tomeres, which showed the disappearance of nuclear membrane had 46condensed chromosomes, and we identified translocation parts on 100% ofthem. ③Embryos which had not showed disappearance of nuclear mem-brane ceased further embryonic development.④The incidence of rate ofblastocysts of embryos after picking blastomeres out was 67% (30/45).⑤The disappearance times of nuclear membrane were 4;5 hours for three,5;7 hours for six, 7;9 hours for nine, 9;10 hours for nine 10;11 hoursfor six, 11;13 hours for three, 13;14 hours for three, and 14;16 hours forsix nuclear membranes after the observation start time (AM9), showing thatthe nuclear membrane disappeared in 67% (30/45) of cells within 5;11hours.

Conclusion: The method we developed identified the translocation partsof chromosomes with nearly 100% probability. This method needs frequentobservation, but it surely is a safe method with no artificial effects on thechromosomes at the stage of investigation and with no losses and damagesof blastomeres during the electrofusion procedure. By this method, we canachieve almost 100% accuracy of preimplantation diagnosis.

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Efficacy of Preimplantation Genetic Diagnosis (PGD) Using Fluores-cent In-Situ Hybridization (FISH) in Balanced Reciprocal or Roberso-nian Translocation Carriers Undergoing Human IVF-ET Program.1C. K. Lim, 1J. H. Jun,1G. J. Song,1J. W. Kim, 2S. Y. Park,3I. S. Kang.1Laboratory of Reproductive Biology and Infertility,2Laboratory of Med-ical Genetics,3Department of Obstetrics and Gynecology, Samsung CheilHospital & Women’s Healthcare Center, Sungkyunkwan University Schoolof Medicine, Seoul, Korea.

Objectives: This study was performed to evaluate efficiency of PGDusing FISH in balanced reciprocal or Robertsonian translocation carriers inhuman IVF-ET program.

Design: A retrospective study of balanced reciprocal or Robertsoniantranslocation carriers undergoing IVF-ET from 1999 to 2000 at our hospitalwas performed.

Materials and Methods: FISH was carried out in total 30 cycles of 20couples with reciprocal translocation (19 cycles of 15 couples) and Robert-sonian translocation (11 cycles of five couples). Two-color FISH analysiswas performed on 259 blastomeres in 25 cycles and three-color FISHanalysis was performed on 57 blastomeres in five cycles. Mieotic segrega-tion pattern was examined in three-color FISH. After FISH analysis, theembryos with normal FISH signals were transferred into the uterus.

Results: In two-color FISH analysis, normal 55 embryos were transferredin 24 cycles and FISH efficiency per embryo was 94.7%. In three-colorFISH analysis, normal nine embryos were transferred in four cycles andFISH efficiency per embryo was 96.2%. The meiotic pattern of adjacent-1,adjacent-2, alternate, 3:1 and chaotic segregation were 17.2% (10/58),20.0% (11/58), 20.7% (12/58) 13.8% (8/58) and 29.3% (17/58), respec-tively. At present, five pregnancies were achieved after 28 cycles of embryotransfer in 19 couples. A girl and a boy were delivered at term. Both of themwere healthy translocation carriers. Among the other three conceptions, onewas confirmed as normal and one was Robrertsonian translocation carrier byamniocentesis. The other conception is not confirmed yet.

Conclusions: The efficiency of PGD using FISH was above 95% and allconceptions after PGD using FISH were confirmed correct as normal ortranslocation carrier. Two or three-color FISH can be successfully appliedfor the patients with translocations of chromosomes.

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Healthy Births and Ongoing Pregnancies Obtained by PreimplantationGenetic Diagnosis with Ca21/Mg21 Free Medium in Patients withAdvanced Maternal Age and Recurrent Implantation Failure. 1S.Kahraman,2M. Bahce, 1H. Samı,2N. Imirzaıoglu, 1K. Yakın, 1E. Donmez.1Reproductive Endocrinology and ART Unit, Istanbul Memorial Hospital,2Genetic Division, G.A.T.A., Istanbul, Turkey.

Objectives: To assess the effect of Ca21/Mg21-free medium on humanembryos and pregnancies during embryo biopsy for preimplantation geneticdiagnosis (PGD).

Design: Prospective clinical trial.Materials and Methods: PGD was executed on third day embryos ob-

tained from 72 couples with 72 cycles using Ca21/Mg21-free medium forthe advanced maternal age (AMA) (n549) and recurrent implantationfailure (RIF) with more than two cycles even though good quality embryoswere transferred (n523). ICSI was performed in these couples due to severemale factor.

Results: From 360 embryos, a successful FISH procedure was carried outon 329 blastomeres (91.3%). The minimum exposure to Ca21/Mg21-freemedium was 5 minutes, while the maximum was 25 minutes before biopsy.In 107 embryos the biopsy procedure lasted less than 3 minutes whilst in222 embryos the duration was longer than 3 minutes. The minimum andmaximum length of the biopsy procedure was found to be 1 minute. 20seconds and 8 minutes respectively. No statistical significant difference wasfound between the rate of normally cleaved and arrested embryos duringfurther development where the duration of the biopsy was less or more than3 minutes (p.0.05). Embryos were observed for their further developmentfor 24 more hours and transferred on the 4th day. From 329 embryos, 84.2%were cleaved while 15.1% were arrested without an increase in the numberof blastomere. A total of 22 pregnancies was achieved from the 70 embryotransfer (31.4%). One pregnancy resulted in a missed abortion and one in ablighted ova. The ongoing pregnancy rate was found to be 28.5%. Thepregnancy rate was 32.5% in the AMA group (36–45 years) and 30% in theRIF group. The chromosomal abnormality rate was found 41.3% in bothgroups. From these two groups 8 patients had centrally located excessivegranulation in their present and previous cycles and 6 males had megalo-head and pin-hear sperm only in their ejaculates. The majority of chromo-somally abnormal embryos had aneuploidy (72.8%). The mean age, the rateof biopsied and chromosomally abnormal and transferred embryos were thesimilar in both groups (p.0.05). The only statistical significant difference ob-served was the fertilization rate (mean 8.16 3.9 vs 10.66 4.1) (p,0.05).

Conclusion: The use of Ca21/Mg21-free medium during the blastomerebiopsy facilitates the procedure while further embryo cleavage, ongoingpregnancies and healthy births are possible.

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Quantitative Fluorescent PCR Using Short Tandem Repeat (STR)Markers in Preimplantation Genetic Diagnosis. 1R. Poverini, 2G. DiCola,1L. Rienzi,1F. Ubaldi,1E. Greco.1Reproductive Medicine, EuropeanHospital, Rome,2Bio-Tech Lab, Parma, Italy.

Objectives: The preimplantation genetic diagnosis of aneuploidy permitsthe investigation of chromosomal status of embryos generated in vitro,allowing the selection of healthy embryos to be transferred. The quantitativefluorescent PCR has been proposed as an new technology in preimplantationgenetic diagnosis; we have developed this technique to establish the numberof 18, 21 and sex chromosomes present in one single blastomere using twodifferent types of short tandem repeat (STR) markers specific for all of thesechromosomes. In order to investigate if the multiplex PCR using (SRT)markers is affordable to generate a successful diagnosis of aneuploidies westudied, in a series of no viable embryos, the chromosomal status using twoblastomeres from each embryos.

Materials and Methods: Embryo biopsy and blastomere collection havebeen performed from 20 monospermic no viable embryos originated frompatients treated with either intracytoplasmic sperm microinjection (ICSI) orin vitro fertilization (IVF). From the same embryo arrested at 3–5 cell stage,three cell were removed by micromanipulation to be tested 18, 21 and sexchromosomes with two different STR specific markers for those chromo-somes. The products of amplification have been analyzed with automaticDNA analyzer (ABI-310 Perkin Elmer).

Results: The quantitative analysis showed 2 peaks with ratio 1:1 innormal embryos, one STR peak in homozygote embryo respectively in 11and 6 cases; 3 embryos have shown trisomy 21 and 18 as proved by the ratiobetween the two peaks of approximately 2:1.

Conclusions: Our findings showed that amplification of quantitative flu-orescent PCR to a single cell may be used to identify aneuploidy of homo-zygoteembryos and normal trisomic DNA samples with a good accuracy.

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New Fixation Technique for Preimplantation Genetics Diagnosis.D.Dozortsev. Department of Obstetrics and Gynecology, Wayne State Uni-versity, Detroit, MI.

S174 Abstracts Vol. 74, No. 3, Suppl. 1, September 2000