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Does laser assisted hatching of residual embryos on day 3 affect blastocyst development?
mately 16 sperm million/ml and 55 million sperm/ml. Laboratories were instructed to perform sperm counting procedures on the survey samples using the lab’s routine methodology, and to report the results, as well the methodology and type of counting chamber used, to the AAB. RESULTS: The number of participating laboratories over the 6 events ranged from 470 –519. The vast majority of laboratories used manual procedures (83.4%) compared with computer assisted semen analysis (CASA; 16.6%). The most common counting chamber used by laboratories performing manual procedures was the hemacytometer (47.0%), followed by the Makler, Micro Cell and Cell Vu chambers (34.6%, 13.6% and 4.8%, respectively). Of those laboratories using CASA procedures, 56.5% used the Makler chamber while the remainder used the Micro Cell chamber (43.5%). In order to determine which counting chamber tended to “overestimate” or “underestimate” sperm counts, the grand mean sperm count was calculated for all participants within each challenge, and these values were compared with the mean results for each counting chamber group. Manual cell counts using the Cell Vu chamber tended to overestimate sperm counts the greatest (mean ⫾ SE; 119.6 ⫾ 12.7%) while CASA cell counts using the Micro Cell chamber tended to underestimate the most (75.3 ⫾ 1.6%). Interestingly, cell counts using the Makler for both the manual and CASA methods came closest to the grand mean (101.8 ⫾ 4.21% and 98.8 ⫾ 2.6%, respectively). Overall, a wide range of variation was noted in the reported results. Coefficients of variation (CV) ranged from a low of 20.4% to a high of 392.9%. Manual counts using the hemacytometer yielded the highest mean CV (124.2 ⫾ 26.0%) while manual counts using the Makler chamber demonstrated the lowest mean CV (32.0 ⫾ 1.7%). CONCLUSION: These data indicate that the majority of laboratories participating in the AAB andrology PT program performed manual counting methods and used the hematytometer for counting sperm. However, this procedure was also associated with the greatest variation. Although disposable counting chambers, such as the Cell Vu and Micro Cell, have become widely used, and may be more convenient compared with other chambers, manual counting methods using the Makler chamber appeared to yield the most reliable and consistent results. Supported by: None
RESULTS:
In the 34 patient cycles included in this study, 67.6% resulted in pregnancy after transfer with an implantation rate of 37%. In these 34 cycles, 157 embryos were designated to the laser-hatching group and 153 were not assisted hatched. Forty-three laser-hatched embryos reached the blastocyst stage (27.4%) and were cryopreserved. Forty non-laser assisted-hatched embryos developed to blastocysts (26.1%) and were cryopreserved. Blastocyst formation rates between the two groups did not reach statistical significance. (Chi-Square, p⫽0.80455246). CONCLUSION: Assisted hatching of non-transferred embryos by retrospective analysis had previously shown a small, but statistically significant enhancement in blastocyst formation (Portmann et al., ASRM, 2003). The results of that study were potentially skewed by the inclusion of higher quality hatched, but non-transferred embryos. In this prospective analysis, and with better randomization, there appears to be no impact of laser hatching upon embryo development to the blastocyst stage. This finding, albeit with a relatively small number of embryos, is reassuring as assisted hatching techniques shift towards greater utilization of the laser. Supported by: None
P-551
P-550 Does laser assisted hatching of residual embryos on day 3 affect blastocyst development? M. P. Portmann, L. S. Morrison, D. R. Prinz, B. A. McGuirk, R. F. Feinberg, M. J. Tucker. Reproductive Associates of Delaware, Newark, DE; Georgia Reproductive Specialists, Atlanta, GA. OBJECTIVE: To prospectively determine the impact of Laser Assisted Hatching (LAH) on blastocyst formation in spare, non-transferred day 3 embryos. DESIGN: Spare, non-transferred embryos were randomized prospectively on day 3 to LAH or non-LAH based on odd/even number designation. All hatched embryos designated for actual or possible transfer were excluded from the analysis. All spare embryos containing at least 2 blastomeres on day 3 were included. For this prospective study, the two-tailed power analysis calculation was based on a prior spare blastocyst formation rate of 35%, with a clinically relevant range deemed to be 20% to 50%. To reach 80% power with an alpha error less than 0.05, our analysis required 138 embryos in the LAH and non-LAH group. The study required approximately 12 months. MATERIALS AND METHODS: Oocytes were retrieved in HTF (InVitrocare—IVC, Frederick, MD), hyaluronidased after 2 to 3 hours incubation and ICSI’ed one to three hours following cumulus-corona removal. Oocytes were placed in IVC-1 (IVC) after ICSI and cultured individually in this media until Day 3. Embryos were placed into CCM (Vitrolife, Denver, CO) on the morning of Day 3 for extended culture. The best embryos were identified and laser hatched prior to transfer using the Zilos laser system from Hamilton Thorne (settings: one pulse; 0.500 milliseconds duration). After transfer, all remaining odd numbered embryos were assisted hatched. Even numbered embryos were left alone. Morphologic assessment occurred on Day 2, 3, 5 and 6. Blastocysts were cryopreserved on Day 5, 6 or 7. All transfers occurred on Day 3 using a Wallace 23cm stylet (Irvine Scientific, Irvine, CA) and Cook Echotip Catheter (Cook OB/GYN, Spencer, IN) under abdominal ultrasound guidance and were performed by the same physician.
S330
Pre-washed frozen donor sperm and frozen donor semen processed immediately prior to insemination have comparable pregnancy rates. C. M. Crane, M. J. Pultorak, J. N. Barnes, K. M. Doody, K. J. Doody. Center for Assisted Reproduction, Bedford, TX. OBJECTIVE: Commercial donor sperm banks now promote pre-washed frozen sperm. This allows intrauterine inseminations to be performed by physicians without access to a reproductive/andrology laboratory. Many of these banks are discussing, or choosing, to discontinue the use of unwashed (regular/ ICI ready) sperm vials. Additionally, those banks that are still offering a choice between regular and pre-washed (IUI ready) vials are now producing fewer regular vials. Previous data has shown a higher progressive motility and pregnancy rate with the use of regular donor sperm vials. The purpose of this study is to do a follow-up comparison of sperm parameters and cycle outcome in women undergoing intrauterine insemination (IUI) with regular and pre-washed donor sperm vials. DESIGN: Retrospective study comparing women undergoing LH timed, Clomid and FSH stimulated intrauterine insemination with regular or prewashed frozen donor sperm from February 1, 2001 through December 31, 2003. MATERIALS AND METHODS: Patients ordered donor sperm from a commercial bank based on donor characteristics and availability. Prewashed specimens were centrifuged over a density gradient and re-suspended in a yolk buffer freezing medium to achieve a desired density of motile sperm prior to cryopreservation. These specimens were thawed and analyzed immediately prior to insemination and required no further preparation. Regular specimens were combined with the freezing medium, centrifuged (no density gradient), and re-suspended as necessary to achieve a desired density of motile sperm prior to freezing. These specimens are thawed approximately 1 hour before the insemination, analyzed, and processed via centrifugation over a density gradient. Regular specimens are additionally analyzed following processing and prior to IUI. All specimens were analyzed by computer assisted semen analysis (CASA-IVOS/Hamilton Thorne). Final pre-insemination sperm parameters and cycle outcomes were recorded.
Vol. 82, Suppl. 2, September 2004
RESULTS:
In the 34 patient cycles included in this study, 67.6% resulted in pregnancy after transfer with an implantation rate of 37%. In these 34 cycles, 157 embryos were designated to the laser-hatching group and 153 were not assisted hatched. Forty-three laser-hatched embryos reached the blastocyst stage (27.4%) and were cryopreserved. Forty non-laser assisted-hatched embryos developed to blastocysts (26.1%) and were cryopreserved. Blastocyst formation rates between the two groups did not reach statistical significance. (Chi-Square, p⫽0.80455246). CONCLUSION: Assisted hatching of non-transferred embryos by retrospective analysis had previously shown a small, but statistically significant enhancement in blastocyst formation (Portmann et al., ASRM, 2003). The results of that study were potentially skewed by the inclusion of higher quality hatched, but non-transferred embryos. In this prospective analysis, and with better randomization, there appears to be no impact of laser hatching upon embryo development to the blastocyst stage. This finding, albeit with a relatively small number of embryos, is reassuring as assisted hatching techniques shift towards greater utilization of the laser. Supported by: None
P-551
P-550 Does laser assisted hatching of residual embryos on day 3 affect blastocyst development? M. P. Portmann, L. S. Morrison, D. R. Prinz, B. A. McGuirk, R. F. Feinberg, M. J. Tucker. Reproductive Associates of Delaware, Newark, DE; Georgia Reproductive Specialists, Atlanta, GA. OBJECTIVE: To prospectively determine the impact of Laser Assisted Hatching (LAH) on blastocyst formation in spare, non-transferred day 3 embryos. DESIGN: Spare, non-transferred embryos were randomized prospectively on day 3 to LAH or non-LAH based on odd/even number designation. All hatched embryos designated for actual or possible transfer were excluded from the analysis. All spare embryos containing at least 2 blastomeres on day 3 were included. For this prospective study, the two-tailed power analysis calculation was based on a prior spare blastocyst formation rate of 35%, with a clinically relevant range deemed to be 20% to 50%. To reach 80% power with an alpha error less than 0.05, our analysis required 138 embryos in the LAH and non-LAH group. The study required approximately 12 months. MATERIALS AND METHODS: Oocytes were retrieved in HTF (InVitrocare—IVC, Frederick, MD), hyaluronidased after 2 to 3 hours incubation and ICSI’ed one to three hours following cumulus-corona removal. Oocytes were placed in IVC-1 (IVC) after ICSI and cultured individually in this media until Day 3. Embryos were placed into CCM (Vitrolife, Denver, CO) on the morning of Day 3 for extended culture. The best embryos were identified and laser hatched prior to transfer using the Zilos laser system from Hamilton Thorne (settings: one pulse; 0.500 milliseconds duration). After transfer, all remaining odd numbered embryos were assisted hatched. Even numbered embryos were left alone. Morphologic assessment occurred on Day 2, 3, 5 and 6. Blastocysts were cryopreserved on Day 5, 6 or 7. All transfers occurred on Day 3 using a Wallace 23cm stylet (Irvine Scientific, Irvine, CA) and Cook Echotip Catheter (Cook OB/GYN, Spencer, IN) under abdominal ultrasound guidance and were performed by the same physician.
S330
Pre-washed frozen donor sperm and frozen donor semen processed immediately prior to insemination have comparable pregnancy rates. C. M. Crane, M. J. Pultorak, J. N. Barnes, K. M. Doody, K. J. Doody. Center for Assisted Reproduction, Bedford, TX. OBJECTIVE: Commercial donor sperm banks now promote pre-washed frozen sperm. This allows intrauterine inseminations to be performed by physicians without access to a reproductive/andrology laboratory. Many of these banks are discussing, or choosing, to discontinue the use of unwashed (regular/ ICI ready) sperm vials. Additionally, those banks that are still offering a choice between regular and pre-washed (IUI ready) vials are now producing fewer regular vials. Previous data has shown a higher progressive motility and pregnancy rate with the use of regular donor sperm vials. The purpose of this study is to do a follow-up comparison of sperm parameters and cycle outcome in women undergoing intrauterine insemination (IUI) with regular and pre-washed donor sperm vials. DESIGN: Retrospective study comparing women undergoing LH timed, Clomid and FSH stimulated intrauterine insemination with regular or prewashed frozen donor sperm from February 1, 2001 through December 31, 2003. MATERIALS AND METHODS: Patients ordered donor sperm from a commercial bank based on donor characteristics and availability. Prewashed specimens were centrifuged over a density gradient and re-suspended in a yolk buffer freezing medium to achieve a desired density of motile sperm prior to cryopreservation. These specimens were thawed and analyzed immediately prior to insemination and required no further preparation. Regular specimens were combined with the freezing medium, centrifuged (no density gradient), and re-suspended as necessary to achieve a desired density of motile sperm prior to freezing. These specimens are thawed approximately 1 hour before the insemination, analyzed, and processed via centrifugation over a density gradient. Regular specimens are additionally analyzed following processing and prior to IUI. All specimens were analyzed by computer assisted semen analysis (CASA-IVOS/Hamilton Thorne). Final pre-insemination sperm parameters and cycle outcomes were recorded.
Vol. 82, Suppl. 2, September 2004