- Email: [email protected]
Multiple rearrangements of mitochondrial DNA and defective oxidative phosphorylation gene expression in unfertilized human oocytes.
than three mature oocytes were included. Cycles undergoing preimplantation genetic diagnosis or coculture because of multiple previous poor outcomes were excluded. For each cycle, the exact time for harvest, hyaluronidase treatment, and injection were entered into a computerized database. The periods between harvest to hyaluronidase, harvest to injection, and hyaluronidase to injection were calculated in minutes. Subsequently, four time groups were analyzed: a) less than 60 minutes; b) 60 to 119 minutes (1-2 hours); c) 120 to 239 minutes (2-4 hours); and d) 240 to 359 minutes (4-6 hours). Clinical pregnancy and implantation were compared using two-sided Chi-Square analysis. Results: Maternal age and number of conceptuses transferred were remarkably similar in each of the time groups. No significant differences in clinical pregnancy or implantation were found between any of the harvest to hyaluronidase or harvest to injection subgroups although a noticeable trend was seen in each for better results with shorter times. Significant differences were found, however, between subgroups of the hyaluronidase to injection category. Results are summarized below. Pregnancy and implantation in relation to hyaluronidase/injection intervals.
Minutes Less than 60 60–119 120–239 240–359 a
No. Cycles
Avg Age
Avg No. Tr
157 437 305 21
32.7 ⫾ 3.5 32.7 ⫾ 3.3 32.7 ⫾ 3.3 32.8 ⫾ 3.4
3.3 ⫾ 0.83 3.2 ⫾ 1.03 3.2 ⫾ 0.97 3.3 ⫾ 1.02
Clin Clin Preg/ Preg/ Sacs/No. Cycle Tr Tr (%) (%) (%) 56.1 54.5 51.5 47.6
56.8 56.4 53.0 52.6
29.8a 30.5b 24.4a,b 23.8
vidual grading parameters because the number of thawed blastocyst transfers is still low. The results were compared using Chi-Square analysis. Results: Generally, there is positive correlation between overall blastocyst quality and implantation rate (example: transfer of a 3AA blastocyst approaches a 70% implantation rate). Examining individual parameters, there was no significant difference between degree of blastocyst expansion and implantation rate (3 ⫽ 67%, 2 ⫽ 58% and 1 ⫽ 53% respectively). Also, no significant differences were found between ICM morphology and implantation rate (A ⫽ 68%, B ⫽ 62% and C ⫽ 61% respectively). In contrast, significantly higher implantation rates were achieved by transferring blastocysts with grade A trophectoderm (76%) compared to B (56%, p ⬍ 0.05) or C (50%, p ⬍ 0.05). A similar trend was observed upon analyzing blastocyst survival after thaw; blastocyst expansion and ICM morphology could not be correlated with blastocyst survival while better TM morphology was positively associated (A ⫽ 85%, B ⫽ 63%, and C ⫽ 62% respectively). The importance of a healthy TM is clearly indicated from these observations. Conclusions: The morphology of the blastocyst, measured with a 3-part grading system, significantly impacted implantation. The quality of the TM influences blastocyst implantation and post-thaw survival, while blastocyst expansion and ICM morphology appear to impact implantation to a lesser degree. The TM is likely the most important individual factor for implantation, as it plays a crucial role in blastocyst attachment, trophoblast development, and subsequent uterine invasion. Following implantation, a good quality ICM will direct embryo development. Supported By: Center for Reproductive Medicine and Infertility.
Monday, October 22, 2001 4:30 P.M.
p ⬍ 0.05; p ⬍ 0.005. b
Conclusions: A clear trend was observed for higher clinical pregnancy and implantation rates with shorter time periods between enzymatic treatment of oocytes and injection. Significance was reached where numbers were large in the implantation category. It may be advisable to perform injection procedures within two hours of cleaning oocytes in preparation for ICSI. Supported By: The Center for Reproductive Medicine and Infertility.
Monday, October 22, 2001 4:15 P.M. O-20 Blastocyst expansion, inner cell mass (ICM) formation, and trophectoderm (TM) quality: is one more important for implantation? N. Zaninovic, R. Berrios, R. N. Clarke, R. Bodine, Z. Ye, L. L. Veeck. Weill Medical Coll of Cornell Univ, New York, NY. Objective: The use of blastocyst culture for in vitro fertilization has been associated with an increase in implantation rates. Better synchronization between preembryo and endometrium and selection of conceptuses with a high implantation potential can be attributed to the success. To correlate morphology with implantation capability, a blastocyst grading system was developed. The system is based on overall expansion, and ICM and TM morphology. Correlation has been demonstrated between this grading system and subsequent implantation rates. However, no report has been published correlating blastocyst expansion, ICM, or TM quality as separate and individual parameters. It may be important to recall that the ICM will form the embryo, while TM, important for implantation, will give rise to the placenta. Design: Retrospective analysis of patients with day 5 transfer or thawed blastocyst transfer. Implantation rates were correlated to expansion, morphology of the ICM and morphology of the TM. Materials/Methods: Blastocyst transfer in 156 patients was performed on day 5. Blastocysts were graded based on blastocele expansion (1 ⫽ no expansion, 2 ⫽ slightly expanded and 3 ⫽ fully expanded); ICM morphology (A ⫽ compacted cells, B ⫽ loose cells, and C ⫽ no visible ICM), and; TM morphology (A ⫽ cohesive epithelium, B ⫽ uneven cells, and C ⫽ few cells). Only patients with known implantation results were included in study. In thawed cycles, only blastocyst survival was correlated with indi-
S8
Abstracts
O-21 Multiple rearrangements of mitochondrial DNA and defective oxidative phosphorylation gene expression in unfertilized human oocytes. R. Hsieh, N. Tsai, H. Au, S. Chang, Y. Cheng, C. Tzeng. Ctr for Reproductive Medicine, Dept of Obstetrics and Gynecology, Taipei Medical Univ Hosp, Taipei, Taiwan. Objective: To detect the mtDNA rearrangement and expression of oxidative phosphorylation genes in unfertilized human oocytes and arrested embryos and to evaluate the relationship between gene expression and oocyte quality. Design: Prospective laboratory research in a university hospital. Materials/Methods: Female patients for IVF including 124 oocytes, 98 embryos and 45 tripronucleate (3PN) embryos from 65 patients. Unfertilized oocytes and poor quality embryos collected at 48 hours after IVF. mtDNA rearrangements were detected by nested PCR and expression of oxidative phosphorylation genes were determined by RT-PCR. Results: Multiple deletions of mtDNA were found in unfertilized oocytes and arrested embryos obtained from IVF patients. There were 13 different deleted mtDNA molecules in unfertilized oocytes and embryos which were confirmed by nested PCR. The rearrangement was not associated with maternal age. However, type I deletion (n ⫽ 9) beared direct repeats flanking the breakpoints at the 5⬘ and 3⬘ ends of the deletion, whereas type II deletion (n ⫽ 4) contained no such feature. The 4977-bp deletion was the most common deletion in human oocytes and embryos. About 66.1% of unfertilized oocytes, 34.8% of arrested or fragmented embryos, and 21.1% of 3PN embryos harbored the 4977-bp deletion of mtDNA. Quantitation of the transcript levels from the mtDNA ND2, CO I, CO II, ATPase 6, CO III, ND3, ND6 and Cyt b genes in oocytes and embryos revealed that the transcript levels were decreased in unfertilized oocytes than in arrested embryos and 3PN embryos. Conclusions: A 4977-bp deletion is demonstrated to be the most common deletion in mtDNA from unfertilized oocytes and poor quality embryos in IVF patients. Aside from 4977-bp deletion, the heteroplasmy still exist in the mutant mtDNA. The frequency of mtDNA deletion in human embryos is lower than that in oocytes, suggesting that oocytes with superior quality are prone to higher fertilization rates. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction. Significantly decreased expression of oxidative phosphorylation genes also play an important role in jeopardizing embryo development. We conclude that the accumulation of modified and mutant mtDNA and defective genes expression may interfere
Vol. 76, No. 3, Suppl. 1, September 2001
with ATP production and thus impair oocyte fertilization and further embryo growth. Supported By: A grant from Taipei Medical University, Taiwan (TMC89Y05-A113).
Monday, October 22, 2001 4:45 P.M. O-22 Live births after ICSI with cryopreserved testicular sperm subjected to motility enhancement. T. S. Thomas, S. S. Howards, B. G. Bateman. Univ of Virginia, Charlottesville, VA. Objective: Intracytoplasmic sperm injection (ICSI) of testicular sperm is most efficacious when the sperm selected for injection is alive. Procurement of motile sperm is a clear means of ensuring vitality. In this study, testicular biopsies were subjected to motility enhancement either by testicular biopsy culture prior to cryopreservation, by exposure to chemical stimulants postthaw, or by the two combined. The results of a trial thaw were used to guide sperm preparation for assisted reproduction. Design: This case management study was performed with 7 couples in which the man was azoospermic because of obstruction. Testis biopsies were divided prior to freezing into cultured and non-cultured groups and further divided after thaw into media with or without a chemical stimulant. The four resulting groups were compared in yield of progressively motile sperm. The optimal procedure was used in subsequent attempts to fertilize the partner’s ova by ICSI. Materials/Methods: Testis biopsy from beneath the tunica albuginea was dissected into small fragments and frozen by a standard method immediately after surgery or after 48-72 hours of in vitro culture. For the trial, cultured and non-cultured specimens were thawed, divided again and washed in the presence or absence of 2 mM 2-deoxyadenosine ⫹ 3.5 mM pentoxifylline (later pentoxifylline alone). Each suspension was concentrated to 50 microliters, placed under oil, and evaluated for progressively motile sperm. On the day of egg retrieval, sperm were prepared by the least invasive method that had produced sufficient motile sperm in the trial. When stimulant was used, sperm were washed exhaustively in a stimulantfree drop of PVP prior to injection. Clinical outcomes were evaluated. Results: Progressively motile testicular sperm were rare in thawed specimens in the absence of biopsy culture. Cultured testicular specimens displayed motile sperm at the edge of the microdrop in 6/6 trial thaws without stimulant. However, progressively motile sperm increased 2.7-fold with stimulant. Thirteen attempts to conceive by ICSI were performed using thawed testicular sperm. The overall rates of 2pn formation, implantation, and clinical pregnancy were 74%, 24%, and 62%. The corresponding rates for ejaculated sperm in our program were 77%, 20% and 41%. Two of 13 attempts were made with sperm from biopsy culture alone and resulted in 2 pregnancies and 3 normal babies. One attempt performed with sperm from non-cultured biopsy exposed to stimulant resulted in a singleton live birth. Five of 10 attempts with sperm from biopsy culture and motility stimulant in combination resulted in clinical pregnancy. Of these, four patients delivered 6 normal offspring and one resulted in early loss. Conclusions: We confirmed reports that in vitro culture of testicular tissue results in stimulation of progressive sperm motility. In only 2 of 13 cases, however, were we confident that this alone would provide enough motile sperm for ICSI after a freeze-thaw. Post-thaw treatment of non-cultured or cultured testicular sperm with stimulant resulted in a significant enhancement of motility and allowed predictable availability of motile sperm for subsequent ICSI. Microinjection of ova with testicular sperm subjected to biopsy culture and/or motility stimulant treatment resulted in the birth of 10 normal infants and one first trimester loss. Supported By: Local funds.
ART: MALE FACTOR Monday, October 22, 2001 2:00 P.M. O-23 Successful formation of moving spermatozoa following the in vitro co-culture of round spermatids with Vero cells. A. Tanaka, I. Tanaka, M.
FERTILITY & STERILITY威
Nagayoshi, S. Awata, Y. Mawatari, H. Kusunoki. Dept of Obstet and Gynecol, Saint Mother Hosp, Fukuoka, Japan; Experimental Farm, Kobe Univ, Kasai, Japan. Objective: For men whose spermatogenesis is arrested at the level of round spermatid (R-ST), the round spermatid injection (ROSI) is the only fertility treatment available. However, ROSI is unlikely to be regarded as a certified treatment and many issues still remain to be solved. Main questions among them are low fertilization rate of ROSI and lack of security when using an immature spermatogenic cell. Therefore, we investigated the in vitro culture of R-ST with co-cultured Vero cells, to make them undergo a series of structural and biochemical modifications to improve the pregnancy rate. Design: Retrospective review of time-dependent structural changes of R-ST following the in vitro co-culture with Vero cells. Materials/Methods: This study deals with three kinds of azoospermic men whose spermatogenesis was arrested at the level of round spermatid (group A), elongating spermatid (group B) or whose testicular spermatozoa were completely abnormal in shape, in spite of their existence (group C). R-ST were collected from testicular biopsy specimens. Testis tissue was gently disentangled in RBC lysing buffer for hemolysis. Each sample was transferred into HTF containing BSA (5 mg/ml) and gently disentangled again. The seminiferous tubules were cut into fine pieces and then rinsed out through pipetting. Debris was filtered out using a nylon mesh filter (pore diameter: 30 –50 m) and the obtained suspension was centrifuged twice at 500 – 800 rpm (300 – 400 G) at 4°C. R-ST were identified from other round cells by their characteristic features (about 7 m diameter, a clear cytoplasm-nucleus boundary, presence of acrosomic granule adjacent to nuclear membrane and a prominent nucleolus) Selected 50⬃100 R-ST were interspersed over cultured Vero cells (Dainippon Pharmaceutical Co., Ltd. Japan) covered by light mineral oil in 100l micro drops on a plastic tissue culture plate in MEM ⫹ FSH (50 IU/L) ⫹ Testosterone (T) (1mol/L) (32.0°C, 5% CO2 in air). At the time of confluence, co-culture was started and continued for 7 days with a change of the medium being performed everyday. Morphological changes were observed on a Nikon inverted research microscope equipped with Hoffman optics. Results: The developmental rate of peripheral displacement of the nucleus, nuclear condensation, flagella development (not moving), moving flagella development, immatured spermatozoa, spermatozoa (not moving) and moving spermatozoa after the in vitro culture with Vero cells in three groups were as follows.A : k12% (12/100), 5% (5/100), 2% (2/100), 1%(1/100), 2%(2/100), 1%(1/100), 1% (1/100) lB : k19.5% (39/200), 13.5% (27/200), 5.5% (11/200), 2.0% (4/200), 10.5% (21/200), 1.0% (2/ 200), 1.0% (2/200) lC : k23.6%(224/950), 15.1%(143/950), 7.6% (72/950), 2.5% (24/950), 12.8% (122/950), 5.1% (48/950), 0.5% (5/950) l Conclusions: Although the number of newly developed normal spermatozoa was small, the moving normally shaped spermatozoa were obtained and late staged spermatids were produced at the high rate from R-ST collected from the non-obstructive azoospermic men following the in vitro co-culture with Vero cells. This in vitro co-culture system of R-ST with Vero cell maybe very useful for the treatment of non-obstructive azoospermic men.
Monday, October 22, 2001 2:15 P.M. O-24 Diagnostic and prognostic value of measurement of reactive oxygen species in neat semen. R. A. Saleh, N. Esfandiari, R. K. Sharma, D. R. Nelson, A. J. Thomas, A. Agarwal. The Cleveland Clinic Foundation, Cleveland, OH. Objective: Oxidative stress (OS)-induced damage to sperm plays a major role in the pathogenesis of male infertility. Seminal OS is more likely caused by excessive reactive oxygen species (ROS) generation, via abnormal sperm and activated leukocytes, rather than reduced antioxidant capacity. We postulate that ROS measurement in neat semen is reflective of OS in vivo as it represents the balance between ROS generation and scavenging. The objective of our study was to examine the correlation of ROS levels in neat semen with standard semen parameters. Design: Prospective study in a male infertility clinic. Materials/Methods: Semen samples obtained from infertile men (n ⫽ 34)
S9
Minutes Less than 60 60–119 120–239 240–359 a
No. Cycles
Avg Age
Avg No. Tr
157 437 305 21
32.7 ⫾ 3.5 32.7 ⫾ 3.3 32.7 ⫾ 3.3 32.8 ⫾ 3.4
3.3 ⫾ 0.83 3.2 ⫾ 1.03 3.2 ⫾ 0.97 3.3 ⫾ 1.02
Clin Clin Preg/ Preg/ Sacs/No. Cycle Tr Tr (%) (%) (%) 56.1 54.5 51.5 47.6
56.8 56.4 53.0 52.6
29.8a 30.5b 24.4a,b 23.8
vidual grading parameters because the number of thawed blastocyst transfers is still low. The results were compared using Chi-Square analysis. Results: Generally, there is positive correlation between overall blastocyst quality and implantation rate (example: transfer of a 3AA blastocyst approaches a 70% implantation rate). Examining individual parameters, there was no significant difference between degree of blastocyst expansion and implantation rate (3 ⫽ 67%, 2 ⫽ 58% and 1 ⫽ 53% respectively). Also, no significant differences were found between ICM morphology and implantation rate (A ⫽ 68%, B ⫽ 62% and C ⫽ 61% respectively). In contrast, significantly higher implantation rates were achieved by transferring blastocysts with grade A trophectoderm (76%) compared to B (56%, p ⬍ 0.05) or C (50%, p ⬍ 0.05). A similar trend was observed upon analyzing blastocyst survival after thaw; blastocyst expansion and ICM morphology could not be correlated with blastocyst survival while better TM morphology was positively associated (A ⫽ 85%, B ⫽ 63%, and C ⫽ 62% respectively). The importance of a healthy TM is clearly indicated from these observations. Conclusions: The morphology of the blastocyst, measured with a 3-part grading system, significantly impacted implantation. The quality of the TM influences blastocyst implantation and post-thaw survival, while blastocyst expansion and ICM morphology appear to impact implantation to a lesser degree. The TM is likely the most important individual factor for implantation, as it plays a crucial role in blastocyst attachment, trophoblast development, and subsequent uterine invasion. Following implantation, a good quality ICM will direct embryo development. Supported By: Center for Reproductive Medicine and Infertility.
Monday, October 22, 2001 4:30 P.M.
p ⬍ 0.05; p ⬍ 0.005. b
Conclusions: A clear trend was observed for higher clinical pregnancy and implantation rates with shorter time periods between enzymatic treatment of oocytes and injection. Significance was reached where numbers were large in the implantation category. It may be advisable to perform injection procedures within two hours of cleaning oocytes in preparation for ICSI. Supported By: The Center for Reproductive Medicine and Infertility.
Monday, October 22, 2001 4:15 P.M. O-20 Blastocyst expansion, inner cell mass (ICM) formation, and trophectoderm (TM) quality: is one more important for implantation? N. Zaninovic, R. Berrios, R. N. Clarke, R. Bodine, Z. Ye, L. L. Veeck. Weill Medical Coll of Cornell Univ, New York, NY. Objective: The use of blastocyst culture for in vitro fertilization has been associated with an increase in implantation rates. Better synchronization between preembryo and endometrium and selection of conceptuses with a high implantation potential can be attributed to the success. To correlate morphology with implantation capability, a blastocyst grading system was developed. The system is based on overall expansion, and ICM and TM morphology. Correlation has been demonstrated between this grading system and subsequent implantation rates. However, no report has been published correlating blastocyst expansion, ICM, or TM quality as separate and individual parameters. It may be important to recall that the ICM will form the embryo, while TM, important for implantation, will give rise to the placenta. Design: Retrospective analysis of patients with day 5 transfer or thawed blastocyst transfer. Implantation rates were correlated to expansion, morphology of the ICM and morphology of the TM. Materials/Methods: Blastocyst transfer in 156 patients was performed on day 5. Blastocysts were graded based on blastocele expansion (1 ⫽ no expansion, 2 ⫽ slightly expanded and 3 ⫽ fully expanded); ICM morphology (A ⫽ compacted cells, B ⫽ loose cells, and C ⫽ no visible ICM), and; TM morphology (A ⫽ cohesive epithelium, B ⫽ uneven cells, and C ⫽ few cells). Only patients with known implantation results were included in study. In thawed cycles, only blastocyst survival was correlated with indi-
S8
Abstracts
O-21 Multiple rearrangements of mitochondrial DNA and defective oxidative phosphorylation gene expression in unfertilized human oocytes. R. Hsieh, N. Tsai, H. Au, S. Chang, Y. Cheng, C. Tzeng. Ctr for Reproductive Medicine, Dept of Obstetrics and Gynecology, Taipei Medical Univ Hosp, Taipei, Taiwan. Objective: To detect the mtDNA rearrangement and expression of oxidative phosphorylation genes in unfertilized human oocytes and arrested embryos and to evaluate the relationship between gene expression and oocyte quality. Design: Prospective laboratory research in a university hospital. Materials/Methods: Female patients for IVF including 124 oocytes, 98 embryos and 45 tripronucleate (3PN) embryos from 65 patients. Unfertilized oocytes and poor quality embryos collected at 48 hours after IVF. mtDNA rearrangements were detected by nested PCR and expression of oxidative phosphorylation genes were determined by RT-PCR. Results: Multiple deletions of mtDNA were found in unfertilized oocytes and arrested embryos obtained from IVF patients. There were 13 different deleted mtDNA molecules in unfertilized oocytes and embryos which were confirmed by nested PCR. The rearrangement was not associated with maternal age. However, type I deletion (n ⫽ 9) beared direct repeats flanking the breakpoints at the 5⬘ and 3⬘ ends of the deletion, whereas type II deletion (n ⫽ 4) contained no such feature. The 4977-bp deletion was the most common deletion in human oocytes and embryos. About 66.1% of unfertilized oocytes, 34.8% of arrested or fragmented embryos, and 21.1% of 3PN embryos harbored the 4977-bp deletion of mtDNA. Quantitation of the transcript levels from the mtDNA ND2, CO I, CO II, ATPase 6, CO III, ND3, ND6 and Cyt b genes in oocytes and embryos revealed that the transcript levels were decreased in unfertilized oocytes than in arrested embryos and 3PN embryos. Conclusions: A 4977-bp deletion is demonstrated to be the most common deletion in mtDNA from unfertilized oocytes and poor quality embryos in IVF patients. Aside from 4977-bp deletion, the heteroplasmy still exist in the mutant mtDNA. The frequency of mtDNA deletion in human embryos is lower than that in oocytes, suggesting that oocytes with superior quality are prone to higher fertilization rates. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction. Significantly decreased expression of oxidative phosphorylation genes also play an important role in jeopardizing embryo development. We conclude that the accumulation of modified and mutant mtDNA and defective genes expression may interfere
Vol. 76, No. 3, Suppl. 1, September 2001
with ATP production and thus impair oocyte fertilization and further embryo growth. Supported By: A grant from Taipei Medical University, Taiwan (TMC89Y05-A113).
Monday, October 22, 2001 4:45 P.M. O-22 Live births after ICSI with cryopreserved testicular sperm subjected to motility enhancement. T. S. Thomas, S. S. Howards, B. G. Bateman. Univ of Virginia, Charlottesville, VA. Objective: Intracytoplasmic sperm injection (ICSI) of testicular sperm is most efficacious when the sperm selected for injection is alive. Procurement of motile sperm is a clear means of ensuring vitality. In this study, testicular biopsies were subjected to motility enhancement either by testicular biopsy culture prior to cryopreservation, by exposure to chemical stimulants postthaw, or by the two combined. The results of a trial thaw were used to guide sperm preparation for assisted reproduction. Design: This case management study was performed with 7 couples in which the man was azoospermic because of obstruction. Testis biopsies were divided prior to freezing into cultured and non-cultured groups and further divided after thaw into media with or without a chemical stimulant. The four resulting groups were compared in yield of progressively motile sperm. The optimal procedure was used in subsequent attempts to fertilize the partner’s ova by ICSI. Materials/Methods: Testis biopsy from beneath the tunica albuginea was dissected into small fragments and frozen by a standard method immediately after surgery or after 48-72 hours of in vitro culture. For the trial, cultured and non-cultured specimens were thawed, divided again and washed in the presence or absence of 2 mM 2-deoxyadenosine ⫹ 3.5 mM pentoxifylline (later pentoxifylline alone). Each suspension was concentrated to 50 microliters, placed under oil, and evaluated for progressively motile sperm. On the day of egg retrieval, sperm were prepared by the least invasive method that had produced sufficient motile sperm in the trial. When stimulant was used, sperm were washed exhaustively in a stimulantfree drop of PVP prior to injection. Clinical outcomes were evaluated. Results: Progressively motile testicular sperm were rare in thawed specimens in the absence of biopsy culture. Cultured testicular specimens displayed motile sperm at the edge of the microdrop in 6/6 trial thaws without stimulant. However, progressively motile sperm increased 2.7-fold with stimulant. Thirteen attempts to conceive by ICSI were performed using thawed testicular sperm. The overall rates of 2pn formation, implantation, and clinical pregnancy were 74%, 24%, and 62%. The corresponding rates for ejaculated sperm in our program were 77%, 20% and 41%. Two of 13 attempts were made with sperm from biopsy culture alone and resulted in 2 pregnancies and 3 normal babies. One attempt performed with sperm from non-cultured biopsy exposed to stimulant resulted in a singleton live birth. Five of 10 attempts with sperm from biopsy culture and motility stimulant in combination resulted in clinical pregnancy. Of these, four patients delivered 6 normal offspring and one resulted in early loss. Conclusions: We confirmed reports that in vitro culture of testicular tissue results in stimulation of progressive sperm motility. In only 2 of 13 cases, however, were we confident that this alone would provide enough motile sperm for ICSI after a freeze-thaw. Post-thaw treatment of non-cultured or cultured testicular sperm with stimulant resulted in a significant enhancement of motility and allowed predictable availability of motile sperm for subsequent ICSI. Microinjection of ova with testicular sperm subjected to biopsy culture and/or motility stimulant treatment resulted in the birth of 10 normal infants and one first trimester loss. Supported By: Local funds.
ART: MALE FACTOR Monday, October 22, 2001 2:00 P.M. O-23 Successful formation of moving spermatozoa following the in vitro co-culture of round spermatids with Vero cells. A. Tanaka, I. Tanaka, M.
FERTILITY & STERILITY威
Nagayoshi, S. Awata, Y. Mawatari, H. Kusunoki. Dept of Obstet and Gynecol, Saint Mother Hosp, Fukuoka, Japan; Experimental Farm, Kobe Univ, Kasai, Japan. Objective: For men whose spermatogenesis is arrested at the level of round spermatid (R-ST), the round spermatid injection (ROSI) is the only fertility treatment available. However, ROSI is unlikely to be regarded as a certified treatment and many issues still remain to be solved. Main questions among them are low fertilization rate of ROSI and lack of security when using an immature spermatogenic cell. Therefore, we investigated the in vitro culture of R-ST with co-cultured Vero cells, to make them undergo a series of structural and biochemical modifications to improve the pregnancy rate. Design: Retrospective review of time-dependent structural changes of R-ST following the in vitro co-culture with Vero cells. Materials/Methods: This study deals with three kinds of azoospermic men whose spermatogenesis was arrested at the level of round spermatid (group A), elongating spermatid (group B) or whose testicular spermatozoa were completely abnormal in shape, in spite of their existence (group C). R-ST were collected from testicular biopsy specimens. Testis tissue was gently disentangled in RBC lysing buffer for hemolysis. Each sample was transferred into HTF containing BSA (5 mg/ml) and gently disentangled again. The seminiferous tubules were cut into fine pieces and then rinsed out through pipetting. Debris was filtered out using a nylon mesh filter (pore diameter: 30 –50 m) and the obtained suspension was centrifuged twice at 500 – 800 rpm (300 – 400 G) at 4°C. R-ST were identified from other round cells by their characteristic features (about 7 m diameter, a clear cytoplasm-nucleus boundary, presence of acrosomic granule adjacent to nuclear membrane and a prominent nucleolus) Selected 50⬃100 R-ST were interspersed over cultured Vero cells (Dainippon Pharmaceutical Co., Ltd. Japan) covered by light mineral oil in 100l micro drops on a plastic tissue culture plate in MEM ⫹ FSH (50 IU/L) ⫹ Testosterone (T) (1mol/L) (32.0°C, 5% CO2 in air). At the time of confluence, co-culture was started and continued for 7 days with a change of the medium being performed everyday. Morphological changes were observed on a Nikon inverted research microscope equipped with Hoffman optics. Results: The developmental rate of peripheral displacement of the nucleus, nuclear condensation, flagella development (not moving), moving flagella development, immatured spermatozoa, spermatozoa (not moving) and moving spermatozoa after the in vitro culture with Vero cells in three groups were as follows.A : k12% (12/100), 5% (5/100), 2% (2/100), 1%(1/100), 2%(2/100), 1%(1/100), 1% (1/100) lB : k19.5% (39/200), 13.5% (27/200), 5.5% (11/200), 2.0% (4/200), 10.5% (21/200), 1.0% (2/ 200), 1.0% (2/200) lC : k23.6%(224/950), 15.1%(143/950), 7.6% (72/950), 2.5% (24/950), 12.8% (122/950), 5.1% (48/950), 0.5% (5/950) l Conclusions: Although the number of newly developed normal spermatozoa was small, the moving normally shaped spermatozoa were obtained and late staged spermatids were produced at the high rate from R-ST collected from the non-obstructive azoospermic men following the in vitro co-culture with Vero cells. This in vitro co-culture system of R-ST with Vero cell maybe very useful for the treatment of non-obstructive azoospermic men.
Monday, October 22, 2001 2:15 P.M. O-24 Diagnostic and prognostic value of measurement of reactive oxygen species in neat semen. R. A. Saleh, N. Esfandiari, R. K. Sharma, D. R. Nelson, A. J. Thomas, A. Agarwal. The Cleveland Clinic Foundation, Cleveland, OH. Objective: Oxidative stress (OS)-induced damage to sperm plays a major role in the pathogenesis of male infertility. Seminal OS is more likely caused by excessive reactive oxygen species (ROS) generation, via abnormal sperm and activated leukocytes, rather than reduced antioxidant capacity. We postulate that ROS measurement in neat semen is reflective of OS in vivo as it represents the balance between ROS generation and scavenging. The objective of our study was to examine the correlation of ROS levels in neat semen with standard semen parameters. Design: Prospective study in a male infertility clinic. Materials/Methods: Semen samples obtained from infertile men (n ⫽ 34)
S9