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P▪51 Polar body diagnosis in translocation carriers
Abstracts - 6th International Symposium on Preimplantation Genetics 2005
P 51 Polar body diagnosis in translocation carriers Clement-Sengewald A1,4, Würfel W2, Seifert B3, Bals-Pratsch M3, Lesoine B4, Berg FD4, Buchholz T1 1Centre for Polar Body Diagnosis, Planegg; 2KinderwunschCentrum, Munich; 3GMP Seifert/Bals-Pratsch; Regensburg; 4GMP Berg/Lesoine, Munich; Germany Objective: To offer an additional option for treatment to patients with balanced translocations after recurrent IVF failures or recurrent abortions. Materials and methods: First and second polar bodies were extracted 6 hours after intracytoplasmic sperm injection (ICSI) from 11 patients (10 with reciprocal translocations, and one with Robertson translocation) after zona opening with acid Tyrode´s solution, and fixed on slides for fluorescence in-situ hybridization (FISH) with two or three commercial probes. Results: One hundred and nine first and second polar bodies were extracted out of 75 oocytes. Clear FISH results could be obtained in 64 oocytes (83%). Twenty-seven oocytes were balanced (43%), and 37 were unbalanced (57%). Fourteen embryos were transferred in nine cycles, resulting in three pregnancies. Unfortunately, all three patients miscarried in the first trimenon. One abortion material was not examined, one was karyotypically normal and one showed a trisomy 9. Conclusions: Using commercial probes, translocation diagnosis is limited. Interchromosomal effects, resulting in an increased rate of aneuploidies in translocation carriers, cannot be excluded.
P 52 PGD for complex cases of structural chromosome aberrations Escudero T, Fischer J, Cinnioglu C*, Munné, S Reprogenetics, 101 Old Short Hills Road, Suite 501, West Orange, NJ, 07052, USA Introduction: It is uncommon for each member of a couple to have a chromosome structural aberration, for an individual to have more than one such finding, or for an individual to have a complex chromosome rearrangement (CCR). These are complex cases and those in these situations are unlikely to have a pregnancy. Materials and methods: A couple in which one member had an inversion and the other had a reciprocal translocation, two patients each with both a Robertsonian and a reciprocal translocation, and a patient with a CCR presented for preimplantation genetic diagnosis (PGD). One hundred and ten embryos were biopsied in 11 PGD cycles for these patients. Results: Testing of embryos found 10 to be normal/ balanced for the respective chromosome aberrations, 83 to be unbalanced, 13 to have abnormalities unrelated to the known aberrations, and three yielded no result. Five of the 11 cycles had no embryo transfer. Only two of the six cycles with transfer were successful, resulting in three children. Conclusions: Though uncommon, complex cases of structural aberrations can lend themselves to PGD.
P 53 Dual colour sperm FISH of a male with a non-Robertsonian translocation 46,XY,t(11;22)(q23;q11) Deveci D, Etem E, Deveci S, Yüce H Introduction: Translocation 46,XY,t(11;22)(q23;q11) is the most common reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility and increased spontaneous abortion rate. Materials and methods: We show the presence of t(11;22) (q23;q11) using standard cytogenetic procedures. Blood samples were obtained from the proband and his family. Fluorescence in situ hybridization (FISH) with specific DNA probes was used to determine the chromosome segregation in sperm translocation of a t(11;22)(q23;q11) in the propositus. The probes selected included a centromere specific for chromosome 11 and a second probe distal to the translocation breakpoint of chromosome 22. Two hundred cells were counted to determine the segregation proportion Results: Parental chromosome analysis performed as result of family history demonstrated that the father carried a balanced reciprocal translocation with the karyotype 46,XY,t(11;22)(q23;q11) in all the metaphases examined. The sperm FISH results showed 3:1 segregation in 26% of spermatozoa in this patient. Conclusions: Molecular cytogenetic analysis of chromosome 11 and 22 using sperm FISH revealed that in this t(11;22) carrier there was 3:1 segregation consistent with the general segregation proportion in balanced chromosomal carriers.
P 54 Development of conventional blastomere metaphase chromosome analysis for PGD Ilkevitch Y, Strelchenko N, Verlinsky Y Reproductive Genetics Institute, Chicago, USA We have previously developed the method for blastomere interphase nucleus conversion into metaphase by its fusion with mouse zygote, which was used for preimplantation genetic diagnostic (PGD) for translocations. Despite acceptable efficiency of obtaining metaphase spreads, this method is still a labour-intensive procedure, so we have now developed a relatively simple non-conversion procedure for visualization of metaphase chromosomes, which is based on the premature chromosome condensation (PCC) of isolated blastomeres obtained from the cleavage-stage human embryo. PCC is induced chemically by a short culture of human blastomeres with phosphatase inhibitor (PP1) Calyculin A (80 nM) or Tautomycin (500 ng/ml) in the presence of nocodazole and cytochalasin D. Our results showed that Calyculin A produced metaphase chromosomes in 47 (50%) of 94 blastomeres cultured for period of 6 h. In contrast, Tautomycin was capable converting the blastomere interphase nucleus into metaphase in only 14 (29%) of 48 blastomeres. So the data suggest that Calyculin may be used for chemical induction of PCC in generating chromosomal preparations from biopsied blastomeres.
P 55 Development of preconception
47
P 51 Polar body diagnosis in translocation carriers Clement-Sengewald A1,4, Würfel W2, Seifert B3, Bals-Pratsch M3, Lesoine B4, Berg FD4, Buchholz T1 1Centre for Polar Body Diagnosis, Planegg; 2KinderwunschCentrum, Munich; 3GMP Seifert/Bals-Pratsch; Regensburg; 4GMP Berg/Lesoine, Munich; Germany Objective: To offer an additional option for treatment to patients with balanced translocations after recurrent IVF failures or recurrent abortions. Materials and methods: First and second polar bodies were extracted 6 hours after intracytoplasmic sperm injection (ICSI) from 11 patients (10 with reciprocal translocations, and one with Robertson translocation) after zona opening with acid Tyrode´s solution, and fixed on slides for fluorescence in-situ hybridization (FISH) with two or three commercial probes. Results: One hundred and nine first and second polar bodies were extracted out of 75 oocytes. Clear FISH results could be obtained in 64 oocytes (83%). Twenty-seven oocytes were balanced (43%), and 37 were unbalanced (57%). Fourteen embryos were transferred in nine cycles, resulting in three pregnancies. Unfortunately, all three patients miscarried in the first trimenon. One abortion material was not examined, one was karyotypically normal and one showed a trisomy 9. Conclusions: Using commercial probes, translocation diagnosis is limited. Interchromosomal effects, resulting in an increased rate of aneuploidies in translocation carriers, cannot be excluded.
P 52 PGD for complex cases of structural chromosome aberrations Escudero T, Fischer J, Cinnioglu C*, Munné, S Reprogenetics, 101 Old Short Hills Road, Suite 501, West Orange, NJ, 07052, USA Introduction: It is uncommon for each member of a couple to have a chromosome structural aberration, for an individual to have more than one such finding, or for an individual to have a complex chromosome rearrangement (CCR). These are complex cases and those in these situations are unlikely to have a pregnancy. Materials and methods: A couple in which one member had an inversion and the other had a reciprocal translocation, two patients each with both a Robertsonian and a reciprocal translocation, and a patient with a CCR presented for preimplantation genetic diagnosis (PGD). One hundred and ten embryos were biopsied in 11 PGD cycles for these patients. Results: Testing of embryos found 10 to be normal/ balanced for the respective chromosome aberrations, 83 to be unbalanced, 13 to have abnormalities unrelated to the known aberrations, and three yielded no result. Five of the 11 cycles had no embryo transfer. Only two of the six cycles with transfer were successful, resulting in three children. Conclusions: Though uncommon, complex cases of structural aberrations can lend themselves to PGD.
P 53 Dual colour sperm FISH of a male with a non-Robertsonian translocation 46,XY,t(11;22)(q23;q11) Deveci D, Etem E, Deveci S, Yüce H Introduction: Translocation 46,XY,t(11;22)(q23;q11) is the most common reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility and increased spontaneous abortion rate. Materials and methods: We show the presence of t(11;22) (q23;q11) using standard cytogenetic procedures. Blood samples were obtained from the proband and his family. Fluorescence in situ hybridization (FISH) with specific DNA probes was used to determine the chromosome segregation in sperm translocation of a t(11;22)(q23;q11) in the propositus. The probes selected included a centromere specific for chromosome 11 and a second probe distal to the translocation breakpoint of chromosome 22. Two hundred cells were counted to determine the segregation proportion Results: Parental chromosome analysis performed as result of family history demonstrated that the father carried a balanced reciprocal translocation with the karyotype 46,XY,t(11;22)(q23;q11) in all the metaphases examined. The sperm FISH results showed 3:1 segregation in 26% of spermatozoa in this patient. Conclusions: Molecular cytogenetic analysis of chromosome 11 and 22 using sperm FISH revealed that in this t(11;22) carrier there was 3:1 segregation consistent with the general segregation proportion in balanced chromosomal carriers.
P 54 Development of conventional blastomere metaphase chromosome analysis for PGD Ilkevitch Y, Strelchenko N, Verlinsky Y Reproductive Genetics Institute, Chicago, USA We have previously developed the method for blastomere interphase nucleus conversion into metaphase by its fusion with mouse zygote, which was used for preimplantation genetic diagnostic (PGD) for translocations. Despite acceptable efficiency of obtaining metaphase spreads, this method is still a labour-intensive procedure, so we have now developed a relatively simple non-conversion procedure for visualization of metaphase chromosomes, which is based on the premature chromosome condensation (PCC) of isolated blastomeres obtained from the cleavage-stage human embryo. PCC is induced chemically by a short culture of human blastomeres with phosphatase inhibitor (PP1) Calyculin A (80 nM) or Tautomycin (500 ng/ml) in the presence of nocodazole and cytochalasin D. Our results showed that Calyculin A produced metaphase chromosomes in 47 (50%) of 94 blastomeres cultured for period of 6 h. In contrast, Tautomycin was capable converting the blastomere interphase nucleus into metaphase in only 14 (29%) of 48 blastomeres. So the data suggest that Calyculin may be used for chemical induction of PCC in generating chromosomal preparations from biopsied blastomeres.
P 55 Development of preconception
47