospective19 and ran-domized20 studies to date have shown no adverse effect on embryo developmentwhen two cells are removed. The evaluation of two blastomeres offers a more reliablediagnosis as mosaic embryos are identified. Moreover, a higher correlation for aneu-ploidy has been demonstrated after re-analysing non-replaced embryos when twoblastomeres are evaluated instead of one blastomere (82% vs 58%).21 Figure 1 illus-trates the removal of two cells from a cleavage-stage embryo.

FISH utilizes coloured labelled probes for specific chromosomes that are hybridizedto the target DNA already fixed into a microscope slide. After hybridization, alllabelled chromosomes are identified and the number of fluorescent signals presentis evaluated. Since only a limited number of chromosomes can be evaluated

PGD for aneuploidy screening: an expensive hoax? 159

simultaneously, two rounds of probes are often applied. Although a third round ofanalysis would enable the evaluation of a higher number of chromosomes, a significantloss of blastomere nuclei because of the repeated washing procedures restricts itsroutine use.22 An alternative strategy is to combine two or more fluorescent labelsin two rounds, thereby making it possible to assess 10 chromosomes with a highrate of hybridization.23 Overlapping signals, however, can result from this strategy,leading to an overdiagnosis of monosomies. In order to avoid this problem, differentfluorochromes must be used for each chromosome.24 On the other hand, signal split-ting can result in the false diagnosis of trisomies. Nonetheless, misdiagnosis remainsinfrequent as only three cases with FISH have been documented in the literature.25

The frequency of numerical chromosomal abnormalities (15e85%)26 is influencedby the type of patients included, the number and type of probes, the number of eval-uated blastomeres, and the quality of the embryos selected for analysis.

The following scoring criteria have been established when two blastomeres areremoved:

� euploid, where both blastomeres have two copies of each analysed chromosome;� aneuploid, where both blastomeres have one chromosome with an abnormal num-

ber of copies;� haploid or polyploid, where both blastomeres have one, three or more copies of

each chromosome;� mosaic, where one blastomere is euploid and the second blastomere has one chro-

mosome with an abnormal number of copies; and� complex abnormalitym, where at least one blastomere has more than one chromo-

some with an abnormal number of copies.

Figure 1. Removal of two blastomeres from a day 3 embryo.


OTHER BIOPSY TECHNIQUES

Polar body biopsy

Polar body biopsy is performed by removing both the first and the second polar bodiesbased on the fact that more than 90% of chromosomal errors originate from maternalmeiosis.27 Although the most significant advantage of this technique is the fact that thereis no detrimental effect on fertilization and embryonic development, the lack of evalua-tion of the paternal inherited genome and the impossibility of detecting disorders arisingin the course of embryo development has made it a less commonly used approach.

Blastocyst biopsy

The chromosomal assessment of trophectoderm cells from human blastocysts consti-tutes one of the most interesting current lines of research as it enables the study ofmultiple cells, thereby offering an improved detection of mosaicism.28,29 Embryos arehatched on day 3 to facilitate the herniation of trophectoderm cells, followed by theremoval of five or six cells on day 5. The largest series reported to date performed173 biopsies in 63 cycles in couples with recurrent implantation failure; an implantationrate of 30% was observed.29 Although the first live births have already been reported30,further studies are necessary to determine the prospective value of this technique.

INDICATIONS

Advanced maternal age

Advanced maternal age defines a group of women with a decreased chance of preg-nancy after in-vitro fertilization (IVF) because of an augmented frequency of numericalchromosomal abnormalities in their embryos (60e70%) as a result of poor-qualityoocytes. Although there is no consensus to date on the definition of these patients,most studies have included women aged over 37 years.

Decreased efficiency for the evaluation of embryo morphology has been docu-mented in women aged over 37 years as 59% of day 3 embryos with eight cells and35% of expanding blastocysts are genetically abnormal.31 In this scenario, improvedembryo selection resulting from the AS might increase implantation and pregnancyrates. Almost all studies to date have been observational with different inclusion cri-teria and control groups; therefore, it is not possible to compare the results (Table 1).A recently performed meta-analysis13 including two randomized studies31,32 reportedno difference in livebirth rates [11% PGD-AS group vs 15% control group; odds ratio(OR) 0.65; 95% confidence interval (CI) 0.36e1.19], ongoing pregnancy rate perwoman (15% PGD-AS group vs 20% control group; OR 0.64; 95% CI 0.37e1.09), clin-ical pregnancy rate (15% PGD-AS group vs 22% control group; OR 0.42; 95% CI0.12e1.51) or miscarriage rate (OR 0.27; 95% CI 0.04e1.82). Although a trend to-wards a lower frequency of multiple pregnancies has been suggested by some obser-vational studies33,34, no significant difference was encountered in the randomizedstudy performed by Staessen et al (OR 0.41; 95% CI 0.12e1.36).31 Nevertheless,the main drawback of this meta-analysis is that the study which had the highest numberof patients (289 cycles analysed)30 replaced more embryos in the control group than inthe PGD-AS group (2.8 vs 2), hence eliminating the benefit of excluding aneuploid


embryos. The design of future randomized trials should therefore include the replace-ment of an equal limited number of embryos in each group. In order to demonstratean improvement in embryo selection, young women in whom single embryo transfercould be performed should also be included. Table 2 summarizes the data provided byrandomized studies.

According to the results provided by observational trials, it seems that the findingof at least one euploid embryo represents the most important prognostic factor, as animplantation rate of ~10% has been observed despite increasing age and embryo an-euploidy rate.34 On the contrary, patients with no euploid embryos available in theirfirst PGD-AS cycle show a significantly lower livebirth rate compared with womenwith one or two euploid embryos (8.5% vs 30%), because more than 90% repeat thisresult on a further attempt.35 In addition, it has also been shown that only patientswho develop eight or more zygotes and have less than two failed IVF attempts achievea higher implantation rate after performing AS.36

In conclusion, it can be stated that when there is no limitation on the number ofembryos to be transferred, performing PGD-AS in women of advanced age doesnot improve IVF outcome.

Table 1. Observational studies evaluating the outcome of women of advanced age after pre-implantation

genetic diagnosis for aneuploidy screening.

Study Inclusion criteria Implantation rate Pregnancy rate per

embryo transfer

Gianaroli et al, 19997 Age >36 years (n¼ 157) 25.8% 39%

Kahraman et al, 200011 Age not stated (n¼ 49) e 32.5%

Munne et al, 200336 Age �35 years (n¼ 138) 17.6% eRubio et al, 200547 Age �38 years (n¼ 341) 26.5% 28.8%

Platteau et al, 200534 Age �37 years (n¼ 279) 10.7% 16.4%

Table 2. Summary of randomized trials evaluating pre-implantation genetic diagnosis for aneuploidy

screening (PGD-AS) in women of advanced age.

Study Werlin et al, 200346 Stevens et al, 200432 Staessen et al, 200431

Patients randomized (n) 19 39 400

Inclusion criteria >38 years of age �35 years of age

with �5 good-quality

embryos on day 3

�37 years of age

undergoing ICSI

Embryos biopsed (n) 41 Not stated 685

Abnormal embryos (%) 53.7 Not stated 63.2

Proportion (%) of women

reaching embryo transfer

Not stated 95/100 41/64

Mean no. of transferred

embryos in PGD-AS/control

Not stated 2.5/2.8 2.0� 0.9/2.8� 1.2

Implantation rate (%)

PGD-AS/control

Not stated 32/not stated 17.1/11.5

Livebirth rate (%)

PDG-AS/control

Not stated Not stated 15/10

ICSI, intracytoplasmic sperm injection.


Unexplained recurrent miscarriage

It has been shown that up to 70% of clinically recognized pregnancy loss occurring before6 weeks of gestation is due to chromosomal errors. After 10 weeks, however, this rate isreduced to 5%37e39 with an incidence of only 0.6% at birth.40,41 Therefore, these dataprovide evidence for significant selection against chromosomally abnormal embryos.

In almost 50% of the cases of recurrent miscarriage (three or more consecutivemiscarriages), no aetiology is found.42 Given the high frequency of embryo chromo-somal abnormalities that has been found (50e60%) in these patients9,10,43,44, perform-ing embryo selection by AS has been proposed as a strategy to enhance the chance ofachieving a live birth. Nevertheless, this procedure remains controversial as a livebirthrate of 70% can be achieved after three miscarriages in the subsequent pregnancy byperforming supportive care alone.45 Only one randomized study has been performed;this included a reduced number of patients (n¼ 19) so no definitive conclusions couldbe drawn.46 Also, in this study, only pregnancy rates per embryo transfer and not mis-carriage rates were reported. When miscarriage rates after PGD-AS have been com-pared with those for patients undergoing PGD for sex-linked diseases, comparableresults have been reported (12.3% PGD-AS vs 8.3% control); however, this controlgroup was inappropriate as these women did not have recurrent miscarriages.47

On the other hand, age appears to play a significant role in the outcome of thesepatients. In fact, a higher ongoing pregnancy rate has been found after performingPGD-AS in women aged less than 37 years (29% vs 5.5); the difference, however, couldbe explained because of the inclusion of women with associated infertility.44 Nonethe-less, Munne et al observed a reduction in the miscarriage rate compared with the ex-pected probability of miscarriage in women over 35 years of age (12% PGD-AS groupvs 45%).10 Table 3 summarizes the data provided by observational studies.

In conclusion, based on the current inconsistencies in the available evidence, as wellas the risks and costs associated with IVF and PGD, AS should not be performed ona routine basis. Randomized trials are needed to establish the definite role of PGD-ASin the management of unexplained recurrent miscarriage.

Recurrent implantation failure

The lack of success after repeated IVF attempts constitutes a distressing situation forcouples and physicians alike; multiple strategies have therefore been evaluated toimprove success rates. Recurrent implantation failure is diagnosed after failure toconceive following three or more IVF cycles or the replacement of 10 or more good-quality embryos. PGD-AS has been advocated as a way of improving embryo selectionin these patients as an increased incidence of numerical chromosomal abnormalitieshas been documented consistently.7,11,12,48 What is more, a correlation betweenthe previous number of unsuccessful IVF treatments and the frequency of chromo-somal abnormalities has been established (50% with three treatments and 67% withmore than five treatments).48

When analysing data on PGD-AS, most of which is from observational studies, itmust be taken into consideration that delivery rates without any added treatmentfor these patients vary between 4% and 33%.49 The largest report of PGD-AS cyclesperformed for this indication encountered a pregnancy rate per ovum pick-up of only18%.50 Nonetheless, some favourable prognostic factors have been identified for thisheterogeneous group of patients, namely age less than 37 years, more than 10 oocytesretrieved, and at least six embryos available for biopsy.51 The single randomized trial


including couples with recurrent implantation failure only reported on the outcome of19 patients and concluded that there was no benefit after AS.46 Table 4 summarizesthe observational studies evaluating the outcome of these couples after PGD-AS.

It can be concluded that there is insufficient evidence to recommend PGD-AS incouples with recurrent implantation failure.

Other indications

PGD-AS can provide important prognostic information for couples undergoing IVF asthe chance of achieving a live birth in the absence of an euploid embryo is significantlydiminished compared with patients in whom one or more euploid embryos are avail-able for replacement (8.5% vs 30%).35 These patients can therefore be well advised toperform a second attempt if there is at least one euploid embryo available, or to un-dergo oocyte donation when aneuploid embryos alone are found.

In recent years, an increased incidence of chromosomal abnormalities in embryoshas been shown in non-obstructive and obstructive azoospermic men.52,53 In thisgroup of patients, which frequently includes young women, AS could be particularlyhelpful when a limited number of embryos has to be replaced (single embryo transfer).Unfortunately, only one small retrospective study has evaluated the influence ofPGD-AS in these patients to date, and this reported no benefit.47 In the case ofteratozoospermia, macrocephalic spermatozoa have also been associated with embryoaneuploidy.54 Although Kahraman et al found a higher implantation rate after PGD-AS,more evidence is needed to advise couples on this matter.54

LIMITATIONS OF PGD-AS

Misdiagnosis

Mosaic embryos are the result of an abnormal mitotic division in a diploid zygote asa consequence of non-disjunction or anaphase lagging55, representing the most

Table 3. Summary of observational studies reporting the pregnancy outcome after pre-implantation

genetic diagnosis (PGD) for aneuploidy screening in couples with unexplained recurrent miscarriage.

Authors Study group Control group Miscarriage rate PR/ET

Rubio et al, 200547 <37 years

(163 cycles)

25 cycles of PGD

for sex-linked

disease

<37 years, 10%;

�37 years, 20%;

control, 8.3%

<37 years,

39%; �37 years,

30%; control, 33%�37 years

(78 cycles)

Platteau et al, 200544 <37 years

(35 cycles)

No Not reported <37 years, 29%;

�37 years, 5.5%

�37 years

(34 cycles)

Munne et al, 200510 <35 years

(21 cycles)

Expected

miscarriage

rate

calculated by

Brigham et al65

<35 years, 23%;

�35 years, 12%;

Expected <35 years 29%

>35 years 45%

<35 years, 57%;

�35 years, 46%

�35 years

(37 cycles)

PR/ET, pregnancy rate per embryo transfer.


prevalent anomaly in pre-implantation embryos (57%).56 Mosaicism constitutes themain source of misdiagnosis (60%)57 as it leads to false-positive and false-negative re-sults, thereby reducing the efficiency of this technique. Although the developmentalpotential of mosaic embryos has been correlated with the type and proportion of an-euploid cells,6,25 it is most likely that the majority of these embryos are lost prior tothe first trimester of pregnancy as mosaicism is only encountered in 5% of spontane-ous abortions.58 Furthermore, the existence of dynamic changes through the processof in-vitro development resulting from the correction of present anomalies or theemergence of new anomalies makes the interpretation of results obtained from a lim-ited number of cells very difficult. Hence, it is possible that a better understanding ofthe fate of these embryos will enable a reduction in the loss of suitable embryos fortransfer.

Other sources of misdiagnosis are due to technical problems such as signal splittingor overlap. Additionally, some probes (Y, 18) tend to produce more errors because ofa larger size.56 Finally, the number of cells evaluated can also influence misdiagnosis, asestimated false-negative and false-positive rates of 22% and 12.6%, respectively, havebeen found when only one blastomere is evaluated, and 14.2% and 6%, respectively,when two blastomeres are evaluated.57

Limited number of chromosomes

FISH only enables the evaluation of a limited number of chromosomes, which probablyresults in an underestimation of the embryo abnormality rate. In fact, the developmentof novel techniques such as comparative genomic hybridization (CGH) that allow allchromosomes to be assessed has revealed that only 25% of embryos are chromoso-mally normal.58e60 However, the long period of time necessary for hybridization (72 h)limits the clinical application of this technique as it requires all embryos to be frozen.Since a significant reduction in the survival rate after thawing of biopsed embryos hasbeen documented61,62, this approach could lead to an unacceptable decrease inpregnancy rates. Nevertheless, the recent development of micro-array CGH usingwhole-genome amplification through PCR for single cell analysis could surpass thislimitation.63,64 However, more studies are required before it can be applied in clinicalpractice.

Table 4. Summary of observational studies evaluating the outcome of patients with recurrent

implantation failure after pre-implantation genetic diagnosis for aneuploidy screening (PGD-AS).

Study Definition Cycles (n) Implantation rate Clinical PR/ET

Gianaroli et al, 19997 �3 failed cycles 27 17.3% 25%

Kahraman et al, 200011 �3 failed cycles 23 Not reported 30.4%

<36 years

Pehlivan et al, 200312 �3 failed cycles 32 24.6% 40.7%

<37 years

Munne et al, 200336 �2 failed cycles 54 14.3% Not reported

�35 years

Platteau et al, 200651 �3 failed cycles 121 19.5% 34%

<37 years

PR/ET, pregnancy rate per embryo transfer.


Follow-up

As for all innovative techniques introduced in clinical practice, follow-up is very impor-tant in ensuring the safety of the procedure. To date, however, only limited data areavailable; they are mainly provided from the ESHRE PGD Consortium, which, todate, has shown no difference when compared with intracytoplasmic sperm injectioncycles.50 Further development of the cumulative information provided by all the cen-tres that perform PGD-AS represents a significant challenge for the near future.

SUMMARY

PDG-AS represents a novel approach for embryo selection in patients for whom re-productive failure is attributable to an increased frequency of chromosomally abnor-mal embryos. Nevertheless, the existence of significant technical limitations andinconsistencies in current evidence makes it mandatory to conduct well-designed ran-domized trials in order to improve patient counselling. Enhanced knowledge concern-ing the frequency of chromosomal abnormalities in embryos in young womenundergoing IVF as well as in natural cycles will provide a better understanding ofthe influence of age and ovarian stimulation in chromosomal abnormalities in embryos.Additionally, the future implementation of new technologies that enable the evaluationof all the chromosomes will possibly overcome the current limitations of AS.

In conclusion, according to currently available literature, it can be stated that ASdoes not improve the outcome of women of advanced age when there is no limitationon the number of embryos to be transferred. In patients who have experienced recur-rent implantation failure or recurrent miscarriage, it appears that this technique canprovide prognostic information based on the finding of euploid embryos to transfer.Additional evidence is needed before other indications for AS can be implementedin routine clinical practice.

Research agenda

� determine which chromosomes should be included when performing FISH, andestablish the value of adding more probes� compare both implantation and ongoing pregnancy rates after one vs two blas-

tomeres have been removed� study the benefits of performing embryo biopsy at the blastocyst stage� evaluate the incorporation of new technologies that enable the assessment of

all chromosomes (CGH)� perform randomized trials in women of advanced age, and patients who have

experienced recurrent miscarriage or recurrent implantation failure while re-placing an equal number of embryos in both groups� evaluate whether PGD-AS can improve embryo selection in young women un-

dergoing single embryo transfer� evaluate the rate of chromosomal abnormality in embryos in natural cycles� assess the safety of this procedure through the long-term follow-up of children

born after PGD-AS


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PGD for aneuploidy screening: an expensive hoax?