Preimplantation diagnosis for aneuploidiesusing fluorescence in situ hybridization orcomparative genomic hybridization

Yury Verlinsky, Ph.D., and Anver Kuliev, M.D., Ph.D.

Reproductive Genetics Institute, Chicago, Illinois

Testing for aneuploidies has become an es-tablished practice of preimplantation geneticdiagnosis (PGD) in many centers, which iscurrently done by fluorescence in situ hybrid-ization (FISH) analysis using the commercialfive-color probe specific for chromosomes 13,16, 18, 21, and 22 (Vysis, Downers Grove, IL).Although this detects most aneuploidies, com-plete karyotyping may be more advantageousand in the future should prevent the transfer ofnonviable embryos that have any chromosomeerrors. This may be achieved by different meth-ods, including the use of additional FISHprobes to increase number of chromosomesselected (1), nuclear transfer technique to con-vert interphase blastomere nuclei into meta-phase (2, 3) and comparative genomic hybrid-ization (CGH).

Three years ago CGH was shown to befeasible to test single blastomeres (4, 5), but itis still a very labor-intensive procedure, takingas much as 5 days to obtain a result, which isincompatible with the current laboratoryframework of PGD. The first practical applica-tion of CGH was obviously performed as fro-zen cycle (6), which involves the risk of losingup to 50% of the preselected embryos afterthawing. Therefore, it is not surprising that thereported study was limited to a very smallseries of 20 patients with a poor IVF prognosis(7). The CGH analysis of 141 embryos fromthese patients resulted in preselection andtransfer of only 20 aneuploidy-free embryos in14 cycles, which yielded three clinical preg-nancies. It is too soon to reach any conclusionbased on the data from this small sample witheven more limited data from the FISH seriesdone in the same study (testing 57 embryosfrom 10 patients). The FISH series resulted in

preselection and transfer of 15 aneuploidy-freeembryos in 8 cycles, which yielded only oneclinical pregnancy. The only conclusion thatcan be derived from this extremely limited datamay be that the standard CGH procedure seemsimpractical for clinical application, requiringfurther improvement of the technique to over-come its limitations.

Recently, there have been efforts to over-come these limitations either by performing thepolar body CGH or by accelerating the proce-dure and completing it in a much shorter timespan. The first practical application of CGHusing the first polar was attempted in a 40-year-old patient undergoing IVF, and resulted in thedetection of a single aneuploidy-free oocytefrom 10 oocytes available for testing. Thetransfer of the embryo resulting from this oo-cyte, however, yielded no clinical pregnancy(8). Limiting the CGH analysis to the first polarbody may have lead to a misdiagnosis. This isdue to undetected meiosis II errors, whichcould have been identified only through se-quential first and second polar body analyses.

Using blastomere biopsy the CGH proce-dure was accelerated with a specific protocolfor the degenerative oligonucleotide primedpolymerase chain reaction (DOP-PCR) (9).Applying a different Taq polymerase and la-beling method in this protocol yielded a three-fold greater DOP-PCR product, in addition toshortening the procedure to 38 hours.

The reported CGH data are not sufficient toreach a conclusion on the aneuploidy detectionrate, although the 60% rate detected in thesample of 126 preimplantation embryos wascomparable to the 67% detected by FISH anal-ysis of 54 embryos in the same sample (7).

Received March 21, 2003;revised and acceptedMarch 21, 2003.Reprint requests: YuryVerlinsky, Ph.D.,Reproductive GeneticsInstitute, 2825 NorthHalsted Street, Chicago,Illinois 60657 (FAX: 773-871 5221; E-mail:rgi@flash.net).

FERTILITY AND STERILITY�VOL. 80, NO. 4, OCTOBER 2003Copyright ©2003 American Society for Reproductive MedicinePublished by Elsevier Inc.Printed on acid-free paper in U.S.A.

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Despite the expected higher detection rates by CGH, it wasin the same range of aneuploidy rates, detected earlier inthousands of oocytes and preimplantation embryos tested byFISH in the practice of PGD (1, 10, 11). For example, theaneuploidy rate of 52% was reported in FISH analysis of6,733 oocytes, using probes specific for five chromosomes(13, 16, 18, 21, and 22) (12). Similarly, a 60% rate wasreported in a comparable sample size of preimplantationembryos tested in PGD for aneuploidy at the cleavage stage(1, 10, 11). However, there was a difference in the type ofaneuploidy, mainly attributable to a high frequency of com-plex errors (involving the errors of different chromosomes)in oocytes, and mosaicism in preimplantation embryos, com-prising almost half of abnormalities at the cleavage stage.Therefore, it cannot be excluded that a complete karyotypingby CGH or other techniques may lead to an increased de-tection rate of embryos with complex errors, rather thansignificantly increasing the overall proportion of embryoswith aneuploidies.

The overall experience of PGD for chromosomal disor-ders performed by FISH presently exceeds 3,000 cases,further confirming the positive impact on the clinical out-come (1, 10, 11, 13–15). This needs to be further quantifiedin randomized controlled studies. A clinical impact of aneu-ploidy testing, in terms of the improved pregnancy andimplantation rates, as well as the improved outcome ofpregnancies through the reduction of spontaneous abortions,has been confirmed not only for IVF patients with advancedmaternal age, but also for patients with other poor prognoses.The available data may suggest that further improvement ofIVF efficiency may be unrealistic without the preselection ofaneuploidy-free oocytes and embryos. This is particularlyrelevant to the current tendency of limiting the number of thetransferred embryos to avoid multiple pregnancies, thereforepreselection of chromosomally normal embryos may be-come a standard IVF practice.

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