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Remohi, Antonio Pellicer, Carmen Rubio. Inst Valenciano de Infertilidad, Valencia, Spain. Objective: The objective of the study was to assess the incidence and distribution of chromosomal abnormalities in day 3 embryos from severe oligozoospermic patients included in a Preimplantation Genetic Diagnosis (PGD) program. Design: This is a prospective study performed on 24 patients undergoing PGD because of implantation failure (IF, n11), recurrent miscarriage (RM, n7) and abnormal FISH (fluorescence “in situ” hybridization) re- sults on spermatozoa (n6). All the sperm samples had 5 mill sperma- tozoa/ml and all the women were 37 years of age. Results were compared to those of a control group formed of 12 PGD cycles in fertile couples undergoing PGD because of sex linked diseases, during the same period of time from 01/1/2001 to 28/2/2003. Materials and Methods: Embryo biopsy was performed at the 6-8 cell stage using acid tyrodes solution and one or two blastomeres were ob- tained. Interphase nuclei were analysed by FISH for chromosomes 13, 16, 18, 21, 22, X and Y (Vysis Ic., Downers Grove. IL). Embryos were co-cultured on a monolayer of endometrial epithelial cells and chromosoma- lly normal embryos were transferred on day 5 of development. Mosaicism was estimated in those embryos in which two cells were analyzed. Percent- ages of chromosomally abnormal embryos (aneuploid, haploid, polyploid and mosaic embryos) were compared among groups using the Fishers exact test p0.05). Results: Please see table 1. p values for table 1 are as follows; a p0.05 vs control group; b p0.05 vs abnormal FISH group Conclusion: In the three study groups a high incidence of chromosomally abnormal embryos was observed. The distribution of abnormalities was different according to the reproductive background of the couple, with significantly increased incidence of mosaic embryos and embryos with aneuploidies for sex chromosomes in couples with abnormal FISH results on sperm compared to the other groups. P-224 Preimplantation Genetic Diagnosis (PGD) for aneuploidy in women under 38 yrs of age on 142 embryos from 13 initiated cycles using Fluorescence In Situ Hybridization (FISH). W G. Kearns, Jennifer Carter, Alana Davis, Kevin S. Richter, James Graham, Robert J. Stillman. Ctr for Preimplantation Genetics (CPG), Rockville, MD; Shady Grove Fertility Reproductive Science Ctr (SGFRSC), Rockville, MD. Objective: It is well recognized that women 37 yrs of age are at high risk of producing aneuploid embryos due to aneuploid oocytes. However, women under 38 yrs, with 2 miscarriages or unsuccessful in vitro fertilization (IVF) cycles and a partner with normal semen parameters, may also be at risk of producing an aneuploid embryo due to the presence of an unexpected number of aneuploid oocytes. We evaluated the usefulness of PGD-IVF for women under 38 experiencing repeated miscarriage or un- successful IVF cycles. Design: Preliminary retrospective study. Materials and Methods: PGD-IVF was performed on couples with a maternal age under 38, with 2 miscarriages or unsuccessful IVF cycles. The control group (IVF) comprised women under 38 with 2 unsuccessful IVF cycles undergoing routine IVF at SGFRSC (n105). PGD was per- formed on blastomeres from 142 cleaving embryos from 13 initiated cycles prior to 13 embryo transfers. The mean maternal age was 33.5 years with a range between 31 and 37. Laser-assisted embryo biopsy and multi-probe, multi-color FISH were used to determine aneuploidy for chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y. One blastomere from each 4-9 cell preimplantation embryo was isolated and fixed onto microscope slides. Hybridization and stringency washes were performed according to routine laboratory protocols. Microscopy was performed using an epi-fluorescent microscope and multiple fluorescent signal detection was accomplished using a SpectralCube imaging system and multiple excitation filters. Images were captured using a cooled CCD camera controlled by Spectral Imaging 2.6 software. Results: Twenty-one percent (30/142) of embryos were diploid and 21 embryos were transferred for 13 couples. Nineteen percent (27/142) of embryos were not diagnosed because of cell fragmentation due to poor embryo quality. Numerical chromosome abnormalities were found in 60% of the analyzed embryos. Of these aneuploid embryos, chromosome abnor- malities ranged from aneuploidy of a single chromosome to abnormalities for all 10 chromosomes. Transfer occurred for all couples. Sixty-two per- cent (8/13) of women achieved a clinical pregnancy (CP) with an implan- tation rate (# sacs/# embryos transferred) (IR) of 43 % (9/21). Low sample sizes in the PGD-IVF group limited the usefulness of statistical compari- sons. Although not significant at the 0.05 alpha level, the p-value of 0.085 comparing implantation rates is suggestive of a benefit of PGD for women 38 yrs. PGD-IVF IR 43% * CP 62% * p 0.085 (vs SGFRSC control) IVF-SGFRSC 2 unsuccessful IVF cycles IR 20% CP 46% Conclusions: Results from this preliminary study, while statistically in- conclusive, suggest that PGD-IVF in couples due to 2 recurrent miscar- riages or unsuccessful IVF cycles may increase the implantation rate and the possibility of achieving a pregnancy unaffected by aneuploidy and a child free of a genetic disorder. P-225 Developmental potential of pre-embryos carrying a chromosomal translocation. Richard J. Bodine, Kangpu Xu, Zhen Ye, Jason Park, Nikica Zaninovic, Lucinda L. Veeck. Cornell Univ Medical Coll, New York, NY. Objective: Preimplantation genetic diagnosis (PGD) offers a means of detecting selected chromosomal abnormalities. Here we examined growth characteristics at different intervals in preembryos possessing normal/bal- anced and unbalanced translocations. Design: A retrospective analysis of 38 treatment cycles (January 2000- Febuary 2003) examining 213 preembryos after PGD was performed for balanced translocation. Materials and Methods: All biopsied preembryos in these treatment cycles were divided into one of two groups: Normal/Balanced or Unbal- anced. Each of the preembryos were then analyzed at three different intervals on Days 2, 3 and 5. On Day 5, preembryos were split into two groups, those that had only developed to the morula stage and those that had developed into blastocysts. Fisher’s Exact Test was used for statistical analysis. Results: There was a significant difference in the rate of development between normal/balanced and unbalanced preembryos and Days 2 and 5. On Day 2, the normal/balanced group had reached the 4-cell stage or better 75% (58/77) of the time while the unbalanced group reached this stage 57% (77/136) of the time, yielding a p-value of 0.0076. On D5, normal/balanced preembryos reached the blastocyst stage 27% (21/77) of the time while the unbalanced group reached it only 9.6% (13/136) of the time (p0.0015). The difference between the normal/balanced and unbalanced groups reach- ing the 8-cell stage on D3 was not statistically significant. Although morulae were more often observed on D5 in the normal/balanced group its value did not reach significance. Conclusions: Preembryos with unbalanced translocations have a high miscarrage rate after implantation and may cause severe birth defects if they implant and go to term. PGD is performed in conjunction with IVF on patients’ preembryos when the parents themselves are known to possess balanced translocations. This study demonstrates that normal/balanced preembryos may actually develop more optimally than do their unbalanced counterparts. Although the results are not statistically significant at each consecutive stage, the percentage of normal/balanced preembryos showing optimum development is always higher than for unbalanced ones. With more data we expect that the stages not achieving statistical significance will S196 Abstracts Vol. 80, Suppl. 3, September 2003

Developmental potential of pre-embryos carrying a chromosomal translocation

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Remohi, Antonio Pellicer, Carmen Rubio. Inst Valenciano de Infertilidad,Valencia, Spain.

Objective: The objective of the study was to assess the incidence anddistribution of chromosomal abnormalities in day 3 embryos from severeoligozoospermic patients included in a Preimplantation Genetic Diagnosis(PGD) program.

Design: This is a prospective study performed on 24 patients undergoingPGD because of implantation failure (IF, n�11), recurrent miscarriage(RM, n�7) and abnormal FISH (fluorescence “in situ” hybridization) re-sults on spermatozoa (n�6). All the sperm samples had � 5 mill sperma-tozoa/ml and all the women were � 37 years of age. Results were comparedto those of a control group formed of 12 PGD cycles in fertile couplesundergoing PGD because of sex linked diseases, during the same period oftime from 01/1/2001 to 28/2/2003.

Materials and Methods: Embryo biopsy was performed at the 6-8 cellstage using acid tyrode�s solution and one or two blastomeres were ob-tained. Interphase nuclei were analysed by FISH for chromosomes 13, 16,18, 21, 22, X and Y (Vysis Ic., Downers Grove. IL). Embryos wereco-cultured on a monolayer of endometrial epithelial cells and chromosoma-lly normal embryos were transferred on day 5 of development. Mosaicismwas estimated in those embryos in which two cells were analyzed. Percent-ages of chromosomally abnormal embryos (aneuploid, haploid, polyploidand mosaic embryos) were compared among groups using the Fisher�s exacttest p�0.05).

Results: Please see table 1.

p values for table 1 are as follows; ap�0.05 vs control group; bp�0.05 vsabnormal FISH group

Conclusion: In the three study groups a high incidence of chromosomallyabnormal embryos was observed. The distribution of abnormalities wasdifferent according to the reproductive background of the couple, withsignificantly increased incidence of mosaic embryos and embryos withaneuploidies for sex chromosomes in couples with abnormal FISH resultson sperm compared to the other groups.

P-224

Preimplantation Genetic Diagnosis (PGD) for aneuploidy in womenunder 38 yrs of age on 142 embryos from 13 initiated cycles usingFluorescence In Situ Hybridization (FISH). W G. Kearns, JenniferCarter, Alana Davis, Kevin S. Richter, James Graham, Robert J. Stillman.Ctr for Preimplantation Genetics (CPG), Rockville, MD; Shady GroveFertility Reproductive Science Ctr (SGFRSC), Rockville, MD.

Objective: It is well recognized that women � 37 yrs of age are at highrisk of producing aneuploid embryos due to aneuploid oocytes. However,women under 38 yrs, with � 2 miscarriages or unsuccessful in vitrofertilization (IVF) cycles and a partner with normal semen parameters, mayalso be at risk of producing an aneuploid embryo due to the presence of anunexpected number of aneuploid oocytes. We evaluated the usefulness ofPGD-IVF for women under 38 experiencing repeated miscarriage or un-successful IVF cycles.

Design: Preliminary retrospective study.Materials and Methods: PGD-IVF was performed on couples with a

maternal age under 38, with � 2 miscarriages or unsuccessful IVF cycles.The control group (IVF) comprised women under 38 with � 2 unsuccessfulIVF cycles undergoing routine IVF at SGFRSC (n�105). PGD was per-formed on blastomeres from 142 cleaving embryos from 13 initiated cyclesprior to 13 embryo transfers. The mean maternal age was 33.5 years with arange between 31 and 37. Laser-assisted embryo biopsy and multi-probe,multi-color FISH were used to determine aneuploidy for chromosomes 13,14, 15, 16, 17, 18, 21, 22, X and Y. One blastomere from each 4-9 cellpreimplantation embryo was isolated and fixed onto microscope slides.

Hybridization and stringency washes were performed according to routinelaboratory protocols. Microscopy was performed using an epi-fluorescentmicroscope and multiple fluorescent signal detection was accomplishedusing a SpectralCube imaging system and multiple excitation filters. Imageswere captured using a cooled CCD camera controlled by Spectral Imaging2.6 software.

Results: Twenty-one percent (30/142) of embryos were diploid and 21embryos were transferred for 13 couples. Nineteen percent (27/142) ofembryos were not diagnosed because of cell fragmentation due to poorembryo quality. Numerical chromosome abnormalities were found in 60%of the analyzed embryos. Of these aneuploid embryos, chromosome abnor-malities ranged from aneuploidy of a single chromosome to abnormalitiesfor all 10 chromosomes. Transfer occurred for all couples. Sixty-two per-cent (8/13) of women achieved a clinical pregnancy (CP) with an implan-tation rate (# sacs/# embryos transferred) (IR) of 43 % (9/21). Low samplesizes in the PGD-IVF group limited the usefulness of statistical compari-sons. Although not significant at the 0.05 alpha level, the p-value of 0.085comparing implantation rates is suggestive of a benefit of PGD forwomen � 38 yrs.PGD-IVF IR � 43%*

CP � 62%*p � 0.085 (vs SGFRSC control)IVF-SGFRSC� 2 unsuccessful IVF cyclesIR � 20%CP � 46%

Conclusions: Results from this preliminary study, while statistically in-conclusive, suggest that PGD-IVF in couples due to � 2 recurrent miscar-riages or unsuccessful IVF cycles may increase the implantation rate and thepossibility of achieving a pregnancy unaffected by aneuploidy and a childfree of a genetic disorder.

P-225

Developmental potential of pre-embryos carrying a chromosomaltranslocation. Richard J. Bodine, Kangpu Xu, Zhen Ye, Jason Park, NikicaZaninovic, Lucinda L. Veeck. Cornell Univ Medical Coll, New York, NY.

Objective: Preimplantation genetic diagnosis (PGD) offers a means ofdetecting selected chromosomal abnormalities. Here we examined growthcharacteristics at different intervals in preembryos possessing normal/bal-anced and unbalanced translocations.

Design: A retrospective analysis of 38 treatment cycles (January 2000-Febuary 2003) examining 213 preembryos after PGD was performed forbalanced translocation.

Materials and Methods: All biopsied preembryos in these treatmentcycles were divided into one of two groups: Normal/Balanced or Unbal-anced. Each of the preembryos were then analyzed at three differentintervals on Days 2, 3 and 5. On Day 5, preembryos were split into twogroups, those that had only developed to the morula stage and those that haddeveloped into blastocysts. Fisher’s Exact Test was used for statisticalanalysis.

Results: There was a significant difference in the rate of developmentbetween normal/balanced and unbalanced preembryos and Days 2 and 5. OnDay 2, the normal/balanced group had reached the 4-cell stage or better 75%(58/77) of the time while the unbalanced group reached this stage 57%(77/136) of the time, yielding a p-value of 0.0076. On D5, normal/balancedpreembryos reached the blastocyst stage 27% (21/77) of the time while theunbalanced group reached it only 9.6% (13/136) of the time (p�0.0015).The difference between the normal/balanced and unbalanced groups reach-ing the 8-cell stage on D3 was not statistically significant. Although morulaewere more often observed on D5 in the normal/balanced group its value didnot reach significance.

Conclusions: Preembryos with unbalanced translocations have a highmiscarrage rate after implantation and may cause severe birth defects if theyimplant and go to term. PGD is performed in conjunction with IVF onpatients’ preembryos when the parents themselves are known to possessbalanced translocations. This study demonstrates that normal/balancedpreembryos may actually develop more optimally than do their unbalancedcounterparts. Although the results are not statistically significant at eachconsecutive stage, the percentage of normal/balanced preembryos showingoptimum development is always higher than for unbalanced ones. Withmore data we expect that the stages not achieving statistical significance will

S196 Abstracts Vol. 80, Suppl. 3, September 2003

do so. The group that demonstrated the greatest statistical significance wasthe most developmentally advanced group of blastocysts.

P-226

Preimplantation Genetic Diagnosis (PGD) for aneuploidy on 381 em-bryos from 39 initiated cycles using Fluorescence In Situ Hybridization(FISH). W G. Kearns, James Graham, Taer Han, Kevin Richter, MichaelJ. Tucker, Eric Widra. Ctr for Preimplantation Genetics (CPG), Rockville,MD; Shady Grove Fertility Reproductive Science Ctr (SGFRSC), Rock-ville, MD; SGFRSC, Georgia Reproductive Specialists, Atlanta, GA; SG-FRSC, Rockville, MD.

Objective: PGD was performed in an attempt to increase the likelihood ofachieving a pregnancy unaffected by aneuploidy and a child free of agenetic disorder following in vitro fertilization (IVF). Patients underwentPGD due to � 2 recurrent miscarriages or unsuccessful in vitro fertilization(IVF) cycles.

Design: Prospective study.Materials and Methods: PGD-IVF was performed on couples with � 2

miscarriages or unsuccessful IVF cycles. The control group (IVF) com-prised couples with � 2 unsuccessful IVF cycles undergoing routine IVF atSGFRSC. PGD was performed on blastomeres from 381 cleaving embryosfrom 39 initiated cycles prior to 32 embryo transfers. The mean maternalage was 38.1 years with a range between 31 and 44. Laser-assisted embryobiopsy and multi-probe, multi-color FISH were used to determine aneu-ploidy for chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y. Oneblastomere from each 4-9 cell preimplantation embryo was isolated andfixed onto microscope slides. Hybridization and stringency washes wereperformed according to routine laboratory protocols. Microscopy was per-formed using an epi-fluorescent microscope and multiple fluorescent signaldetection was accomplished using a SpectralCube imaging system andmultiple excitation filters. Images were captured using a cooled CCDcamera controlled by Spectral Imaging 2.6 software.

Results: Seventeen percent (64/381) of embryos were diploid and 52embryos were transferred for 32 couples. Fourteen percent (53/381) ofembryos were not diagnosed because of cell fragmentation due to poorembryo quality. Numerical chromosome abnormalities were found in 69%of embryos tested. Of these aneuploid embryos, chromosome abnormalitiesranged from aneuploidy of a single chromosome to abnormalities of all 10chromosomes. No transfer occurred for seven couples due to a chromosomeabnormality in all embryos analyzed. Forty-one percent (13/32) of womenachieved a clinical pregnancy with an implantation rate of 32% (16/50).Statistical comparisons of implantation and pregnancy rates were madebetween PGD-CPG and IVF-SGFRSC within each age group. Implantationrates were significantly higher (p � 0.038) for the PGD group within the 35to 37 yr old age category. Although the comparisons of implantation rateswere not significant within the other age categories at the current lowsample sizes for the PGD group, the higher implantation rates for each agegroup is promising.

Conclusions: Results from this preliminary study, while statistically in-conclusive, suggest that PGD-IVF in couples due to � 2 recurrent miscar-riages or unsuccessful IVF cycles may increase the implantation rate and thepossibility of achieving a pregnancy unaffected by aneuploidy and a childfree of a genetic disorder.

P-227

Embryo development post laser biopsy for PGD. Jennifer Carter, JamesGraham, Taer Han, Alana Davis, William G. Kearns, Michael Tucker.Shady Grove Fertility Reproductive Science Ctr, Rockville, MD; ShadyGrove Ctr for Preimplantation Genetics, Rockville, MD; Georgia Repro-ductive Specialists, Atlanta, GA.

Objective: To assess the effect of laser biopsy for preimplantation geneticdiagnosis (PGD) on embryo development.

Design: A retrospective analysis comparing development betweenmatched biopsied and non-biopsied embryos.

Materials and methods: There were 36 patients in the biopsy group and56 controls. Embryo development was observed from day 3 through day 5in both groups, with the biopsy group having undergone blastomere biopsyof one to two cells on day 3, and the control group undergoing no laser orbiopsy procedures. Zona pellucida (ZP) dissection was undertaken with aClass 1 1.48�m wavelength IR laser, using a double pulsed 1.0msec burstwith 140mW power at the target (Zilos Laser System, Hamilton ThorneResearch, Beverly, MA, USA). Transfer of genetically normal embryos wascarried out on day 5, and no further development was recorded for themajority of embryos due to embryo arrest or genetically abnormal resultsyielding embryos unsuitable for cryopreservation. Control patients werematched with the biopsy group for both age and insemination method(Conv�conventional IVF; ICSI�intracytoplasmic sperm injection). Bothgroups consisted of patients undergoing IVF treatment during the laboratoryseries from August 2002 to April 2003. Maternal ages given for the patientgroups are averages; actual ranges were 26-44 years for the biopsy group,and 26-43 years for the control group.

Results: # Embryos refers to the average number that underwent biopsyin the biopsy group, and the number continuing development through day 5in both groups. % Prog refers to the percentage of embryos that showedforward development from day 3 to day 5; % Arrest refers to the percentageof embryos that showed no change in development; and % Deg refers to thepercentage of embryos that degenerated during this time.

Conclusions: Laser-assisted biopsy with the removal of one to twoblastomeres for the purpose of PGD analysis does not appear to have adetrimental effect on the continued general development of those embryos,when compared to embryos from patients of comparable ages and insemi-nation methods not undergoing any laser or biopsy procedures.

P-228

A prospective, randomized study to compare acidified Tyrode’s zonadrilling with laser-assisted hatching for day-3 embryo biopsy followedby blastocyst culture. Amy E. Jones, Graham W. Wright, Cathleen A.Davidson-Garcia, Thomas A. Elliott, Hilton I. Kort, Zsolt P. Nagy. Repro-ductive Biology Assoc, Atlanta, GA.

Objectives: Removal of a single blastomere for preimplantation geneticdiagnosis (PGD) from day-3 embryo is a well established method. However,it is unknown whether the method of choice for zona pellucida (ZP) drillingfor the biopsy procedure will influence further embryonic development. Themost commonly applied technique for perforation of the ZP is zona drillingusing acidified Tyrode’s solution (AT), while only a few centers employlaser-assisted hatching (LAH). Because of the lack of studies comparing thetwo methods, many centers are reluctant to switch to the simpler and fasterlaser drilling. Thus, in the present study we assessed two zona drillingmethods in terms of blastocyst development rates using sister embryos.

Design: Prospective, randomized study. Sister embryos of 14 patientswere randomly assigned to (1) AT zona drilling or to (2) laser zona drillingon day 3. After biopsy, subsequent embryo culture until the blastocyst stage(day 5) was performed.

Materials and Methods: A total of 14 IVF-PGD cycles were included intothe study that was performed between January and March of 2003. Patientsunderwent controlled ovarian hyperstimulation using recombinant FSHafter pituitary down-regulation with long leuprolide acetate protocol. Em-bryos from the same patients (eligible for biopsy: �5 cells and �30%fragmentation) were randomly divided into two groups on day 3. In Group1 embryos were submitted to Acidified Tyrode’s zona drilling and in Group2 embryos underwent laser-assisted hatching using a 1.48 �m diode laser(Octax). In both groups an opening of the zona of 15 to 20 micro-meter wasformed followed by the removal of a single blastomere. Embryos werecultured sequentially in Sage cleavage and blastocyst medium until day 5.For statistical analysis One-way ANOVA, Kruskal-Wallis and chi-squaretests were applied whenever appropriate.

Results: The mean age (�S.D.) of the women in this study was 36.9(�3.9). A total of 285 oocytes were recovered, and 175 oocytes fertilized

FERTILITY & STERILITY� S197