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Translocation Testing Prior to Embryo Transfer
Tuesday, October 24, 2000 2:30 P.M. O-133 Translocation Testing Prior to Embryo Transfer. S. Munne´, T. Escudero, M. Sandalinas, J. Cohen, 1M. Lee, 2L. Gianaroli. The Institute for Reproductive Medicine and Science of Saint Barnabas, Livingston, NJ 07052, 1The New England Clinic of Reproductive Medicine, Inc. Reading, MA 01867, 2SISMER, Bologna, Italy. Objective: To analyze the outcome of 72 cycles where preimplantation genetic diagnosis of translocation was performed. Design: Retrospective analysis of 54 couples (72 cycles) with one partner carrying a chromosome translocation. Material and methods: Translocation testing was performed using different approaches of fluorescence in-situ hybridization. The first approach was the use of chromosome painting probes in polar bodies. The second method used subtelomeric probes, enumerator probes or breakpoint-spanning probes in blastomeres. The third approach used chromosome painting probes on metaphase chromosomes after fusion of blastomeres with cow eggs. Results: Translocation testing can achieve a significant reduction in spontaneous abortion from 95% to 9%, however, the establishment of pregnancy was highly correlated with 50% or more embryos being chromosomally normal. Robertsonian translocation patients produce less abnormal gametes and more pregnancies than patients with reciprocal translocations. The new protocol for testing of translocations using telomeric probes has proved reliable with a 6% error rate. Conclusions: Translocation testing significantly reduces spontaneous abortion, both after polar body and blastomere biopsy. Pregnancy outcome strongly depends on the ratio of normal embryos. Robertsonian translocations are less likely to cause abnormalities in embryos than reciprocal translocations.
Tuesday, October 24, 2000 2:45 P.M. O-134 Association Between Spindle Assembly Checkpoint Gene Expression and Maternal Age in Human Oocytes. N. Steuerwald, 1,2D. Wells, 1S. Munne, 1T. Escudero, 1J. Cohen, 1C. A. Brenner. 1Institute for Reproductive Medicine and Science of Saint Barnabas, NJ, 2University College London, Department of Obstetrics/Gynecology. Objective: Maternal aging remains the overwhelming factor in the aetiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at metaphase. MAD2 and BUB1 genes encode conserved kinetochore-associated proteins believed to be major components of this regulatory pathway. A failure in this surveillance system could lead to genomic instability, which may underlie the increased incidence of aneuploidy in oocytes of older women. Design: The concentrations of MAD2 and BUB1 transcripts in human oocytes at GV, MI and MII stages of maturation were determined by real-time rapid cycle fluorescent RT-PCR. The incidence of aneuploidy was established by multi-probe FISH analysis. Materials and Methods: Nonviable human mature and immature oocytes (n568) were obtained from patients undergoing IVF. Rapid fluorescence monitored cycling was used to examine expression levels of the checkpoint and housekeeping genes in individual human oocytes. Unknown concentrations were extrapolated from standards co-amplified producing a standard curve. Polar body biopsy, fixation and multi-probe FISH analysis was performed to determine chromosomal status of MII oocytes. Results: Regression analysis confirmed that a significant linear correlation exists between maternal age and MAD2 transcript concentration for GV, MI and MII stages of maturation. Likewise, a similar trend emerged when the BUB1 transcript copy numbers were plotted versus maternal age but only for GV and MI stages. No association was apparent between maternal age and the concentration of b-actin mRNA. Experiments are in progress comparing the incidence of aneuploidy and checkpoint transcript concentrations. However, preliminary results suggest that an association may exist between aneuploidy status and MAD2 transcript concentrations.
FERTILITY & STERILITYt
Conclusions: The observations reported here suggest that the MAD2 and BUB1 transcripts may degrade as the oocyte ages. Since the concentration of housekeeping transcripts remained constant throughout each stage of maturation examined, it appears that this phenomenon is not universal of all transcripts in oocytes from older women. Potentially, the degradation of these messages may impair spindle checkpoint function in older oocytes and be a contributing factor in age-related aneuploidy. Understanding the checkpoint defects associated with chromosomal abnormalities may have profound therapeutic consequences for older women undergoing assisted reproduction.
Tuesday, October 24, 2000 3:00 P.M. O-135 Analysis of Oct-4 Expression and Ploidy in Individual Human Blastomeres. C. Hansis, Y. X. Tang, L. Chi, J. A. Grifo, L. C. Krey. Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Oct-4, a marker for totipotent cells, is upregulated by 101 fold in the inner cell mass (ICM) of human blastocysts. Different levels of Oct-4 expression in the blastomeres of 4 – 8 cell embryos might predict development towards ICM (Oct-4 1) or trophectoderm (Oct-4 2). The object of this study is to determine simultaneously Oct-4 expression and chromosome number in individual blastomeres of human embryos. Design: Human blastomeres were individually separated into nuclear and cytoplasmic lysate fractions. The nucleus was submitted to FISH for chromosomes X, Y and 18 and the lysate was analyzed by RT-PCR for Oct-4 expression. Materials and Methods: Fifteen embryos (4 – 8 cells) were discarded with patient consent on day 3 or 4 and dissociated individually into 85 blastomeres by removing the zona pellucida with Acid Tyrode’s solution. Individual blastomeres were incubated in a cell membrane lysis buffer; the nucleus was identified by phase contrast microscopy, transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X, Y and 18 CEP direct-labeled probes (Vysis) for 30min in HYBrite. Oct-4 expression was assessed by submitting the cytosol to single cell, nested RT-PCR reaction (Gibco BRL; Boehringer Mannheim). The sensitivity of this assay was determined by a dilution series of positive blastomeres; b-actin served as a control gene. Results: b-Actin was expressed in 94% of the blastomeres tested and 46% of these were also positive for Oct-4. In these blastomeres Oct-4 expression was up-regulated by a mean of 10-fold. The frequency of cells expressing Oct-4 decreased with increasing blastomere number. Although all embryos derived from 2pn zygotes, only 60% of the blastomeres were euploid. Oct-4 expression was detected in euploid and in multinucleated blastomeres. Oct-4 was also detected in 2 of 3 cells with an excess chromosome X or 18 but not in 5 cells that lacked a normal complement of chromosomes X, Y or 18. Conclusions: Individual blastomeres can be evaluated for Oct-4 expression and ploidy thereby providing the means for a more complete understanding of mosaic aneuploidy. Significantly, the nuclear and cytosolic fractions can also be assayed for any combination of ploidy, genetic mutations and their respective expression vectors by simultaneous FISH, genomic PCR and RT-PCR, respectively. In addition, further characterization of gene expression in the cytosolic fractions of Oct-4 1 and Oct-4 2 cells will provide information about key steps in differentiation during early embryonic development.
ASSISTED REPRODUCTIVE TECHNOLOGY FROZEN EMBRYO TRANSFER Tuesday, October 24, 2000 3:45 P.M. O-136 Increased Clinical Pregnancy Rates from Frozen Thawed Human Blastocysts Using a Rapid Dilution and Transfer Protocol. 1L. Scott, 2M. P. Leondires, 2R. J. Alvero, 2B. Miller. 1The ART Institute of Washington DC at Walter Reed Army Medical Center Washington, DC and 2Combined Federal Program in Reproductive Endocrinology, Bethesda, MD.
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Tuesday, October 24, 2000 2:45 P.M. O-134 Association Between Spindle Assembly Checkpoint Gene Expression and Maternal Age in Human Oocytes. N. Steuerwald, 1,2D. Wells, 1S. Munne, 1T. Escudero, 1J. Cohen, 1C. A. Brenner. 1Institute for Reproductive Medicine and Science of Saint Barnabas, NJ, 2University College London, Department of Obstetrics/Gynecology. Objective: Maternal aging remains the overwhelming factor in the aetiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at metaphase. MAD2 and BUB1 genes encode conserved kinetochore-associated proteins believed to be major components of this regulatory pathway. A failure in this surveillance system could lead to genomic instability, which may underlie the increased incidence of aneuploidy in oocytes of older women. Design: The concentrations of MAD2 and BUB1 transcripts in human oocytes at GV, MI and MII stages of maturation were determined by real-time rapid cycle fluorescent RT-PCR. The incidence of aneuploidy was established by multi-probe FISH analysis. Materials and Methods: Nonviable human mature and immature oocytes (n568) were obtained from patients undergoing IVF. Rapid fluorescence monitored cycling was used to examine expression levels of the checkpoint and housekeeping genes in individual human oocytes. Unknown concentrations were extrapolated from standards co-amplified producing a standard curve. Polar body biopsy, fixation and multi-probe FISH analysis was performed to determine chromosomal status of MII oocytes. Results: Regression analysis confirmed that a significant linear correlation exists between maternal age and MAD2 transcript concentration for GV, MI and MII stages of maturation. Likewise, a similar trend emerged when the BUB1 transcript copy numbers were plotted versus maternal age but only for GV and MI stages. No association was apparent between maternal age and the concentration of b-actin mRNA. Experiments are in progress comparing the incidence of aneuploidy and checkpoint transcript concentrations. However, preliminary results suggest that an association may exist between aneuploidy status and MAD2 transcript concentrations.
FERTILITY & STERILITYt
Conclusions: The observations reported here suggest that the MAD2 and BUB1 transcripts may degrade as the oocyte ages. Since the concentration of housekeeping transcripts remained constant throughout each stage of maturation examined, it appears that this phenomenon is not universal of all transcripts in oocytes from older women. Potentially, the degradation of these messages may impair spindle checkpoint function in older oocytes and be a contributing factor in age-related aneuploidy. Understanding the checkpoint defects associated with chromosomal abnormalities may have profound therapeutic consequences for older women undergoing assisted reproduction.
Tuesday, October 24, 2000 3:00 P.M. O-135 Analysis of Oct-4 Expression and Ploidy in Individual Human Blastomeres. C. Hansis, Y. X. Tang, L. Chi, J. A. Grifo, L. C. Krey. Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Oct-4, a marker for totipotent cells, is upregulated by 101 fold in the inner cell mass (ICM) of human blastocysts. Different levels of Oct-4 expression in the blastomeres of 4 – 8 cell embryos might predict development towards ICM (Oct-4 1) or trophectoderm (Oct-4 2). The object of this study is to determine simultaneously Oct-4 expression and chromosome number in individual blastomeres of human embryos. Design: Human blastomeres were individually separated into nuclear and cytoplasmic lysate fractions. The nucleus was submitted to FISH for chromosomes X, Y and 18 and the lysate was analyzed by RT-PCR for Oct-4 expression. Materials and Methods: Fifteen embryos (4 – 8 cells) were discarded with patient consent on day 3 or 4 and dissociated individually into 85 blastomeres by removing the zona pellucida with Acid Tyrode’s solution. Individual blastomeres were incubated in a cell membrane lysis buffer; the nucleus was identified by phase contrast microscopy, transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X, Y and 18 CEP direct-labeled probes (Vysis) for 30min in HYBrite. Oct-4 expression was assessed by submitting the cytosol to single cell, nested RT-PCR reaction (Gibco BRL; Boehringer Mannheim). The sensitivity of this assay was determined by a dilution series of positive blastomeres; b-actin served as a control gene. Results: b-Actin was expressed in 94% of the blastomeres tested and 46% of these were also positive for Oct-4. In these blastomeres Oct-4 expression was up-regulated by a mean of 10-fold. The frequency of cells expressing Oct-4 decreased with increasing blastomere number. Although all embryos derived from 2pn zygotes, only 60% of the blastomeres were euploid. Oct-4 expression was detected in euploid and in multinucleated blastomeres. Oct-4 was also detected in 2 of 3 cells with an excess chromosome X or 18 but not in 5 cells that lacked a normal complement of chromosomes X, Y or 18. Conclusions: Individual blastomeres can be evaluated for Oct-4 expression and ploidy thereby providing the means for a more complete understanding of mosaic aneuploidy. Significantly, the nuclear and cytosolic fractions can also be assayed for any combination of ploidy, genetic mutations and their respective expression vectors by simultaneous FISH, genomic PCR and RT-PCR, respectively. In addition, further characterization of gene expression in the cytosolic fractions of Oct-4 1 and Oct-4 2 cells will provide information about key steps in differentiation during early embryonic development.
ASSISTED REPRODUCTIVE TECHNOLOGY FROZEN EMBRYO TRANSFER Tuesday, October 24, 2000 3:45 P.M. O-136 Increased Clinical Pregnancy Rates from Frozen Thawed Human Blastocysts Using a Rapid Dilution and Transfer Protocol. 1L. Scott, 2M. P. Leondires, 2R. J. Alvero, 2B. Miller. 1The ART Institute of Washington DC at Walter Reed Army Medical Center Washington, DC and 2Combined Federal Program in Reproductive Endocrinology, Bethesda, MD.
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