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6.002 Case report: meiotic segregation pattern and preimplantation diagnosis in a Robertsonian translocation (13;21)
Abstracts - PGDIS: 8th International Symposium on PGD
6.001 Sperm FISH analysis of a familial complex chromosome rearrangement: predictive value on the outcome of PGD Loup V1, Bernicot TI2, Janssens P2, Pellestor F3, Lefort G2, Hamamah S3, Anahory T3 1CHU Arnaud de Villeneuve; 2Génétique, CHU Arnaud de Villeneuve; 3Biologie de la Reproduction/unité DPI, CHU Arnaud de Villeneuve, Montpellier, France Objective: Complex chromosome rearrangements (CCR) are balanced and unbalanced structural aberrations involving three or more breakpoints on two or more chromosomes. These CCR result in a high risk of abnormalities and they can have a significant impact on fertility. In this report, we analysed meiotic segregation in the spermatozoa of a patient with a familial CCR t(1;13;19)(p31;q31;q13) using fluorescence in-situ hybridization (FISH) techniques, in order to evaluate the reproductive possibility of this patient and to assess the usefulness of the PGD procedure. Materials/Methods: A t(1;13;19)(p31;q31;q13) was first identified in a 37-year-old man using conventional and molecular cytogenetic methods. This patient was included in an ICSI (intracytoplasmic sperm injection) programme because of oligoasthenoteratozoospermia. The sperm sample was prepared for FISH protocol. PRINS and FISH techniques were combined to allow the use of five fluorochromes on the same sperm cell preparation. The PRINS procedure used the primer J52 specific for the satellite III sequence of chromosome 1 labelled with biotin and detected with Alexafluor-350conjugated streptavidin. The FISH technique was performed using four probes: two sub-telomeric probes, 1pter (labelled in spectrum Orange) and 19qter (labelled in spectrum Aqua), the LSI FKHR probe (a mixture of two probes labelled in spectrum Green and in spectrum Orange) and the LSI 13q34 (labelled in spectrum Green) Results: Segregation analysis was performed on a total of 800 sperm nuclei. Normal and balanced spermatozoa were identified in 119 cases (14.9%) and unbalanced spermatozoa were observed in 681 cases (85.1%). The most frequent modes of segregation were 4:2 and 3:3. Conclusion: To the best of our knowledge, this is the first report on chromosome segregation analysis in human spermatozoa in a carrier of CCR using five fluorochromes. These results permit us to have a better understanding of mechanisms of meiotic segregation, thus leading to the improvement of genetic counselling for PGD. 6.002 Case report: meiotic segregation pattern and preimplantation diagnosis in a Robertsonian translocation (13;21) Fernández S1,2, Colomar A1,2, Toro E1,2, Casanovas A1, Serra O1, López-Teijón M1, Velilla E1,2 1Institut Marquès, Barcelona; 2Centre for Embryo Medicine, Madrid, Spain Objective: The aim of this study is to describe the meiotic segregation pattern of a rare male Robertsonian translocation carrier and the outcome of the PGS cycle. Materials/Methods: A couple (female, 34 years old; male, 36 years old) presented with a 2-year history of primary infertility. The male partner was a carrier of a familiar Robertsonian translocation (13; 21) and was diagnosed as having oligoasthenoteratozoospermia (0.13 million/ml and motility of 18.4%). To analyse the chromosome segregation
pattern, a sperm FISH analysis for chromosomes 13 and 21 was performed and chromosomes 18, X, Y were added to the FISH analysis to detect the interchromosomal effect (ICE). With the aim of selecting normal or balanced embryos in an IVF-PGS cycle, chromosomes 13 and 21 were studied and in order to detect the interchromosomal effect chromosomes X, Y, 15, 16, 17, 18 and 22 were included in the PGS cycle. Results: The segregation analysis of normal or balanced spermatozoa for chromosomes 13 and 21, resulting from alternant segregation pattern, was 91.8%, similar to that observed in the literature (Anton et al., 2004). The unbalanced chromosomal constitution resulting from adjacent segregation (nullisomies and disomies, 3:0 or diploids and others) was 8.2%. An interchromosomal effect has been detected by some authors (Anton et al., 2004; Ogur et al., 2006). The first IVFPGS cycle for this couple analysed two embryos and resulted in one normal or balanced embryo that was transferred at the morulae stage on day 4 of development without pregnancy. A second IVF-PGD cycle was performed; 18 metaphase II oocytes were collected, 12 were fertilized, eight embryos were analysed by FISH and three normal or balanced embryos were detected. Three embryos were transferred on day 4 of development: two embryos initiated compactation and one embryo was at the morulae stage. Pregnancy was achieved and two fetal heart beats were detected. Two healthy babies were born: one male (1990 g) and one female (2300 g). Conclusion: Robertsonian translocations (13; 21) represent only 2% of the overall Robertsonian translocations. Although it has been poorly reported, this translocation presents a similar segregation pattern to other Robertsonian rearrangements. It is recommended that an aneuploidy panel be added to the PGS cycle. 6.003 FISH analysis of chromosome segregation in spermatozoa of a t(1;15)(q21;p12) carrier: predictive test for PGD Loginova J, Chiryaeva O, Chanisheva O Russian–Finnish Clinic ‘AVA-PETER’, Saint-Petersburg, Russia Introduction: Balanced reciprocal translocations (RT) are the most common structural abnormalities. Men carrying RT often have fertility problems because of oligozoospermia or azoospermia. Even if their sperm count is normal, RT carriers are at increased risk for spontaneous abortions and chromosomally unbalanced offspring due to the production of spermatozoa with chromosomal imbalances. The frequency of unbalanced spermatozoa in RT carriers is 50% on average, but varies from 19% to more than 80% in different patients. The frequency depends on the chromosomes involved in the translocation, breakpoints and the patient’s genetic background. Often for RT carriers, IVF/intracytoplasmic sperm injection (ICSI) treatment with PGD is the only chance to have a healthy baby. An estimation of the overall sperm chromosomal imbalance of such patients could help to give couples valuable genetic counselling for making a resolve about PGD practicability. The aim of this study is to analyse meiotic segregation of chromosomes 1 and 15 in RT carrier spermatozoa to provide objective information for genetic counselling. Materials/Methods: A 41-year-old male patient – a carrier of the translocation 46,XY,t(1;15)(q21;p12), and his 40-year-old wife (46,XX) had infertility for 10 years, one miscarriage and two unsuccessful ICSI cycles without PGD. The male patient displayed cryptozoospermia – sperm count: 0.1 s 106/ml.
S-43 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
6.001 Sperm FISH analysis of a familial complex chromosome rearrangement: predictive value on the outcome of PGD Loup V1, Bernicot TI2, Janssens P2, Pellestor F3, Lefort G2, Hamamah S3, Anahory T3 1CHU Arnaud de Villeneuve; 2Génétique, CHU Arnaud de Villeneuve; 3Biologie de la Reproduction/unité DPI, CHU Arnaud de Villeneuve, Montpellier, France Objective: Complex chromosome rearrangements (CCR) are balanced and unbalanced structural aberrations involving three or more breakpoints on two or more chromosomes. These CCR result in a high risk of abnormalities and they can have a significant impact on fertility. In this report, we analysed meiotic segregation in the spermatozoa of a patient with a familial CCR t(1;13;19)(p31;q31;q13) using fluorescence in-situ hybridization (FISH) techniques, in order to evaluate the reproductive possibility of this patient and to assess the usefulness of the PGD procedure. Materials/Methods: A t(1;13;19)(p31;q31;q13) was first identified in a 37-year-old man using conventional and molecular cytogenetic methods. This patient was included in an ICSI (intracytoplasmic sperm injection) programme because of oligoasthenoteratozoospermia. The sperm sample was prepared for FISH protocol. PRINS and FISH techniques were combined to allow the use of five fluorochromes on the same sperm cell preparation. The PRINS procedure used the primer J52 specific for the satellite III sequence of chromosome 1 labelled with biotin and detected with Alexafluor-350conjugated streptavidin. The FISH technique was performed using four probes: two sub-telomeric probes, 1pter (labelled in spectrum Orange) and 19qter (labelled in spectrum Aqua), the LSI FKHR probe (a mixture of two probes labelled in spectrum Green and in spectrum Orange) and the LSI 13q34 (labelled in spectrum Green) Results: Segregation analysis was performed on a total of 800 sperm nuclei. Normal and balanced spermatozoa were identified in 119 cases (14.9%) and unbalanced spermatozoa were observed in 681 cases (85.1%). The most frequent modes of segregation were 4:2 and 3:3. Conclusion: To the best of our knowledge, this is the first report on chromosome segregation analysis in human spermatozoa in a carrier of CCR using five fluorochromes. These results permit us to have a better understanding of mechanisms of meiotic segregation, thus leading to the improvement of genetic counselling for PGD. 6.002 Case report: meiotic segregation pattern and preimplantation diagnosis in a Robertsonian translocation (13;21) Fernández S1,2, Colomar A1,2, Toro E1,2, Casanovas A1, Serra O1, López-Teijón M1, Velilla E1,2 1Institut Marquès, Barcelona; 2Centre for Embryo Medicine, Madrid, Spain Objective: The aim of this study is to describe the meiotic segregation pattern of a rare male Robertsonian translocation carrier and the outcome of the PGS cycle. Materials/Methods: A couple (female, 34 years old; male, 36 years old) presented with a 2-year history of primary infertility. The male partner was a carrier of a familiar Robertsonian translocation (13; 21) and was diagnosed as having oligoasthenoteratozoospermia (0.13 million/ml and motility of 18.4%). To analyse the chromosome segregation
pattern, a sperm FISH analysis for chromosomes 13 and 21 was performed and chromosomes 18, X, Y were added to the FISH analysis to detect the interchromosomal effect (ICE). With the aim of selecting normal or balanced embryos in an IVF-PGS cycle, chromosomes 13 and 21 were studied and in order to detect the interchromosomal effect chromosomes X, Y, 15, 16, 17, 18 and 22 were included in the PGS cycle. Results: The segregation analysis of normal or balanced spermatozoa for chromosomes 13 and 21, resulting from alternant segregation pattern, was 91.8%, similar to that observed in the literature (Anton et al., 2004). The unbalanced chromosomal constitution resulting from adjacent segregation (nullisomies and disomies, 3:0 or diploids and others) was 8.2%. An interchromosomal effect has been detected by some authors (Anton et al., 2004; Ogur et al., 2006). The first IVFPGS cycle for this couple analysed two embryos and resulted in one normal or balanced embryo that was transferred at the morulae stage on day 4 of development without pregnancy. A second IVF-PGD cycle was performed; 18 metaphase II oocytes were collected, 12 were fertilized, eight embryos were analysed by FISH and three normal or balanced embryos were detected. Three embryos were transferred on day 4 of development: two embryos initiated compactation and one embryo was at the morulae stage. Pregnancy was achieved and two fetal heart beats were detected. Two healthy babies were born: one male (1990 g) and one female (2300 g). Conclusion: Robertsonian translocations (13; 21) represent only 2% of the overall Robertsonian translocations. Although it has been poorly reported, this translocation presents a similar segregation pattern to other Robertsonian rearrangements. It is recommended that an aneuploidy panel be added to the PGS cycle. 6.003 FISH analysis of chromosome segregation in spermatozoa of a t(1;15)(q21;p12) carrier: predictive test for PGD Loginova J, Chiryaeva O, Chanisheva O Russian–Finnish Clinic ‘AVA-PETER’, Saint-Petersburg, Russia Introduction: Balanced reciprocal translocations (RT) are the most common structural abnormalities. Men carrying RT often have fertility problems because of oligozoospermia or azoospermia. Even if their sperm count is normal, RT carriers are at increased risk for spontaneous abortions and chromosomally unbalanced offspring due to the production of spermatozoa with chromosomal imbalances. The frequency of unbalanced spermatozoa in RT carriers is 50% on average, but varies from 19% to more than 80% in different patients. The frequency depends on the chromosomes involved in the translocation, breakpoints and the patient’s genetic background. Often for RT carriers, IVF/intracytoplasmic sperm injection (ICSI) treatment with PGD is the only chance to have a healthy baby. An estimation of the overall sperm chromosomal imbalance of such patients could help to give couples valuable genetic counselling for making a resolve about PGD practicability. The aim of this study is to analyse meiotic segregation of chromosomes 1 and 15 in RT carrier spermatozoa to provide objective information for genetic counselling. Materials/Methods: A 41-year-old male patient – a carrier of the translocation 46,XY,t(1;15)(q21;p12), and his 40-year-old wife (46,XX) had infertility for 10 years, one miscarriage and two unsuccessful ICSI cycles without PGD. The male patient displayed cryptozoospermia – sperm count: 0.1 s 106/ml.
S-43 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008