- Email: [email protected]
Microsurgical embryonic fragment removal (MsFR) in patients with multiple IVF implantation failures
hours and mature oocytes were selected for ICSI. Fertilization was assessed 19 –21 hours after ICSI for the appearance of 2 distinct pronuclei and 2 polar bodies. Resulting day 3 embryos were transferred after exposure to protease solution for 24 hours (biochemical assisted hatching). The Serum was collected from the patient 24 hours after HCG administration. FF collected from the patients of stimulation cycles was used under informed consent. RESULTS: There were no differences between group A and group B either in maturation rate (49.7% vs. 46.3%) or fertilization rate (83.4% vs. 83.3%, respectively). The pregnancy rate in fresh embryo transfer of group A (8.3%; 1/12) was similar to that of group B (12.0%; 3/25). In frozen cycles, there were no differences of pregnancy rate between group A (50.0%; 2/4) and group B (17.6%; 3/17). Serum supplementation was same maturation rate or fertilization rates compared with Human mature follicular fluid. There was no significant difference between serum and FF. CONCLUSION: No difference between group A and group B either in maturation rates or fertilization rates. Clinical outcome such as pregnancy rates were also similar between group A and B. The results of the present study suggested that supplementation of patient serum with maturation media for IVM-IVF provided similar effect as well as follicular fluid from stimulated patients. Patient serum provides not only identical effect as follicular fluid, but also no risks of transferring of infectious diseases. Supported by: None
P-539 Cryo-banking of great ape spermatozoa using human protocols and variation among males. T. J. Kuehl, T. Bowsher, J. Perlman, R. L. Lefever, J. L. Dye, M. L. Brown. Scott & White Clinic, Temple, TX; Dallas Zoo, Dallas, TX; M.D. Anderson, Bastrop, TX. OBJECTIVE: To develop a resource of great ape sperm cells and test the hypothesis that great ape males, like human males, differ in production of sperm cells and in rates of sperm cell survival after exposure to similar processing for cryopreservation. DESIGN: Prospective trial in a cohort of normal male chimpanzees. MATERIALS AND METHODS: Seven male chimpanzees ranging in age from 9.9 to 24.2 years were trained to provide semen samples using an artificial vagina. Samples were removed from the plastic sleeve of the device and placed into conical centrifuge tubes. The liquid fraction of semen was removed within 30 minutes and mixed with an equal volume of human commercial transport medium containing egg yolk and buffer (Refrigeration Medium, Irvine Scientific). Samples were transported to the andrology lab for semen analysis and cryopreservation. Briefly, the diluted semen was assayed, then mixed with an equal volume of glycerol containing cryopreservation medium (Freezing Medium, Irvine Scientific) and loaded into half cc straws with labeled handles. Straws were placed into a controlledrate, nitrogen vapor freezing machine for slow cooling to -7 degrees C, seeding, cooling to -80 degrees C at -10 degrees/min and plunging into liquid nitrogen. A straw from each sample was subsequently thawed, diluted with culture medium, concentrated by centrifugation, and diluted to the original volume of the straw for assay of motility via CASA system. RESULTS: During a 20-month interval 93 semen samples from seven males were obtained for analysis and potential cryopreservation. Individual males provided between 5 and 29 samples. Males differed significantly (p⬍0.000005) in the number of sperm cells provided in each sample from averages of 79, 88, 454, 1858, 1138, 678, and 1640 million and in the percentage of motile cells available for cryopreservation from averages of 48, 4, 32, 27, 13, 18, and 47 percent. Between 5 and 26 samples were cryopreserved for each male. The ratio of progressive motility after thaw and washing compared to pre-freeze levels differed (p⫽0.03) between males (0.43, 0.16, 0.33, 0.23, 0.41, 0.30, and 0.17) and was not related to number of sperm in the ejaculate or the initial motility. Currently, 531 straws with estimated yields after thaw of 3,686 million motile cells have been banked for research uses and seven males are trained to provide samples on demand. These procedures have been extended to another great ape species, the bonobo. CONCLUSION: Male chimpanzees of varying ages can be trained to provide semen samples for analysis, research, and cryopreservation. Once trained, animals can be induced with rewards to provide samples on a regular basis. A cryo-bank has been developed with sperm cells from seven males. Even with delays of 3 to 6 hours between collection and initiation of processing for cryopreservation, progressively motile sperm cells survive
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standardized cryopreservation and thawing protocols. However, sperm survival rates vary significantly among males. Supported by: Noble Centennial Endowment and NIH.
P-540 Influence of transfer catheter placement techniques on in vitro fertilization outcome. G. D. Adamson, M. Gvakharia. Fertility Physicians of Northern California, San Jose and Palo Alto, CA. OBJECTIVE: It has been reported that embryo transfer techniques may vary among physicians and that this variability may have an effect on the outcome of an in vitro fertilization cycle. The aim of this study was to compare clinical outcomes among reproductive endocrinologists (REI) in our practice based on different techniques of actual catheter placement during embryo transfer procedure. DESIGN: Retrospective analysis of in vitro fertilization cycles performed in our center during 2002 and 2003. MATERIALS AND METHODS: During 2002–2003, five REI’s used three different techniques of actual introduction of Wallace embryo transfer catheter into uterine cavity. For technique I (physicians A and B), a pre-loaded catheter was directly introduced into the uterine cavity. If this was unsuccessful, outer sheath was introduced through 2/3 of cervical canal, followed by the internal catheter. For technique II (physicians C and D), the outer sheath was introduced through 2/3 of cervical canal, followed by the loaded internal catheter. For technique III (physician E), the outer sheath of trail Wallace catheter was introduced through 2/3 of cervical canal, the trial catheter was then removed while outer sheath was left in place and preloaded internal catheter was threaded through the trial catheter sheath. For this study, 756 patients were allocated into three groups based on transfer technique used. There were 315, 249 and 192 patients in groups I, II, and III, respectively. Embryos were assessed by cell number and by grade (Grade 1⫽0 –10% fragmentation, Grade 2⫽10 –25% fragmentation, Grade 3⫽26 – 50% fragmentation, Grade 4⬎50% fragmentation). Results were analyzed using Chi-Square test with Yates’ correction. RESULTS: There were no differences among study groups in terms of mean maternal age, number of retrieved oocytes or embryo quality. Clinical pregnancy rates among groups I, II, and III were 36%, 35% and 31 %, respectively (P⬎0.05). CONCLUSION: There was no difference in pregnancy rates among different REI’s in our practice. Based on our results, we conclude that while minimizing trauma during embryo transfer technique is crucial for the outcome, the actual technique of transfer catheter insertion has no measurable effect on pregnancy rates. Supported by: na
P-541 Microsurgical embryonic fragment removal (MsFR) in patients with multiple IVF implantation failures. J. R. Alegretti, T. Rocca, E. L. Motta, J. M. Oliveira, J. Fioravanti, P. C. Serafini. Centro Reproduc¸ ao Huntington Brazil, Sao Paulo, Brazil. OBJECTIVE: The presence of high amounts of embryonic fragmentation correlates negatively with implantation (IR) and pregnancy rates (PR). Fragmentation of blastomeric origin may extract considerable amounts of cytoplasm from the blastomeres resulting in blastomeres with deficiency of important cytoplasm contents. The relevance of these injuries is supported by previous research where embryonic fragmentation ⬎10% impaired the success of IVF. Although few investigators have shown an improved IR and PR after MsFR, this technique has not gained much popularity. We aimed to evaluate the impact of MsFR performed on embryos just before embryo
Vol. 82, Suppl. 2, September 2004
transfer in a subgroup of patients with impaired chances of pregnancy due to age and multiple IVF failures. DESIGN: Case controlled retrospective study. MATERIALS AND METHODS: Informed consent was obtained from all patients before MsFR. The study was comprised of 2 groups who failed at least 2 IVF treatments: A) 20 women aged 28 – 42 (36.1⫾3.9) yr who had 2.8⫾1.2 IVF treatments. MsFR was performed in 76 embryos that exhibited fragmentation ranging from 15–50%; B) 20 women aged 28 – 43 (35.1⫾4.4) yr who had 2.5⫾1.4 IVFs. 87 embryos from these patients were transferred without MsFR. The pairing of the groups was based on age, infertility factors, quality of embryos, and the interval of the procedures. The technique utilized for MsFR included an initial assisted hatching (AH) step using acid solutions, followed by the removal of some or all fragments after placing a new AH micropipette. Fragments were finally removed via controlled gentle mouth suction. Study endpoints included # of embryonic cells at ET, degree of fragmentation, improvement after MsFR, IR and PR. The Mann-Whitney and chi-square were used as appropriate. Levels ⬍0.05 were considered significant. RESULTS: All types of fragmentation were removed and there were no visible blastomere damage after MsFR. A statistically significant improvement on embryonic morphology was observed after MsFR (p⬍0.001) as noted by the improvement in the standard morphologic classification used (documented photographic embryonic improvement of 37%). There were no differences regarding PR (A⫽45% and B⫽20%, p⫽0.177); however, IR were significantly greater in group B (A⫽ 26.5% and B⫽49.6%, p⫽0.011). Conversely, the incidence of single gestation was 78% in group A and 25% in group B. Additional results are shown in the Table. CONCLUSION: These findings support the applicability of MsFR to selected patients. The significant morphologic improvement in the embryonic appearance along with a tendency to an improved PR may imply that MsFR provided intracellular mechanisms to “fight” cell derangement and death. Although some indicators showed benefits to MsFR, the fact that the IR observed in the MsFR group is perplexing. The small sample size, lack of strict criteria for fragment removal, use of chemical/mechanic technique instead of hydraulic, and lack of biomarkers for this microsurgical approach provide some reasoning for such evidence. Supported by: None.
P-542 Time- and media-dependent improvements in motility of testicular spermatozoa retrieved from non-obstructive azoospermic men. D. E. Davis, T. G. Schuster, M. R. Hiner, D. A. Ohl, G. D. Smith. University of Michigan, Ann Arbor, MI. OBJECTIVE: Testicular-derived spermatozoa motility, reflecting viability, is key to intracytoplasmic sperm injection success. Experiments were conducted to compare number of motile sperm isolated from testicular aspirations incubated in different media over time. DESIGN: Prospective side-by-side comparative laboratory study. MATERIALS AND METHODS: Diagnostic and/or therapeutic fine needle testicular aspirations were performed on 14 men with non-obstructive azoospermia. Seminiferous tubules were manually dissected and seminiferous epithelial content dispersed into a single cell suspension. Number of non-motile and motile sperm/20 high-powered fields was assessed initially at the time of cell dispersion and following incubation at 37°C for 24 and 48 hours in either HEPES buffered human tubal fluid ⫹ protein (H-HTF) in atmospheric conditions or Ham’s F10 ⫹ protein (F10) in 5% CO2 and air. Statistical analyses were performed with paired Student’s t-test and ANOVA with repeat measures. RESULTS: Initially following seminiferous tubule micro-dissection, total sperm found was 93 ⫾ 22 (mean ⫾ SE) and mean number of motile sperm/20 high-powered fields was 3 ⫾ 2. In H-HTF sperm motility increased in 8 of 14 samples after 24 hours of culture (18 ⫾ 9) and 11 of 14 samples after 48 hours of culture (19 ⫾ 9), although these increases were not significant. Mean number of motile sperm/20 high-powered fields significantly increased in all 14 samples following incubation in F10 for 24
FERTILITY & STERILITY威
hours (30 ⫾ 11, paired t-test, P⫽0.03) and 48 hours of culture (33 ⫾ 13, P⫽0.04). Testicular sperm motility increased significantly after incubation in F10 compared to HTF at both 24 and 48 hours of culture (repeated measures ANOVA, P⫽0.05). CONCLUSION: Incubation of spermatozoa from fine needle testicular aspirations facilitates motile spermatozoa isolation. More motile spermatozoa were isolated after incubation in F10 than in H-HTF. There was no significant increase in motile sperm isolated between 24 and 48 hours. Following seminiferous tubule micro-dissection, culture of testicular spermatozoa for 24 hours in Ham’s F10 is an efficient means of collecting motile spermatozoa for ICSI. Supported by: None.
P-543 Maker chromosome identification in infertility. A. Abderazek, H. Kayed, R. Mansour, O. Kamal,, M. Aboulghar. Egyptian IVF-ET Center, Cairo, Egypt. OBJECTIVE: A marker chromosome is a structurally abnormal chromosome in which no part can be identified. So far the identification of marker chromosomes has been of interest for cancer cytogenetics only. Molecular cytogenetics (fluorescence in situ hybridization:FISH) has been considered a very powerful research tool, in the understanding of many aspects of human biology. The aim of this study was to identify the origin of chromosome markers detected by G-banding in patients with infertility entering ICSI program. This is of great importance for genetic counseling and offering these patients chance to enter Perimplantation Genetic diagnosis (PGD) program. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 3540 patients, between August 2000 ⫺ January 2004, underwent chromosome analysis and genetic counseling before entering our IVF/ICSI program. Chromosomal analysis was indicated for couples with severe male factor infertility, azoospermia and/or other factors. Chromosomal analysis of peripheral blood lymphocytes culture was performed by standard cytogenetic technique, and banding using Giemsa trypsin banding (GTG) was done. In all cases, 12 metaphases were karyotyped. When any apparent chromosomal aberrations were detected, 20 metaphases were analyzed. Computerized Karyotyper (Cytovision 2.7) was used for observation and detection of chromosomal abnormalities. FISH with alpha satellite DNA (centromeric) and whole chromosome painting probes for chromosomes 13,14,15, 21 and 22 were used because the markers appeared to be a part of acrocentric chromosomes. RESULTS: Cytogenetic results were successfully obtained from all patients. Overall, 95.02 % of patients (3250 patients) had a normal karyotype, and 4.98% (170 patients) had abnormal karyotypes. In 6 cases (0.001 %), 4 males and 2 females a marker chromosome was identified. All of these marker chromosomes by G-banding appeared to be part of acrocentric chromosomes, so by using probes for these chromosomes we could identify these markers to be parts of chromosomes 13,15, and 22. Two males and one female had partial trisomy 13, a male and a female had partial trisomy 15, and one male had partial trisomy 22. CONCLUSION: The combined use of classical banding and FISH techniques is a useful tool in the identification of the origin of the unknown chromosome markers. It could be of significant importance for diagnosis and offering PGD for these patients to prevent the transmission of this anomaly to their offspring. Supported by: None
P-544 A microfluidic chemotaxis system to select motile and mature sperm. L. Karakoc Sokmensuer, S. Palaniappan, M. Toner, T. L. Toth, D. L. Wright. Massachusetts General Hospital, Boston, MA. OBJECTIVE: The evolution of IVF technology has been weighted heavily in the area of embryo culture with limited attention to sperm processing. Many of the current sperm processing techniques may be less than desirable due to damaging effects inflicted on the sample. This study describes the development of a microfluidic chemotaxis device for the purpose of selecting a highly motile, mature sperm fraction for potential use in IVF/ICSI.
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P-539 Cryo-banking of great ape spermatozoa using human protocols and variation among males. T. J. Kuehl, T. Bowsher, J. Perlman, R. L. Lefever, J. L. Dye, M. L. Brown. Scott & White Clinic, Temple, TX; Dallas Zoo, Dallas, TX; M.D. Anderson, Bastrop, TX. OBJECTIVE: To develop a resource of great ape sperm cells and test the hypothesis that great ape males, like human males, differ in production of sperm cells and in rates of sperm cell survival after exposure to similar processing for cryopreservation. DESIGN: Prospective trial in a cohort of normal male chimpanzees. MATERIALS AND METHODS: Seven male chimpanzees ranging in age from 9.9 to 24.2 years were trained to provide semen samples using an artificial vagina. Samples were removed from the plastic sleeve of the device and placed into conical centrifuge tubes. The liquid fraction of semen was removed within 30 minutes and mixed with an equal volume of human commercial transport medium containing egg yolk and buffer (Refrigeration Medium, Irvine Scientific). Samples were transported to the andrology lab for semen analysis and cryopreservation. Briefly, the diluted semen was assayed, then mixed with an equal volume of glycerol containing cryopreservation medium (Freezing Medium, Irvine Scientific) and loaded into half cc straws with labeled handles. Straws were placed into a controlledrate, nitrogen vapor freezing machine for slow cooling to -7 degrees C, seeding, cooling to -80 degrees C at -10 degrees/min and plunging into liquid nitrogen. A straw from each sample was subsequently thawed, diluted with culture medium, concentrated by centrifugation, and diluted to the original volume of the straw for assay of motility via CASA system. RESULTS: During a 20-month interval 93 semen samples from seven males were obtained for analysis and potential cryopreservation. Individual males provided between 5 and 29 samples. Males differed significantly (p⬍0.000005) in the number of sperm cells provided in each sample from averages of 79, 88, 454, 1858, 1138, 678, and 1640 million and in the percentage of motile cells available for cryopreservation from averages of 48, 4, 32, 27, 13, 18, and 47 percent. Between 5 and 26 samples were cryopreserved for each male. The ratio of progressive motility after thaw and washing compared to pre-freeze levels differed (p⫽0.03) between males (0.43, 0.16, 0.33, 0.23, 0.41, 0.30, and 0.17) and was not related to number of sperm in the ejaculate or the initial motility. Currently, 531 straws with estimated yields after thaw of 3,686 million motile cells have been banked for research uses and seven males are trained to provide samples on demand. These procedures have been extended to another great ape species, the bonobo. CONCLUSION: Male chimpanzees of varying ages can be trained to provide semen samples for analysis, research, and cryopreservation. Once trained, animals can be induced with rewards to provide samples on a regular basis. A cryo-bank has been developed with sperm cells from seven males. Even with delays of 3 to 6 hours between collection and initiation of processing for cryopreservation, progressively motile sperm cells survive
S326
standardized cryopreservation and thawing protocols. However, sperm survival rates vary significantly among males. Supported by: Noble Centennial Endowment and NIH.
P-540 Influence of transfer catheter placement techniques on in vitro fertilization outcome. G. D. Adamson, M. Gvakharia. Fertility Physicians of Northern California, San Jose and Palo Alto, CA. OBJECTIVE: It has been reported that embryo transfer techniques may vary among physicians and that this variability may have an effect on the outcome of an in vitro fertilization cycle. The aim of this study was to compare clinical outcomes among reproductive endocrinologists (REI) in our practice based on different techniques of actual catheter placement during embryo transfer procedure. DESIGN: Retrospective analysis of in vitro fertilization cycles performed in our center during 2002 and 2003. MATERIALS AND METHODS: During 2002–2003, five REI’s used three different techniques of actual introduction of Wallace embryo transfer catheter into uterine cavity. For technique I (physicians A and B), a pre-loaded catheter was directly introduced into the uterine cavity. If this was unsuccessful, outer sheath was introduced through 2/3 of cervical canal, followed by the internal catheter. For technique II (physicians C and D), the outer sheath was introduced through 2/3 of cervical canal, followed by the loaded internal catheter. For technique III (physician E), the outer sheath of trail Wallace catheter was introduced through 2/3 of cervical canal, the trial catheter was then removed while outer sheath was left in place and preloaded internal catheter was threaded through the trial catheter sheath. For this study, 756 patients were allocated into three groups based on transfer technique used. There were 315, 249 and 192 patients in groups I, II, and III, respectively. Embryos were assessed by cell number and by grade (Grade 1⫽0 –10% fragmentation, Grade 2⫽10 –25% fragmentation, Grade 3⫽26 – 50% fragmentation, Grade 4⬎50% fragmentation). Results were analyzed using Chi-Square test with Yates’ correction. RESULTS: There were no differences among study groups in terms of mean maternal age, number of retrieved oocytes or embryo quality. Clinical pregnancy rates among groups I, II, and III were 36%, 35% and 31 %, respectively (P⬎0.05). CONCLUSION: There was no difference in pregnancy rates among different REI’s in our practice. Based on our results, we conclude that while minimizing trauma during embryo transfer technique is crucial for the outcome, the actual technique of transfer catheter insertion has no measurable effect on pregnancy rates. Supported by: na
P-541 Microsurgical embryonic fragment removal (MsFR) in patients with multiple IVF implantation failures. J. R. Alegretti, T. Rocca, E. L. Motta, J. M. Oliveira, J. Fioravanti, P. C. Serafini. Centro Reproduc¸ ao Huntington Brazil, Sao Paulo, Brazil. OBJECTIVE: The presence of high amounts of embryonic fragmentation correlates negatively with implantation (IR) and pregnancy rates (PR). Fragmentation of blastomeric origin may extract considerable amounts of cytoplasm from the blastomeres resulting in blastomeres with deficiency of important cytoplasm contents. The relevance of these injuries is supported by previous research where embryonic fragmentation ⬎10% impaired the success of IVF. Although few investigators have shown an improved IR and PR after MsFR, this technique has not gained much popularity. We aimed to evaluate the impact of MsFR performed on embryos just before embryo
Vol. 82, Suppl. 2, September 2004
transfer in a subgroup of patients with impaired chances of pregnancy due to age and multiple IVF failures. DESIGN: Case controlled retrospective study. MATERIALS AND METHODS: Informed consent was obtained from all patients before MsFR. The study was comprised of 2 groups who failed at least 2 IVF treatments: A) 20 women aged 28 – 42 (36.1⫾3.9) yr who had 2.8⫾1.2 IVF treatments. MsFR was performed in 76 embryos that exhibited fragmentation ranging from 15–50%; B) 20 women aged 28 – 43 (35.1⫾4.4) yr who had 2.5⫾1.4 IVFs. 87 embryos from these patients were transferred without MsFR. The pairing of the groups was based on age, infertility factors, quality of embryos, and the interval of the procedures. The technique utilized for MsFR included an initial assisted hatching (AH) step using acid solutions, followed by the removal of some or all fragments after placing a new AH micropipette. Fragments were finally removed via controlled gentle mouth suction. Study endpoints included # of embryonic cells at ET, degree of fragmentation, improvement after MsFR, IR and PR. The Mann-Whitney and chi-square were used as appropriate. Levels ⬍0.05 were considered significant. RESULTS: All types of fragmentation were removed and there were no visible blastomere damage after MsFR. A statistically significant improvement on embryonic morphology was observed after MsFR (p⬍0.001) as noted by the improvement in the standard morphologic classification used (documented photographic embryonic improvement of 37%). There were no differences regarding PR (A⫽45% and B⫽20%, p⫽0.177); however, IR were significantly greater in group B (A⫽ 26.5% and B⫽49.6%, p⫽0.011). Conversely, the incidence of single gestation was 78% in group A and 25% in group B. Additional results are shown in the Table. CONCLUSION: These findings support the applicability of MsFR to selected patients. The significant morphologic improvement in the embryonic appearance along with a tendency to an improved PR may imply that MsFR provided intracellular mechanisms to “fight” cell derangement and death. Although some indicators showed benefits to MsFR, the fact that the IR observed in the MsFR group is perplexing. The small sample size, lack of strict criteria for fragment removal, use of chemical/mechanic technique instead of hydraulic, and lack of biomarkers for this microsurgical approach provide some reasoning for such evidence. Supported by: None.
P-542 Time- and media-dependent improvements in motility of testicular spermatozoa retrieved from non-obstructive azoospermic men. D. E. Davis, T. G. Schuster, M. R. Hiner, D. A. Ohl, G. D. Smith. University of Michigan, Ann Arbor, MI. OBJECTIVE: Testicular-derived spermatozoa motility, reflecting viability, is key to intracytoplasmic sperm injection success. Experiments were conducted to compare number of motile sperm isolated from testicular aspirations incubated in different media over time. DESIGN: Prospective side-by-side comparative laboratory study. MATERIALS AND METHODS: Diagnostic and/or therapeutic fine needle testicular aspirations were performed on 14 men with non-obstructive azoospermia. Seminiferous tubules were manually dissected and seminiferous epithelial content dispersed into a single cell suspension. Number of non-motile and motile sperm/20 high-powered fields was assessed initially at the time of cell dispersion and following incubation at 37°C for 24 and 48 hours in either HEPES buffered human tubal fluid ⫹ protein (H-HTF) in atmospheric conditions or Ham’s F10 ⫹ protein (F10) in 5% CO2 and air. Statistical analyses were performed with paired Student’s t-test and ANOVA with repeat measures. RESULTS: Initially following seminiferous tubule micro-dissection, total sperm found was 93 ⫾ 22 (mean ⫾ SE) and mean number of motile sperm/20 high-powered fields was 3 ⫾ 2. In H-HTF sperm motility increased in 8 of 14 samples after 24 hours of culture (18 ⫾ 9) and 11 of 14 samples after 48 hours of culture (19 ⫾ 9), although these increases were not significant. Mean number of motile sperm/20 high-powered fields significantly increased in all 14 samples following incubation in F10 for 24
FERTILITY & STERILITY威
hours (30 ⫾ 11, paired t-test, P⫽0.03) and 48 hours of culture (33 ⫾ 13, P⫽0.04). Testicular sperm motility increased significantly after incubation in F10 compared to HTF at both 24 and 48 hours of culture (repeated measures ANOVA, P⫽0.05). CONCLUSION: Incubation of spermatozoa from fine needle testicular aspirations facilitates motile spermatozoa isolation. More motile spermatozoa were isolated after incubation in F10 than in H-HTF. There was no significant increase in motile sperm isolated between 24 and 48 hours. Following seminiferous tubule micro-dissection, culture of testicular spermatozoa for 24 hours in Ham’s F10 is an efficient means of collecting motile spermatozoa for ICSI. Supported by: None.
P-543 Maker chromosome identification in infertility. A. Abderazek, H. Kayed, R. Mansour, O. Kamal,, M. Aboulghar. Egyptian IVF-ET Center, Cairo, Egypt. OBJECTIVE: A marker chromosome is a structurally abnormal chromosome in which no part can be identified. So far the identification of marker chromosomes has been of interest for cancer cytogenetics only. Molecular cytogenetics (fluorescence in situ hybridization:FISH) has been considered a very powerful research tool, in the understanding of many aspects of human biology. The aim of this study was to identify the origin of chromosome markers detected by G-banding in patients with infertility entering ICSI program. This is of great importance for genetic counseling and offering these patients chance to enter Perimplantation Genetic diagnosis (PGD) program. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 3540 patients, between August 2000 ⫺ January 2004, underwent chromosome analysis and genetic counseling before entering our IVF/ICSI program. Chromosomal analysis was indicated for couples with severe male factor infertility, azoospermia and/or other factors. Chromosomal analysis of peripheral blood lymphocytes culture was performed by standard cytogenetic technique, and banding using Giemsa trypsin banding (GTG) was done. In all cases, 12 metaphases were karyotyped. When any apparent chromosomal aberrations were detected, 20 metaphases were analyzed. Computerized Karyotyper (Cytovision 2.7) was used for observation and detection of chromosomal abnormalities. FISH with alpha satellite DNA (centromeric) and whole chromosome painting probes for chromosomes 13,14,15, 21 and 22 were used because the markers appeared to be a part of acrocentric chromosomes. RESULTS: Cytogenetic results were successfully obtained from all patients. Overall, 95.02 % of patients (3250 patients) had a normal karyotype, and 4.98% (170 patients) had abnormal karyotypes. In 6 cases (0.001 %), 4 males and 2 females a marker chromosome was identified. All of these marker chromosomes by G-banding appeared to be part of acrocentric chromosomes, so by using probes for these chromosomes we could identify these markers to be parts of chromosomes 13,15, and 22. Two males and one female had partial trisomy 13, a male and a female had partial trisomy 15, and one male had partial trisomy 22. CONCLUSION: The combined use of classical banding and FISH techniques is a useful tool in the identification of the origin of the unknown chromosome markers. It could be of significant importance for diagnosis and offering PGD for these patients to prevent the transmission of this anomaly to their offspring. Supported by: None
P-544 A microfluidic chemotaxis system to select motile and mature sperm. L. Karakoc Sokmensuer, S. Palaniappan, M. Toner, T. L. Toth, D. L. Wright. Massachusetts General Hospital, Boston, MA. OBJECTIVE: The evolution of IVF technology has been weighted heavily in the area of embryo culture with limited attention to sperm processing. Many of the current sperm processing techniques may be less than desirable due to damaging effects inflicted on the sample. This study describes the development of a microfluidic chemotaxis device for the purpose of selecting a highly motile, mature sperm fraction for potential use in IVF/ICSI.
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