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Quantitative Fluorescent PCR Using Short Tandem Repeat (STR) Markers in Preimplantation Genetic Diagnosis
tomeres, which showed the disappearance of nuclear membrane had 46 condensed chromosomes, and we identified translocation parts on 100% of them. ③Embryos which had not showed disappearance of nuclear membrane ceased further embryonic development. ④The incidence of rate of blastocysts 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 hours for six, 11;13 hours for three, 13;14 hours for three, and 14;16 hours for six nuclear membranes after the observation start time (AM9), showing that the nuclear membrane disappeared in 67% (30/45) of cells within 5;11 hours. Conclusion: The method we developed identified the translocation parts of chromosomes with nearly 100% probability. This method needs frequent observation, but it surely is a safe method with no artificial effects on the chromosomes at the stage of investigation and with no losses and damages of blastomeres during the electrofusion procedure. By this method, we can achieve almost 100% accuracy of preimplantation diagnosis.
P-248 Efficacy of Preimplantation Genetic Diagnosis (PGD) Using Fluorescent In-Situ Hybridization (FISH) in Balanced Reciprocal or Robersonian Translocation Carriers Undergoing Human IVF-ET Program. 1 C. K. Lim, 1J. H. Jun, 1G. J. Song, 1J. W. Kim, 2S. Y. Park, 3I. S. Kang. 1 Laboratory of Reproductive Biology and Infertility, 2Laboratory of Medical Genetics, 3Department of Obstetrics and Gynecology, Samsung Cheil Hospital & Women’s Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea. Objectives: This study was performed to evaluate efficiency of PGD using FISH in balanced reciprocal or Robertsonian translocation carriers in human IVF-ET program. Design: A retrospective study of balanced reciprocal or Robertsonian translocation carriers undergoing IVF-ET from 1999 to 2000 at our hospital was performed. Materials and Methods: FISH was carried out in total 30 cycles of 20 couples with reciprocal translocation (19 cycles of 15 couples) and Robertsonian translocation (11 cycles of five couples). Two-color FISH analysis was performed on 259 blastomeres in 25 cycles and three-color FISH analysis was performed on 57 blastomeres in five cycles. Mieotic segregation pattern was examined in three-color FISH. After FISH analysis, the embryos with normal FISH signals were transferred into the uterus. Results: In two-color FISH analysis, normal 55 embryos were transferred in 24 cycles and FISH efficiency per embryo was 94.7%. In three-color FISH analysis, normal nine embryos were transferred in four cycles and FISH 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), respectively. At present, five pregnancies were achieved after 28 cycles of embryo transfer in 19 couples. A girl and a boy were delivered at term. Both of them were healthy translocation carriers. Among the other three conceptions, one was confirmed as normal and one was Robrertsonian translocation carrier by amniocentesis. The other conception is not confirmed yet. Conclusions: The efficiency of PGD using FISH was above 95% and all conceptions after PGD using FISH were confirmed correct as normal or translocation carrier. Two or three-color FISH can be successfully applied for the patients with translocations of chromosomes.
P-249 Healthy Births and Ongoing Pregnancies Obtained by Preimplantation Genetic Diagnosis with Ca21/Mg21 Free Medium in Patients with Advanced Maternal Age and Recurrent Implantation Failure. 1S. Kahraman, 2M. Bahc¸e, 1H. S¸amı, 2N. Imirzaıog˘lu, 1K. Yakın, 1E. Do¨nmez. 1 Reproductive Endocrinology and ART Unit, Istanbul Memorial Hospital, 2 Genetic Division, G.A.T.A., Istanbul, Turkey. Objectives: To assess the effect of Ca21/Mg21-free medium on human embryos and pregnancies during embryo biopsy for preimplantation genetic diagnosis (PGD). Design: Prospective clinical trial. Materials and Methods: PGD was executed on third day embryos ob-
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Abstracts
tained from 72 couples with 72 cycles using Ca21/Mg21-free medium for the advanced maternal age (AMA) (n549) and recurrent implantation failure (RIF) with more than two cycles even though good quality embryos were transferred (n523). ICSI was performed in these couples due to severe male factor. Results: From 360 embryos, a successful FISH procedure was carried out on 329 blastomeres (91.3%). The minimum exposure to Ca21/Mg21-free medium was 5 minutes, while the maximum was 25 minutes before biopsy. In 107 embryos the biopsy procedure lasted less than 3 minutes whilst in 222 embryos the duration was longer than 3 minutes. The minimum and maximum length of the biopsy procedure was found to be 1 minute. 20 seconds and 8 minutes respectively. No statistical significant difference was found between the rate of normally cleaved and arrested embryos during further development where the duration of the biopsy was less or more than 3 minutes (p.0.05). Embryos were observed for their further development for 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 number of blastomere. A total of 22 pregnancies was achieved from the 70 embryo transfer (31.4%). One pregnancy resulted in a missed abortion and one in a blighted ova. The ongoing pregnancy rate was found to be 28.5%. The pregnancy rate was 32.5% in the AMA group (36 – 45 years) and 30% in the RIF group. The chromosomal abnormality rate was found 41.3% in both groups. From these two groups 8 patients had centrally located excessive granulation in their present and previous cycles and 6 males had megalohead and pin-hear sperm only in their ejaculates. The majority of chromosomally abnormal embryos had aneuploidy (72.8%). The mean age, the rate of biopsied and chromosomally abnormal and transferred embryos were the similar in both groups (p.0.05). The only statistical significant difference observed was the fertilization rate (mean 8.1 6 3.9 vs 10.6 6 4.1) (p,0.05). Conclusion: The use of Ca21/Mg21-free medium during the blastomere biopsy facilitates the procedure while further embryo cleavage, ongoing pregnancies and healthy births are possible.
P-250 Quantitative Fluorescent PCR Using Short Tandem Repeat (STR) Markers in Preimplantation Genetic Diagnosis. 1R. Poverini, 2G. Di Cola, 1L. Rienzi, 1F. Ubaldi, 1E. Greco. 1Reproductive Medicine, European Hospital, Rome, 2Bio-Tech Lab, Parma, Italy. Objectives: The preimplantation genetic diagnosis of aneuploidy permits the investigation of chromosomal status of embryos generated in vitro, allowing the selection of healthy embryos to be transferred. The quantitative fluorescent PCR has been proposed as an new technology in preimplantation genetic diagnosis; we have developed this technique to establish the number of 18, 21 and sex chromosomes present in one single blastomere using two different types of short tandem repeat (STR) markers specific for all of these chromosomes. In order to investigate if the multiplex PCR using (SRT) markers is affordable to generate a successful diagnosis of aneuploidies we studied, in a series of no viable embryos, the chromosomal status using two blastomeres from each embryos. Materials and Methods: Embryo biopsy and blastomere collection have been performed from 20 monospermic no viable embryos originated from patients treated with either intracytoplasmic sperm microinjection (ICSI) or in 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 sex chromosomes with two different STR specific markers for those chromosomes. The products of amplification have been analyzed with automatic DNA analyzer (ABI-310 Perkin Elmer). Results: The quantitative analysis showed 2 peaks with ratio 1:1 in normal embryos, one STR peak in homozygote embryo respectively in 11 and 6 cases; 3 embryos have shown trisomy 21 and 18 as proved by the ratio between the two peaks of approximately 2:1. Conclusions: Our findings showed that amplification of quantitative fluorescent PCR to a single cell may be used to identify aneuploidy of homozygote embryos and normal trisomic DNA samples with a good accuracy.
P-251 New Fixation Technique for Preimplantation Genetics Diagnosis. D. Dozortsev. Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI.
Vol. 74, No. 3, Suppl. 1, September 2000
P-248 Efficacy of Preimplantation Genetic Diagnosis (PGD) Using Fluorescent In-Situ Hybridization (FISH) in Balanced Reciprocal or Robersonian Translocation Carriers Undergoing Human IVF-ET Program. 1 C. K. Lim, 1J. H. Jun, 1G. J. Song, 1J. W. Kim, 2S. Y. Park, 3I. S. Kang. 1 Laboratory of Reproductive Biology and Infertility, 2Laboratory of Medical Genetics, 3Department of Obstetrics and Gynecology, Samsung Cheil Hospital & Women’s Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea. Objectives: This study was performed to evaluate efficiency of PGD using FISH in balanced reciprocal or Robertsonian translocation carriers in human IVF-ET program. Design: A retrospective study of balanced reciprocal or Robertsonian translocation carriers undergoing IVF-ET from 1999 to 2000 at our hospital was performed. Materials and Methods: FISH was carried out in total 30 cycles of 20 couples with reciprocal translocation (19 cycles of 15 couples) and Robertsonian translocation (11 cycles of five couples). Two-color FISH analysis was performed on 259 blastomeres in 25 cycles and three-color FISH analysis was performed on 57 blastomeres in five cycles. Mieotic segregation pattern was examined in three-color FISH. After FISH analysis, the embryos with normal FISH signals were transferred into the uterus. Results: In two-color FISH analysis, normal 55 embryos were transferred in 24 cycles and FISH efficiency per embryo was 94.7%. In three-color FISH analysis, normal nine embryos were transferred in four cycles and FISH 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), respectively. At present, five pregnancies were achieved after 28 cycles of embryo transfer in 19 couples. A girl and a boy were delivered at term. Both of them were healthy translocation carriers. Among the other three conceptions, one was confirmed as normal and one was Robrertsonian translocation carrier by amniocentesis. The other conception is not confirmed yet. Conclusions: The efficiency of PGD using FISH was above 95% and all conceptions after PGD using FISH were confirmed correct as normal or translocation carrier. Two or three-color FISH can be successfully applied for the patients with translocations of chromosomes.
P-249 Healthy Births and Ongoing Pregnancies Obtained by Preimplantation Genetic Diagnosis with Ca21/Mg21 Free Medium in Patients with Advanced Maternal Age and Recurrent Implantation Failure. 1S. Kahraman, 2M. Bahc¸e, 1H. S¸amı, 2N. Imirzaıog˘lu, 1K. Yakın, 1E. Do¨nmez. 1 Reproductive Endocrinology and ART Unit, Istanbul Memorial Hospital, 2 Genetic Division, G.A.T.A., Istanbul, Turkey. Objectives: To assess the effect of Ca21/Mg21-free medium on human embryos and pregnancies during embryo biopsy for preimplantation genetic diagnosis (PGD). Design: Prospective clinical trial. Materials and Methods: PGD was executed on third day embryos ob-
S174
Abstracts
tained from 72 couples with 72 cycles using Ca21/Mg21-free medium for the advanced maternal age (AMA) (n549) and recurrent implantation failure (RIF) with more than two cycles even though good quality embryos were transferred (n523). ICSI was performed in these couples due to severe male factor. Results: From 360 embryos, a successful FISH procedure was carried out on 329 blastomeres (91.3%). The minimum exposure to Ca21/Mg21-free medium was 5 minutes, while the maximum was 25 minutes before biopsy. In 107 embryos the biopsy procedure lasted less than 3 minutes whilst in 222 embryos the duration was longer than 3 minutes. The minimum and maximum length of the biopsy procedure was found to be 1 minute. 20 seconds and 8 minutes respectively. No statistical significant difference was found between the rate of normally cleaved and arrested embryos during further development where the duration of the biopsy was less or more than 3 minutes (p.0.05). Embryos were observed for their further development for 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 number of blastomere. A total of 22 pregnancies was achieved from the 70 embryo transfer (31.4%). One pregnancy resulted in a missed abortion and one in a blighted ova. The ongoing pregnancy rate was found to be 28.5%. The pregnancy rate was 32.5% in the AMA group (36 – 45 years) and 30% in the RIF group. The chromosomal abnormality rate was found 41.3% in both groups. From these two groups 8 patients had centrally located excessive granulation in their present and previous cycles and 6 males had megalohead and pin-hear sperm only in their ejaculates. The majority of chromosomally abnormal embryos had aneuploidy (72.8%). The mean age, the rate of biopsied and chromosomally abnormal and transferred embryos were the similar in both groups (p.0.05). The only statistical significant difference observed was the fertilization rate (mean 8.1 6 3.9 vs 10.6 6 4.1) (p,0.05). Conclusion: The use of Ca21/Mg21-free medium during the blastomere biopsy facilitates the procedure while further embryo cleavage, ongoing pregnancies and healthy births are possible.
P-250 Quantitative Fluorescent PCR Using Short Tandem Repeat (STR) Markers in Preimplantation Genetic Diagnosis. 1R. Poverini, 2G. Di Cola, 1L. Rienzi, 1F. Ubaldi, 1E. Greco. 1Reproductive Medicine, European Hospital, Rome, 2Bio-Tech Lab, Parma, Italy. Objectives: The preimplantation genetic diagnosis of aneuploidy permits the investigation of chromosomal status of embryos generated in vitro, allowing the selection of healthy embryos to be transferred. The quantitative fluorescent PCR has been proposed as an new technology in preimplantation genetic diagnosis; we have developed this technique to establish the number of 18, 21 and sex chromosomes present in one single blastomere using two different types of short tandem repeat (STR) markers specific for all of these chromosomes. In order to investigate if the multiplex PCR using (SRT) markers is affordable to generate a successful diagnosis of aneuploidies we studied, in a series of no viable embryos, the chromosomal status using two blastomeres from each embryos. Materials and Methods: Embryo biopsy and blastomere collection have been performed from 20 monospermic no viable embryos originated from patients treated with either intracytoplasmic sperm microinjection (ICSI) or in 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 sex chromosomes with two different STR specific markers for those chromosomes. The products of amplification have been analyzed with automatic DNA analyzer (ABI-310 Perkin Elmer). Results: The quantitative analysis showed 2 peaks with ratio 1:1 in normal embryos, one STR peak in homozygote embryo respectively in 11 and 6 cases; 3 embryos have shown trisomy 21 and 18 as proved by the ratio between the two peaks of approximately 2:1. Conclusions: Our findings showed that amplification of quantitative fluorescent PCR to a single cell may be used to identify aneuploidy of homozygote embryos and normal trisomic DNA samples with a good accuracy.
P-251 New Fixation Technique for Preimplantation Genetics Diagnosis. D. Dozortsev. Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI.
Vol. 74, No. 3, Suppl. 1, September 2000