PRENATAL DIAGNOSISPrenat Diagn 2003; 23: 678–684.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pd.660

Comparison between fluorescence in situ hybridization(FISH) and quantitative-fluorescent polymerase chainreaction (QF-PCR) for the detection of aneuploidies in singleblastomeres

Takeshi Sato1*, Katsuo Ikuta1, Jon Sherlock2, Matteo Adinolfi2 and Kaoru Suzumori1

1Department of Obstetrics and Gynaecology, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan2The Galton Laboratory and Department of Obstetrics and Gynaecology, University College London, London, UK

Objectives The aim of our investigation was to compare the efficiencies of the fluorescence in situhybridization (FISH) and the quantitative-fluorescent PCR (QF-PCR) methods for the detection of sexingand numerical chromosome disorders in single blastomeres collected from the same preimplantation humanembryos.

Methods FISH analysis was carried out on 145 blastomeres from the 79 surplus embryos with probes specificfor chromosomes 13, 18, 21, X, and Y. QF-PCR was performed with each one or two of the primers specificfor the same chromosomes on 151 blastomeres from the same embryos obtained from patients undergoing IVFtreatment.

Results Analyses were possible on 135 blastomeres (93%) by FISH and on 117 blastomeres (77%) byQF-PCR. Of 65 embryos, which could be analyzed by both methods, 20 embryos (31%) were diagnosed asabnormal.

Conclusion The present study shows that FISH tests are more accurate than QF-PCR assays for the detectionof numerical chromosome disorders when performed on single blastomeres. Copyright 2003 John Wiley &Sons, Ltd.

KEY WORDS: fluorescence in situ hybridization (FISH); quantitative-fluorescent polymerase chain reaction (QF-PCR); preimplantation genetic diagnosis (PGD); blastomere; aneuploidy

INTRODUCTION

In recent years, the introduction of in vitro fertil-ization (IVF) and the molecular analysis of singlecells has made it possible to perform preimplanta-tion screening of human embryos for a variety ofgenetic disorders (Harper and Wells, 1999; ESHREPGD Consortium Steering Committee, 2000). Preim-plantation genetic diagnosis (PGD) offers the advan-tage of allowing the selection and transfer of appar-ently normal embryos, so that couples at risk oftransmitting inherited disorders to their children canstart a pregnancy without the anxiety of a possibletermination.

Basically two approaches have been employed forPGD. The first is fluorescence in situ hybridization(FISH), which has been used for embryo sexing, analysisof chromosome abnormalities such as translocations inblastomeres (Munne et al., 1998b, 2000; Scriven et al.,2000; Lersch et al., 2000; Fung et al., 2001), and to tryand improve the IVF pregnancy rates for patients withadvanced reproductive age or with previous IVF failures

*Correspondence to: Takeshi Sato, Department of Obstetrics andGynaecology, Graduate School of Medical Science, Nagoya CityUniversity, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan. E-mail: og.sato@med.nagoya-cu.ac.jp

by transferring embryos diagnosed as normal, so-calledaneuploidy screening (PGD-AS) (Munne et al., 1998a,1999; Magli et al., 1998; Gianaroli et al., 1999; Wilton,2002).

The other method is the polymerase chain reaction(PCR), which has mainly been used for the detection ofsingle-gene disorders (Liu et al., 1995; Sermon et al.,1997, 2002; Ao et al., 1998; Blaszczyk et al., 1998;Strom et al., 1998; Piyamongkol et al., 2001a,b).

As a variation of PCR, the quantitative-fluorescentPCR (QF-PCR) is based on the amplification of aDNA sequence by PCR while incorporating a fluo-rochrome in the final product, which is then analyzedusing a DNA scanner (Mansfield, 1993; Pertl et al.,1994, 1996). Although this method has been employedextensively for the prenatal detection of numerical chro-mosome disorders (Pertl et al., 1996, 1999; Verma et al.,1998; Sherlock et al., 1998; Mann et al., 2001; Adi-nolfi and Sherlock, 2001) and has the advantage thatit can be used on DNA extracted from a small num-ber of cells (Sherlock et al., 1998), only a few PGDstudies of numerical chromosome disorders have beenreported using this technique (Findlay et al., 1995,1998a,b).

The aim of the present investigation was to evalu-ate the efficiency of FISH and QF-PCR methods forthe detection of sexing and numerical chromosome

Copyright 2003 John Wiley & Sons, Ltd. Received: 21 December 2002Accepted: 29 April 2003

COMPARISON BETWEEN FISH AND QF-PCR FOR DETECTION OF ANEUPLOIDIES IN SINGLE BLASTOMERES 679

Figure 1—Electrophoretogram of a QF-PCR amplification using D21S11, AMXY and X22. The x -axis displays the computed length of the PCRproducts in base pairs as determined automatically using an internal lane standard; the y-axis displays fluorescent intensity (in arbitrary units). Thissample displays two different-sized peaks with a ratio of 2 : 1 for the chromosome 21 marker D21S11, indicating trisomy 21 (diallelic trisomicpattern). Amplification of marker AMXY and X22 resulted in a single peak and two different-sized peaks with a ratio of 1 : 1, corresponding totwo X chromosomes in a female sample

aberrations in single blastomeres collected from thesame preimplantation human embryo.

MATERIALS AND METHODS

Human preimplantation embryos at the 4- to 8-cellstage—estimated as grade I–IV by Veeck’s classification

Figure 2—In situ hybridization with probes specific for chromosomes13, 18, 21, X, and Y. This nucleus shows two signals for chromosome13 (small green), 2 for 18 (blue), 3 for 21 (red), and 2 for X (largegreen), indicating that this blastomere is from a female embryo withtrisomy 21

(1991)—were obtained from patients undergoing IVFtreatment for infertility at the Nagoya City Univer-sity Medical School. In this hospital, up to threemorphologically acceptable embryos developing fromdipronucleated zygotes are replaced into patients onday 3, and the surplus morphologically good embryosare cryopreserved, with the approval of the couple.Thus, morphologically low-grade embryos are essen-tially discarded. Written consent from patients, con-cerning donation of these clinically unusable embryos,was obtained. Another source of samples were embryosthat had been cryopreserved and were going to bediscarded according to the rules of the Research Eth-ical Committee of Nagoya City University MedicalSchool that had approved the plan of the presentresearch. Embryo biopsy was performed on day 3.A hole was drilled through the zona pellucida withacidic Tyrode’s solution (pH 2.4) with a flat-endednarrow pipette, 4 µm in diameter; blastomeres wereremoved by aspiration using a pipette with a 30 to40 µm diameter. For QF-PCR assays, about half of theblastomeres retrieved from each embryo were trans-ferred individually into microtubes containing a smallvolume of distilled water, frozen immediately, andstored until analysis. Other blastomeres from the sameembryo were fixed individually on glass slides for FISHtests.

For the QF-PCR analysis, DNA was extracted fromsingle blastomeres using the QIAamp extraction kit(QIAGEN, UK) (Sherlock et al., 1998; Cirigliano et al.,1999a).

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680 T. SATO ET AL.

Table 1—Primers used in this study

Marker Sequence of primers Chromosome location

AMXY (F) 5′-CTGATGGTTGGCCTCAAGCCT-3′ X and YAMXY (R) 5′-ATGAGGAAACCAGGGTTCCA-3′X22 (F) 5′-TAATGAGAGTTGGAAAGAAA-3′ Xq/Yq pseudoautosomal region PAR2X22 (R) 5′-CCCATTGTTGCTACTTGAGA-3′D13S631 (F) 5′-GGCAACAAGAGCAAAACTCT-3′ 13q31–32D13S631 (R) 5′-TAGCCCTCACCATGATTGG-3′D18S535 (F) 5′-TCATGTGACAAAAGCCACAC-3′ 18q12.2–12.3D18S535 (R) 5′-AGACAGAAATATAGATGAGAATGCA-3′D21S11 (F) 5′-TATGTGAGTCAATTCCCCAAGTGA-3′ 21q21D21S11 (R) 5′-GTTGTATTAGTCAATGTTCTCCAG-3′D21S1412 (F) 5′-CGGAGGTTGCAGTGAGTT-3′ 21(S171–S198)D21S1412 (R) 5′-GGGAAGGCTATGGAGGAGA-3′D21S1414 (F) 5′-AAATTAGTGTCTGGCACCCAGTA-3′ 21q21D21S1414 (R) 5′-CCATTCCCCAAGTGAATTGCCTTC-3′

QF-PCR was performed as previously described(Sherlock et al., 1998; Cirigliano et al., 1999a). Briefly,PCR amplification was carried out using genomic DNA,200 µM dNTPs, 5 to 20 pmol of each primer (Table 1),1X Taq polymerase buffer (1.5 µM MgCl2), and 1.5U Taq polymerase (Promega, USA). To perform fluo-rescent PCR analysis, each forward primer was labeledfluorescently. The reverse primers were unlabeled. Thesingle-cell DNA and buffer were amplified for 35 cycles.The relative fluorescent intensities of the amplificationproducts were calculated using Genescan 672 software(Applied Biosystems).

Since a large proportion of normal samples are het-erozygous for each of the highly polymorphic autosomicshort tandem repeat (STR) markers, the QF-PCR prod-ucts are expected to show two peaks of similar flu-orescence intensity (ratio close to 1 : 1) characterizingthe two different alleles at each STR locus; only afew normal samples should be homozygous for an STRautosomic marker (Adinolfi et al., 1997; Sherlock et al.,1998).

Trisomic samples are expected to fall into two majorgroups: those with three different-sized STR productswith similar fluorescent intensities and a ratio of 1 : 1 : 1(trisomic triallelic); and a second group (trisomic dial-lelic) characterized by the presence of two peaks with aratio close to 2 : 1 (two copies of the same STR alleleand a third different allele). Only a few trisomic samplesare expected to have identical STR markers on all threechromosomes and to show a single peak (Adinolfi et al.,1997; Sherlock et al., 1998).

In the present investigation, samples with two peaksand ratios 1 : 1 to 1.3 : 1 were considered to be normalheterozygous, while ratios of 1 : 1 : 1 or over 1.6 : 1 wereassumed to be diagnostic for trisomies. It was difficultto diagnose the samples with two peaks and ratios1.3 : 1 to 1.6 : 1, but these samples might be normalheterozygous as we reported previously (Adinolfi et al.,1997; Sherlock et al., 1998). However, these sampleswere treated as undiagnosable in this study.

For the sex chromosomes, the amelogenin (AMXY)and the X22 markers were amplified. The AMXYprimers are not polymorphic and can detect the pres-ence of chromosomes X and Y as well as selected sex

chromosome abnormalities (Cirigliano et al., 1999a,b).X22 is a highly polymorphic pentanucleotide marker thatmaps on the pseudoautosomal region PAR2 (Xq/Yq) ofthe sex chromosomes, so that samples from heterozy-gous female or male subjects are expected to show twopeaks of equal intensity (ratio 1 : 1); in contrast, sam-ples from homozygous individuals should produce asingle PCR product. Thus, AMXY and X22 are usefulmarkers for the detection of sex chromosome disorders(Cirigliano et al., 1999a,b).

For FISH analysis, blastomeres were fixed individu-ally on glass slides using Tarkowski’s technique (1966)modified by Yoshizawa (1997). Each blastomere waswashed in phosphate buffered saline (PBS) and, after 5-min exposure, was suspended into a hypotonic solution(1% sodium citrate in water) containing bovine serumalbumin (6 mg-BSA/mL) to prevent attachment to thedish; the blastomere was then fixed mildly with 2.5%fixative (acetic acid 1: methanol 3) in a hypotonic solu-tion (1% sodium citrate in water). Then, the blastomerewas transferred onto a glass slide with a minimal vol-ume of hypotonic solution, and a few drops of fixative(acetic acid 1: methanol 3) were dropped on top of thecell. The fixative was spread by continuous blowing, andthe disappearance of the cytoplasm was observed undera stereoscope.

DNA probes specific for chromosomes 13, 18, 21,X, and Y (Vysis Inc. Downers Grove, IL) were usedin this study. The hybridization targets of the probesspecific for chromosomes 18, X, and Y were α-satelliterepeat mapping in the centromeric region; those forchromosomes 13 and 21 were 13q14 and 21q22.13 to21q22.2 regions.

Each nucleus fixed on the glass slide was denaturedby incubation at 75 ◦C in 70% formamide (FA), 2Xsaline sodium citrate (SSC) (pH 7.0) for 5 min. Then,hybridization solution containing the predenatured DNAprobes was added to the slide, covered with a glasscoverslip, and sealed with rubber cement. Subsequently,the slides were placed in a humidified chamber at37 ◦C for 1 to 4 h to allow hybridization to occur.After washing in 50% FA, 2X SSC, pH 7.0, at 45 ◦Cfor 10 min and additional washing in 2X SSC, 0.1%Nonidet-P40 (NP-40) at 45 ◦C for 5 min, the nucleus

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was counterstained with 4’, 6-diamino-2-phenylindole(DAPI II; 0.5 mg/mL; Vysis) in antifade solution.

Nuclear fluorescent signals were examined using anOPTIPHOTO-2 (Nikon, Japan) epifluorescence micro-scope equipped with a triple–band pass filter forDAPI/Spectrum Green/Spectrum Orange and a sin-gle–band pass filter for Spectrum Aqua with the CYTO-VISION imaging system (Applied Imaging Inc., USA).

In the present investigation, three sets of experimentswere carried out. In group 1, only the sex chromo-somes were analyzed. Primers for AMXY and X22 wereemployed for QF-PCR and probes specific for chromo-some X and Y for FISH. Blastomeres of the secondgroup were analyzed by QF-PCR using primers D21S11and D21S1412 and a chromosome 21–specific probe forFISH. In group 3, sex chromosomes and chromosomes13, 18, and 21 were analyzed. Primers for AMXY,D13S631, D18S535, and D21S1414 were employed ina multiplex QF-PCR assay; probes specific for chromo-some X, Y, 13, 18, and 21 were used simultaneouslyfor FISH.

RESULTS

A total of 157 blastomeres from 41 embryos weretested in the first study group. Eighty blastomeres weresubjected to QF-PCR; of these, 64 (80%) from 34embryos (83%) could be analyzed. In the remaining16 blastomeres, satisfactory results were not obtainedbecause of total amplification failure or contamination.Seventy-seven blastomeres were investigated by FISH,and of these, 71 (92%) from 40 embryos (98%) couldbe analyzed. In the remaining 6 blastomeres, no signalswere found (Table 2). Of 33 embryos analyzed by bothmethods, 11 were diagnosed as males, 15 as females,1 as XXY, and 1 as monosomy Y on the basis of thecombined results. Discordant results between QF-PCRand FISH were observed in the remaining 5 embryos; 2being regarded as possible mosaic, 2 as QF-PCR, and1 as FISH technical errors. In 23 blastomeres from 10embryos, the AMXY and X22 markers were amplifiedby QF-PCR. Of these, 18 blastomeres from 9 embryosshowed concordant QF-PCR fetal sexing results.

A total of 125 blastomeres from 33 embryos weretested in the second group. Of 64 blastomeres subjectedto QF-PCR, 47 (73%) from 27 embryos (82%) wereanalyzable; of 61 blastomeres tested by FISH, 57 (93%)

Table 2—Summary of group 1

No. of embryos(blastomeres)

Total 41 (157)Analyzable by QF-PCR 34 (64/80)Analyzable by FISH 40 (71/77)Analyzable by both methods 33Male 11Female 15XXY 1Monosomy Y 1Other 5

Table 3—Summary of group 2

No. of embryos(blastomeres)

Total 33 (125)Analyzable by QF-PCR 27 (47/64)Analyzable by FISH 32 (57/61)Analyzable by both methods 27Disomy 18Trisomy 1Monosomy 1Other (or mosaic) 7

from 32 embryos (97%) could be analyzed (Table 3).Of the 27 embryos analyzed by both methods, 18 werediagnosed as disomic and 1 as trisomic diallelic since2 blastomeres produced QF-PCR fluorescent peaks withratios close to 2 : 1 and the other 2 blastomeres showed 3signals by FISH. One embryo appeared to be monosomicand seven mosaics, based on the results of both methods.Of these 7 mosaic embryos, 5 appeared to be mosaicswith disomic and trisomic single cells and the other 2were mosaics with disomic, monosomic, and trisomicblastomeres.

Fourteen blastomeres from five embryos were testedin the third group. Six out of seven blastomeres testedby QF-PCR and all seven analyzed by FISH weresuccessful (Tables 4 and 5, Figures 1 and 2).

Two of the five embryos were identified as femalesby QF-PCR (only X-derived peaks) and FISH (two Xsignals). Two others were identified as males with X- andY-derived peaks by QF-PCR and 1 X and 1 Y signal byFISH. The remaining sample was classified as XO withonly an X-derived peak by QF-PCR and 1 X signal by

Table 4—Results of QF-PCR in group 3

Chromosomes

Embryo andblastomere no. Sex (AMXY)

13No. of peaks; ratio

18No. of peaks; ratio

21No. of peaks; ratio

E1-1 Female Disomy (2; 1.3 : 1) Disomy (2; 1 : 1) Trisomy (2; 2.2 : 1)E2-1 Male Disomy (2; 1 : 1) Undiagnosable (2; 1.5 : 1) Disomy (2; 1 : 1)E3-1 Female Disomy (2; 1 : 1) Trisomy (2; 1 : 2) Trisomy (2; 2.1 : 1)E4-1 Female Disomy (2; 1 : 1) Disomy (2; 1 : 1) Disomy (2; 1 : 1)E4-2 Female Disomy (2; 1 : 1) Trisomy (2; 1.6 : 1) Disomy (2; 1 : 1)E5-1 Male Disomy (2; 1 : 1) Disomy (2; 1 : 1) Trisomy (2; 1.7 : 1)

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Table 5—Results of FISH in group 3

Chromosomes

Embryo andblastomere no.

SexDiag. (no. of signal X, Y)

13Diag. (no. of signal)

18Diag. (no. of signal)

21Diag. (no. of signal)

E1-3 Female (2,0) Disomy (2) Disomy (2) Trisomy (3)E1-4 Female (2,0) Disomy (2) Disomy (2) Disomy (2)E2-2 Male (1,1) Disomy (2) Disomy (2) Disomy (2)E3-2 Female (2,0) Disomy (2) Trisomy (3) Disomy (2)E4-3 Monosomy X (1,0) Disomy (2) Disomy (2) Disomy (2)E4-4 Monosomy X (1,0) Disomy (2) Disomy (2) Disomy (2)E5-2 Male (1,1) Disomy (2) Disomy (2) Disomy (2)

FISH. The results of the sex chromosome analysis byboth methods were thus concordant for all five embryos(Tables 4 and 5). The results of testing chromosomes13, 18, and 21 by QF-PCR and FISH are summarized inTables 4 and 5. Two blastomeres from embryo E1 (E1-1and E1-3) were diagnosed as trisomic for chromosome21, one by QF-PCR and the other by FISH, but theother blastomere (E1-4) was normal by FISH. Thisembryo could have been a mosaic. Embryo E2 appearedto be normal disomic according to the FISH results,but the ratio of 1.5 by QF-PCR for chromosome 18was unsatisfactory for diagnosis. Blastomere E3-2 (fromembryo E3) was found to be trisomic for chromosome18 by FISH. On the other hand, E3-1, analyzed by QF-PCR, seemed to be trisomic for chromosome 18 andfor chromosome 21. Single blastomeres (E4-2, E5-1)from embryos E4 and E5, tested by QF-PCR, suggestedaneuploidy, while FISH showed blastomeres E4-3, E4-4,and E5-2 to be normal.

DISCUSSION

Previous investigations of human preimplantation emb-ryos produced by IVF have documented a high incidenceof chromosome aberrations, assumed to be the majorcause of implantation failure and early abortion (Munneet al., 1993, 1995a,b, 1999; Delhanty et al., 1993, 1997;Harper et al., 1995).

Although FISH analysis with direct fluorescence-labeled DNA probes may be associated with detectionerrors due to failure of hybridization with one of thetwo chromosomes, it has the advantage of allowing highspecificity and sensitivity, rapid performance, and thepossibility of using several probes simultaneously. Thus,FISH has been employed as the method of selection forPGD of aneuploidy to try and improve IVF pregnancyrates (Magli et al., 1998; Munne et al., 1998a,b, 1999,2000; Gianaroli et al., 1999; Wilton, 2002).

Fluorescent PCR has been employed to detect single-gene disorders (Sermon et al., 1999; Van de Velde et al.,1999; Ioulianos et al., 2000; De Rycke et al., 2001;Eftedal et al., 2001; Moutou et al., 2001; Piyamongkolet al., 2001a,b), but limited investigations by QF-PCRhave been performed for the diagnosis of aneuploidieson single blastomeres (Findlay et al., 1995, 1998a,b).

Rapid prenatal diagnosis of aneuploidies by QF-PCRhas the advantage that several samples can be analyzed

in a few hours, but tests on single cells are hamperedby technical difficulties. Potential QF-PCR errors forthe detection of aneuploidies in single cells includeallelic dropout (ADO), that is, amplification failureof one allele, and preferential amplification, that is,hypoamplification of one allele compared to the other(Sherlock et al., 1998). Using single cells, ADO wasobserved in previous PGD investigations (Apessos et al.,2001; Rechitsky et al., 2001; Abou-Sleiman et al., 2002;Chamayou et al., 2002; Hussey et al., 2002; Moutouet al., 2002, 2003; Girardet et al., 2003). However,ADO can be minimized by using the DNA extractionmethod employed by Sherlock et al. (1998) and inthe present study. On the other hand, Sherlock et al.(1998) have shown that preferential amplification occursin about 30% of single cells analyzed by multiplexQF-PCR, since the ratio of fluorescent peaks may behigher than the 1 : 1 to 1.3 : 1 expected from cells withnormal chromosomes. Single trisomic diallelic cells withexpected ratios of 2 : 1 may also produce ratios closeto those of normal diploid cells. Accordingly, Sherlocket al. (1998) have suggested that only trisomic triallelicpatterns with ratios close to 1 : 1 : 1 can provide clear-cut results for the detection of trisomies in single cells.However, it should also be noticed that triallelic patternsmay result from ADO, although such cases are rare.

Homozygosity for autosomal markers is another factoraffecting the interpretation of the QF-PCR results. In thepresent investigations, several blastomeres were found toproduce QF-PCR patterns with single fluorescent peaksor patterns with doubtful ratios. Although the single flu-orescent peaks could have resulted from homozygosity,their unexpected high frequency suggested that ADOmight have affected the results, for which one possiblecause might be the storage of the samples, because ADOmay increase in stored samples more than in fresh ones.

On the other hand, preferential amplification may havecaused the discordant results between QF-PCR and FISHtests of embryos E2, E4, and E5 in the third set ofexperiments.

Another limitation of QF-PCR in PGD is that it isdifficult to detect more than tetrasomies and polyploidiesbecause this method is based on the ratios of theintensity of the fluorescent PCR products. The incidenceof these abnormalities have been shown to be higher inblastomeres than in later embryonic or fetal cells (Munneet al., 1993, 1995a,b, 1999).

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In the present study, 77% of blastomeres could beexamined with QF-PCR and 93% with FISH; resultswith the 2 methods often demonstrating satisfactorycoincidence, especially for sex chromosome analysis.

Findlay et al. (1998b) reported FISH and fluorescentPCR as efficient methods for blastomere sexing by com-paring the accuracy of FISH, primed in situ synthesis(PRINS), and conventional and fluorescent PCR withsingle buccal cells and blastomeres. In their report, theanalyzable rates of sexing in single blastomeres were95% and 93% respectively for fluorescent PCR andFISH. In the present study, a similar rate for sexing wasattained using FISH (93% of blastomeres analyzed ingroups 1 and 3), but by QF-PCR (77%) it was consider-ably lower. A possible cause of this low rate of successcould be linked to the storage of the samples, but thepresent results are close to those previously publishedby Sherlock et al. (1998) on testing single cells.

In conclusion, our investigation shows that at presentFISH is the best method for the detection of numer-ical chromosome aberrations in single blastomeres,since preferential amplification may affect the QF-PCR.Instead, the fluorescent PCR tests provide clear resultswhen applied to the detection of single-gene defects,since quantification of the PCR products is not required(Sherlock et al., 1998). Furthermore, it can analyze chro-mosomal disorders and single-gene defects simultane-ously, for which FISH cannot be used.

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