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Majority of ectopic pregnancies that fail methotrexate are euploid by comparative genomic hybridization (CGH)
MATERIALS AND METHODS: The Institutional Review Board approved the study and all the patients provided informed consent. 889 vasectomies for voluntary sterilization purposes were performed from January 1999 to March 2003. Computer-assisted semen analysis was performed on all specimens, with a Motion Analysis (VP 50) semen analyzer. We divided the patients into four groups: non-smokers (Group A; n ⫽ 522), mild smokers (Group B, ⬍ ⫺ 10 cigarettes/day; n ⫽ 143), moderate smokers (Group C, ⫺ 11–20 cigarettes/day; n ⫽ 154), and heavy smokers (Group D, ⬎ ⫺ 20 cigarettes/day; n ⫽ 70). We evaluated sperm concentration, motility, motion parameters, and hormonal levels in these men at the time of their visit. Data was evaluated with ANOVA. RESULTS: No differences were seen across Groups A to D in sperm concentration (109.18 ⫾ 76.21, 107.49 ⫾ 86.62, 124.51 ⫾ 87.3, and 118.41 ⫾ 85.21; P ⫽ 0.071), motility (60.35 ⫾ 15.67, 59.8 ⫾ 15.79, 60.82 ⫾ 14.42, and 53.9 ⫾ 13.18; P ⫽ 0.678), follicle-stimulating hormone (3.67 ⫾ 2.10, 4.021 ⫾ 2.10, 3.40 ⫾ 2.4, and 3.53 ⫾ 1.57; P ⫽ 0.562), luteinizing hormone (3.32 ⫾ 1.6, 3.66 ⫾ 4.02, 3.03 ⫾ 1.57, and 2.63 ⫾ 1.17; P ⫽ 0.213), and serum total testosterone (558.59 ⫾ 178.4, 595.26 ⫾ 175.33, 561.23 ⫾ 166.62, and 659.92 ⫾ 212.67; P ⫽ 0.266). Also, sperm motion characteristics did not differ across the groups: curvilinear velocity (71.99 ⫾ 16.44, 71.008 ⫾ 16.37, 72.81 ⫾ 14.41, and 70.2 ⫾ 13.38; P ⫽ 0.828), linear velocity (40.58 ⫾ 10.33, 39.43 ⫾ 10.72, 39.60 ⫾ 9.752, and 38.85 ⫾ 7.50; P ⫽ 0.796), linearity (55.72 ⫾ 7.86, 54.82 ⫾ 8.73, 53.4 ⫾ 9.16, and 54.63 ⫾ 8.38; P ⫽ 0.211), lateral head displacement (3.03 ⫾ 0.92, 3.1 ⫾ 0.88, 3.28 ⫾ 0.99, and 3.001 ⫾ 0.94; P ⫽ 0.473), and beat cross frequency (23.79 ⫾ 6.48, 24.01 ⫾ 4.96, 23.47 ⫾ 7.28, and 21.74 ⫾ 7.96; P ⫽ 0.499). Semen volume was the only semen parameter, which tends to decrease according to the number of cigarettes smoked (2.82 ⫾ 1.44, 2.42 ⫾ 1.403, 2.30 ⫾ 1.151, and 2.08 ⫾ 0.82; P ⫽ 0.004). CONCLUSION: Semen volume tends to decrease according to the number of cigarettes smoked. A decrease in semen volume may be the first sign of semen quality impairment in cigarette smokers even before a decline in sperm concentration, percent sperm motility and normal sperm morphology. Supported by: None.
P-423 Modulation of mitochondrial mediated apoptosis in ejaculated human spermatozoa and its impact on sperm motility. S. Grunewald, U. Paasch, T. M. Said, R. K. Sharma, A. Agarwal, H.-J. Glander. Universitatsklinikum Leipzig, Leipzig, Germany; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Intact mitochondrial metabolism is a prerequisite for motility and fertilizing potential of the spermatozoa. Mitochondria are highly susceptible to cellular stress and respond to membrane changes similar to those seen after apoptosis and/or necrosis. The aim of our study was to examine the effect of specific mitochondrial inducer of apoptosis (betulinic acid, BA) and inhibitor of apoptosis (bongkregic acid, BOA) on sperm motility. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Washed spermatozoa from 10 infertile male patients were incubated with 100 mol/L BA or 100 mol/L BOA for 1, 3, and 6 hours. Controls consisted of spermatozoa with an equal volume of phosphate buffered saline (PBS). The percentages of motile spermatozoa and other sperm motion characteristics were measured by computer assisted sperm motion analyzer (CASA). RESULTS: BA resulted in a significant decrease of motility and straight line velocity (VSL), which resulted in a complete loss of motility after 3 hours of incubation. In contrast, percent motility and VSL were significantly improved (up to 24.7% increase in progressive motile sperm within the first hour) in spermatozoa incubated with BOA. However, the efficacy of the motility-improving effect decreased with time (table). CONCLUSION: Both antagonists and agonists of mitochondria derived apoptotic pathways have a significant impact on sperm motility. The administration of BOA may help maintain sperm motility and velocity particularly if the incubation time does not exceed 3 hours and might be therefore used for therapeutic interventions to influence the motility of spermatozoa. In contrast, disruption of motility by BA may be useful as an affective spermicidal. Supported by: None.
FERTILITY & STERILITY威
Table: Percentage of progressive motile sperm and their straightline velocity (VSL) in neat semen samples and after incubation with BA and BOA.
*P⬍0.05 compared to control. P-424 Apoptosis signal transduction in ejaculated human spermatozoa following physiological and pathological stimuli. S. Grunewald, T. M. Said, U. Paasch, R. K. Sharma, A. Agarwal, H.-J. Glander. Universitatsklinikum Leipzig, Leipzig, Germany; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The presence of Fas (CD95) and caspase (CP) 8 activation (type I apoptosis), and CP-9 activation (type II apoptosis), along with the activation of their shared effector CP3 and CP1 have been reported in human spermatozoa. The objective of our study was to examine the role of receptor (type I) and mitochondrial (type II) as well as CP1 derived apoptosis signaling in ejaculated human spermatozoa in response to type I and II apoptosis inducers (Fas, CD95 and Betulinic acid, BA), oxidative stress (hypochlorous acid, HOCl) and capacitation. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen specimens collected from 15 healthy donors were separated into 7 aliquots. Two aliquots were incubated with 2 g/mL CD95 antibody and 60 g/mL Betulinic acid. Another aliquot was subjected to incubation with HOCl (10-3 mol/L) and incubated for 1h. The remaining pellet was re-suspended in 1 mL of BWW ⫹ 3% BSA at 5% CO2 to induce capacitation for 3h. Controls were incubated under identical conditions with equal volumes of PBS. Using carboxyfluorescein derivatives, the levels of active caspase 1, 3, 8 and 9 were estimated with flow cytometry. RESULTS: Both CD95 and HOCl treatment did not activate any of the measured caspases. BA resulted in a significant increase in activation of CP9 and CP3 compared with controls (43.6 ⫾ 13.4 vs. 25.5 ⫾ 10.7, 45.3 ⫾ 17.8 vs. 26.1 ⫾ 8.2; P ⬍ 0.001). Following capacitation, compared with controls, a significant increase was seen in CP1 (44.1 ⫾ 16.1 vs. 36.1 ⫾ 12.2, P⬍0.05); CP9 (31.3 ⫾ 15.0 vs. 21.3 ⫾ 12.1, P⬍0.05) and CP3 activation (39.9 ⫾ 17.6 vs. 28.9 ⫾ 12.4; P ⬍ 0.05). CONCLUSION: Ejaculated human spermatozoa may not display the complete signaling pathways in response to both physiological and pathological apoptotic stimuli. Mitochondria derived type II pathway appears to be predominant pathway in both stimuli and therefore should be the target of intervention. Supported by: None. P-425 Majority of ectopic pregnancies that fail methotrexate are euploid by comparative genomic hybridization (CGH). L. McKenzie, H. El-Zimaity, S. Krotz, P. Amato, S. A. Carson, J. E. Buster. Baylor College of Medicine, Houston, TX. OBJECTIVE: To determine the chromosomal complement of ectopic pregnancies that fail methotrexate therapy by comparative genomic hybridization DESIGN: Examination of archived ectopic pregnancy tissue by comparative genomic hybridization (CGH). MATERIALS AND METHODS: Seventeen patients were identified that demonstrated surgically confirmed tubal rupture after MTX therapy. Pathology tissue blocks were obtained on 17 identified patients. Gestational age, means of conception (spontaneous versus assisted reproductive technology), ultrasound findings, history of ectopic pregnancy, history of sexually transmitted disease, age, gravidity, parity, time from first MTX to rupture, and surgical implantation site were recorded from patients’ charts. Human chorionic gonadotropin (hCG) levels and serial trends were recorded in detail. Genomic anlysis was performed by comparative genomic hybridization and restricted to the chorionic villi using coning cyclindars.
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RESULTS: Of the 17 tissue blocks available, 3 blocks did not contain villi, and were therefore not amenable for study. In fifteen cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH after degenerate oligonucleotide-primed PCR. Eleven of fifteen were euploid. Four of fifteen ectopic gestations were aneuploid. Three of the four aneuploid gestations were mosaic, with normal 46,XX cell lines present. The four aneuploid gestations did not differ clinically from the euploid gestations in terms of patient age, gravidity, history of ectopic pregnancy or pelvic adhesive disease, gestational age at diagnosis, hCG at diagnosis, peak hCG, days to operative management, or peak hCG change (P⬎0.05 for all parameters). CONCLUSION: In this sample, the majority of ectopic pregnancies that fail methotrexate are euploid. Our results also confirm the feasibility of CGH for archived tissues. Supported by: None.
P-426 Withdrawn P-427 Levels of antioxidant enzyme in infertile patients with normal and abnormal semen parameters and fertile men. F. F. Pasqualotto, S. S. Allamaneni, E. B. Pasqualotto, C. Oliveira, M. Salvador, A. Agarwal. University of Caxias do Sul, Caxias do Sul, Brazil; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Semen contains many enzymatic and non-enzymatic antioxidants to counter act the harmful effects of excessive reactive oxygen species. Superoxide dismutase (SOD) and catalase are important antioxidant enzymes that can quench excess free radicals such as: superoxide anion and hydrogen peroxide respectively. The objective of our study was to evaluate and compare the seminal antioxidant enzymatic activity (SOD and catalase levels) among fertile and infertile men. DESIGN: Prospective study. MATERIAL AND METHODS: Ten fertile donors and 63 infertile patients were included in the study. Infertile patients were subdivided into patients with normal (n ⫽ 19) or abnormal semen parameters (n ⫽ 44). Semen analysis was performed according to the WHO guidelines. Superoxide dismutase and catalase levels were determined with a spectrophotometer. RESULTS: Significantly lower levels of SOD and catalase were seen in infertile patients compared to fertile donors (P ⬍0.0001, Table). A significant difference was noted in the median levels of SOD between patients with normal and abnormal semen parameters (P ⫽ 0.021). Similar difference was also noted with catalase (P ⫽ 0.032). Sperm morphology by WHO criteria was significantly correlated with levels of SOD (r ⫽ 0.434, P ⫽ 0.000) and catalase (r ⫽ 0.395, P ⫽ 0.001). SOD was also correlated with sperm concentration (r ⫽ 0.303, P ⫽ 0.012) and percentage motility (r ⫽ 0.295, P ⫽ 0.014). Levels of catalase and SOD in all subjects showed a significant correlation (r ⫽ 0.461, P ⫽ 0.000). CONCLUSIONS: Decreased antioxidant enzyme levels are associated with infertility. The SOD and catalase levels were significantly less in patients with abnormal semen parameters. High degree of correlation between sperm morphology and antioxidant enzymes suggests the ability of abnormal spermatozoa to produce reactive oxygen species resulting in reduced levels of antioxidant enzyme. Supported by: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq). Table : Levels of superoxide dismutase (SOD) and catalase in different study population
Values presented as median (25th, 75th percentiles). P ⬍ 0.05 was considered significant by Mann-Whitney U test aP value between fertile donors and infertile patients bP value between infertile patients with normal and abnormal semen parameters
P-428 Genetic screening of 2061 infertile couples undergoing treatments for infertility. S. Bianchi, A. Cesana, P. V. Novara, E. Albani, G. Morreale, P. E. Levi Setti. ISTITUTO CLINICO HUMANITAS, Rozzano (mi), Italy. OBJECTIVE: Research on genetic causes of male and female infertility rapidly expanded in the last years, following the development of in vitro fertilising techniques. The aim of this study is to asses the rate of chromosomal aberrations, cystic fibrosis transmembrane conductance regulator (CFTR) mutations and chromosome Y microdeletions in infertile couples prior to planned assisted reproduction treatments. DESIGN: Retrospective clinic and cytogenetic study. MATERIALS AND METHODS: 2061 infertile couples were analysed. Karyotype analysis was performed in all patients using cytogenetic tests based on fluorescence in situ hybridization. Results were described according to the International System for Human Cytogenetic Nomenclature. CFTR gene alterations were investigated in 677 females and in men whose partner was positive for any mutation. CFTR gene was analyzed for the 29 most common mutations and the poly-T polymorphism in intron 8 (5T/7T/ 9T) by the reverse-hybridization technique, using the INNO-LIPA CF2 test. 633 males were tested for microdeletions detection on the Y chromosome. Microdeletions were detected by separate multiplex-polymerase chain reaction (PCR) reactions using primer pairs for 21 different single tagged sites (STSs) of all three azoospermia factor regions (AZFa, AZFb and AZFc). RESULTS:
a 11 were 47,XXY, 3 were 47,XYY and 1 had a mosaicism (47,XXY/ b 46,XY) 1 was 45,X, 18 were 45,X/46,XX, 3 were 47,XXX/46,XX c 7,XX⫹18/46,XX d17 had at least one heterozygosis mutation, 8 presented polyT polymorphism, 1 had both of them 1Jacobs PA et al., Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J Med Genet (1992) 29:103–108 2Nielsen J and Wohlert M, Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Human Genetics (1991) 87:81– 83 3Lenzi A et al., ICSI E PATOLOGIA GENETICA, pp.57– 65, Sterilita` maschile: trattamento con la intracytoplasmic sperm injection, 1996
CONCLUSION: We have shown that a significant number of genetic aberrations is present in infertile couples, according with the frequency of alterations reported by other laboratories. As a consequence a genetic counselling, that should also include AZF and CFTR genotyping, is recommended before assisted reproductive treatments, as parental factors can be transferred to offspring that would most likely not have been conceived by natural means. Supported by: None.
P-429 TCDD induce oxidative stress and the mechanism in human trophoblast cells. T. L. Liao, H. T. Chao, S. H. Kao, C. R. Tzeng. Graduated
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Vol. 82, Suppl. 2, September 2004
P-423 Modulation of mitochondrial mediated apoptosis in ejaculated human spermatozoa and its impact on sperm motility. S. Grunewald, U. Paasch, T. M. Said, R. K. Sharma, A. Agarwal, H.-J. Glander. Universitatsklinikum Leipzig, Leipzig, Germany; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Intact mitochondrial metabolism is a prerequisite for motility and fertilizing potential of the spermatozoa. Mitochondria are highly susceptible to cellular stress and respond to membrane changes similar to those seen after apoptosis and/or necrosis. The aim of our study was to examine the effect of specific mitochondrial inducer of apoptosis (betulinic acid, BA) and inhibitor of apoptosis (bongkregic acid, BOA) on sperm motility. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Washed spermatozoa from 10 infertile male patients were incubated with 100 mol/L BA or 100 mol/L BOA for 1, 3, and 6 hours. Controls consisted of spermatozoa with an equal volume of phosphate buffered saline (PBS). The percentages of motile spermatozoa and other sperm motion characteristics were measured by computer assisted sperm motion analyzer (CASA). RESULTS: BA resulted in a significant decrease of motility and straight line velocity (VSL), which resulted in a complete loss of motility after 3 hours of incubation. In contrast, percent motility and VSL were significantly improved (up to 24.7% increase in progressive motile sperm within the first hour) in spermatozoa incubated with BOA. However, the efficacy of the motility-improving effect decreased with time (table). CONCLUSION: Both antagonists and agonists of mitochondria derived apoptotic pathways have a significant impact on sperm motility. The administration of BOA may help maintain sperm motility and velocity particularly if the incubation time does not exceed 3 hours and might be therefore used for therapeutic interventions to influence the motility of spermatozoa. In contrast, disruption of motility by BA may be useful as an affective spermicidal. Supported by: None.
FERTILITY & STERILITY威
Table: Percentage of progressive motile sperm and their straightline velocity (VSL) in neat semen samples and after incubation with BA and BOA.
*P⬍0.05 compared to control. P-424 Apoptosis signal transduction in ejaculated human spermatozoa following physiological and pathological stimuli. S. Grunewald, T. M. Said, U. Paasch, R. K. Sharma, A. Agarwal, H.-J. Glander. Universitatsklinikum Leipzig, Leipzig, Germany; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The presence of Fas (CD95) and caspase (CP) 8 activation (type I apoptosis), and CP-9 activation (type II apoptosis), along with the activation of their shared effector CP3 and CP1 have been reported in human spermatozoa. The objective of our study was to examine the role of receptor (type I) and mitochondrial (type II) as well as CP1 derived apoptosis signaling in ejaculated human spermatozoa in response to type I and II apoptosis inducers (Fas, CD95 and Betulinic acid, BA), oxidative stress (hypochlorous acid, HOCl) and capacitation. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen specimens collected from 15 healthy donors were separated into 7 aliquots. Two aliquots were incubated with 2 g/mL CD95 antibody and 60 g/mL Betulinic acid. Another aliquot was subjected to incubation with HOCl (10-3 mol/L) and incubated for 1h. The remaining pellet was re-suspended in 1 mL of BWW ⫹ 3% BSA at 5% CO2 to induce capacitation for 3h. Controls were incubated under identical conditions with equal volumes of PBS. Using carboxyfluorescein derivatives, the levels of active caspase 1, 3, 8 and 9 were estimated with flow cytometry. RESULTS: Both CD95 and HOCl treatment did not activate any of the measured caspases. BA resulted in a significant increase in activation of CP9 and CP3 compared with controls (43.6 ⫾ 13.4 vs. 25.5 ⫾ 10.7, 45.3 ⫾ 17.8 vs. 26.1 ⫾ 8.2; P ⬍ 0.001). Following capacitation, compared with controls, a significant increase was seen in CP1 (44.1 ⫾ 16.1 vs. 36.1 ⫾ 12.2, P⬍0.05); CP9 (31.3 ⫾ 15.0 vs. 21.3 ⫾ 12.1, P⬍0.05) and CP3 activation (39.9 ⫾ 17.6 vs. 28.9 ⫾ 12.4; P ⬍ 0.05). CONCLUSION: Ejaculated human spermatozoa may not display the complete signaling pathways in response to both physiological and pathological apoptotic stimuli. Mitochondria derived type II pathway appears to be predominant pathway in both stimuli and therefore should be the target of intervention. Supported by: None. P-425 Majority of ectopic pregnancies that fail methotrexate are euploid by comparative genomic hybridization (CGH). L. McKenzie, H. El-Zimaity, S. Krotz, P. Amato, S. A. Carson, J. E. Buster. Baylor College of Medicine, Houston, TX. OBJECTIVE: To determine the chromosomal complement of ectopic pregnancies that fail methotrexate therapy by comparative genomic hybridization DESIGN: Examination of archived ectopic pregnancy tissue by comparative genomic hybridization (CGH). MATERIALS AND METHODS: Seventeen patients were identified that demonstrated surgically confirmed tubal rupture after MTX therapy. Pathology tissue blocks were obtained on 17 identified patients. Gestational age, means of conception (spontaneous versus assisted reproductive technology), ultrasound findings, history of ectopic pregnancy, history of sexually transmitted disease, age, gravidity, parity, time from first MTX to rupture, and surgical implantation site were recorded from patients’ charts. Human chorionic gonadotropin (hCG) levels and serial trends were recorded in detail. Genomic anlysis was performed by comparative genomic hybridization and restricted to the chorionic villi using coning cyclindars.
S285
RESULTS: Of the 17 tissue blocks available, 3 blocks did not contain villi, and were therefore not amenable for study. In fifteen cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH after degenerate oligonucleotide-primed PCR. Eleven of fifteen were euploid. Four of fifteen ectopic gestations were aneuploid. Three of the four aneuploid gestations were mosaic, with normal 46,XX cell lines present. The four aneuploid gestations did not differ clinically from the euploid gestations in terms of patient age, gravidity, history of ectopic pregnancy or pelvic adhesive disease, gestational age at diagnosis, hCG at diagnosis, peak hCG, days to operative management, or peak hCG change (P⬎0.05 for all parameters). CONCLUSION: In this sample, the majority of ectopic pregnancies that fail methotrexate are euploid. Our results also confirm the feasibility of CGH for archived tissues. Supported by: None.
P-426 Withdrawn P-427 Levels of antioxidant enzyme in infertile patients with normal and abnormal semen parameters and fertile men. F. F. Pasqualotto, S. S. Allamaneni, E. B. Pasqualotto, C. Oliveira, M. Salvador, A. Agarwal. University of Caxias do Sul, Caxias do Sul, Brazil; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Semen contains many enzymatic and non-enzymatic antioxidants to counter act the harmful effects of excessive reactive oxygen species. Superoxide dismutase (SOD) and catalase are important antioxidant enzymes that can quench excess free radicals such as: superoxide anion and hydrogen peroxide respectively. The objective of our study was to evaluate and compare the seminal antioxidant enzymatic activity (SOD and catalase levels) among fertile and infertile men. DESIGN: Prospective study. MATERIAL AND METHODS: Ten fertile donors and 63 infertile patients were included in the study. Infertile patients were subdivided into patients with normal (n ⫽ 19) or abnormal semen parameters (n ⫽ 44). Semen analysis was performed according to the WHO guidelines. Superoxide dismutase and catalase levels were determined with a spectrophotometer. RESULTS: Significantly lower levels of SOD and catalase were seen in infertile patients compared to fertile donors (P ⬍0.0001, Table). A significant difference was noted in the median levels of SOD between patients with normal and abnormal semen parameters (P ⫽ 0.021). Similar difference was also noted with catalase (P ⫽ 0.032). Sperm morphology by WHO criteria was significantly correlated with levels of SOD (r ⫽ 0.434, P ⫽ 0.000) and catalase (r ⫽ 0.395, P ⫽ 0.001). SOD was also correlated with sperm concentration (r ⫽ 0.303, P ⫽ 0.012) and percentage motility (r ⫽ 0.295, P ⫽ 0.014). Levels of catalase and SOD in all subjects showed a significant correlation (r ⫽ 0.461, P ⫽ 0.000). CONCLUSIONS: Decreased antioxidant enzyme levels are associated with infertility. The SOD and catalase levels were significantly less in patients with abnormal semen parameters. High degree of correlation between sperm morphology and antioxidant enzymes suggests the ability of abnormal spermatozoa to produce reactive oxygen species resulting in reduced levels of antioxidant enzyme. Supported by: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq). Table : Levels of superoxide dismutase (SOD) and catalase in different study population
Values presented as median (25th, 75th percentiles). P ⬍ 0.05 was considered significant by Mann-Whitney U test aP value between fertile donors and infertile patients bP value between infertile patients with normal and abnormal semen parameters
P-428 Genetic screening of 2061 infertile couples undergoing treatments for infertility. S. Bianchi, A. Cesana, P. V. Novara, E. Albani, G. Morreale, P. E. Levi Setti. ISTITUTO CLINICO HUMANITAS, Rozzano (mi), Italy. OBJECTIVE: Research on genetic causes of male and female infertility rapidly expanded in the last years, following the development of in vitro fertilising techniques. The aim of this study is to asses the rate of chromosomal aberrations, cystic fibrosis transmembrane conductance regulator (CFTR) mutations and chromosome Y microdeletions in infertile couples prior to planned assisted reproduction treatments. DESIGN: Retrospective clinic and cytogenetic study. MATERIALS AND METHODS: 2061 infertile couples were analysed. Karyotype analysis was performed in all patients using cytogenetic tests based on fluorescence in situ hybridization. Results were described according to the International System for Human Cytogenetic Nomenclature. CFTR gene alterations were investigated in 677 females and in men whose partner was positive for any mutation. CFTR gene was analyzed for the 29 most common mutations and the poly-T polymorphism in intron 8 (5T/7T/ 9T) by the reverse-hybridization technique, using the INNO-LIPA CF2 test. 633 males were tested for microdeletions detection on the Y chromosome. Microdeletions were detected by separate multiplex-polymerase chain reaction (PCR) reactions using primer pairs for 21 different single tagged sites (STSs) of all three azoospermia factor regions (AZFa, AZFb and AZFc). RESULTS:
a 11 were 47,XXY, 3 were 47,XYY and 1 had a mosaicism (47,XXY/ b 46,XY) 1 was 45,X, 18 were 45,X/46,XX, 3 were 47,XXX/46,XX c 7,XX⫹18/46,XX d17 had at least one heterozygosis mutation, 8 presented polyT polymorphism, 1 had both of them 1Jacobs PA et al., Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J Med Genet (1992) 29:103–108 2Nielsen J and Wohlert M, Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Human Genetics (1991) 87:81– 83 3Lenzi A et al., ICSI E PATOLOGIA GENETICA, pp.57– 65, Sterilita` maschile: trattamento con la intracytoplasmic sperm injection, 1996
CONCLUSION: We have shown that a significant number of genetic aberrations is present in infertile couples, according with the frequency of alterations reported by other laboratories. As a consequence a genetic counselling, that should also include AZF and CFTR genotyping, is recommended before assisted reproductive treatments, as parental factors can be transferred to offspring that would most likely not have been conceived by natural means. Supported by: None.
P-429 TCDD induce oxidative stress and the mechanism in human trophoblast cells. T. L. Liao, H. T. Chao, S. H. Kao, C. R. Tzeng. Graduated
S286
Vol. 82, Suppl. 2, September 2004