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Effect of semen sample collection site on semen analysis parameters and intrauterine insemination (IUI) pregnancy rates
density gradient temperatures (4 C, 25 C or 37 C) with 3-layers: 95%/70%/ 45% (Suprasperm, Origio). DGS, samples were washed 2X, with final pellets suspended in 0.3mL WM for monitoring of motility and progression immediately, 3, 24 and 48 hrs post preparation. Two complete replicates were averaged and analyzed using c2. RESULTS: Drops in motility were observed at 3 and 24 hrs across each temperature treatment, and significantly different (p<0.05) only after 48 hrs. The 4 C gradient resulted in an 82.6% drop in motility after 48 hrs compared to 65.4% and 63.0% decreases for 37 C and 25 C, respectively. CONCLUSION: DGS with 4 C gradient significantly effected sperm motility only after 48 hrs. It is possible cold gradient placed increased stress on the oxidative metabolism and energy needed for motility over an extended period. This study demonstrated the importance of validating manufacturer protocols and ensuring the quality of their products in producing acceptable results. We also demonstrated the importance of temperature QC during procedures. No differences were noted between experimental replicates testing the repeatability of sample processing. We continue longitudinal analyses for temperature related changes in motility, progression and recovery during sperm processing.
P-506 Wednesday, October 27, 2010 MODIFIABLE FACTORS THAT MAY IMPROVE MOTILE YIELD IN SPERM CRYOPRESERVATION. J. M. Hotaling, C. H. Muller, E. R. Pagel, H. J. Christianson, T. J. Walsh. Department of Urology, University of Washington, Seattle, WA. OBJECTIVE: To identify modifiable, laboratory factors that may improve the yield of motile sperm following cryopreservation of ejaculated samples. DESIGN: Retrospective review, Academic Medical Center. MATERIALS AND METHODS: We performed retrospective review of laboratory data from the University of Washington Male Infertility Laboratory from 1994-2009. 1000 semen samples from 423 men that underwent cryopreservation and at least one test thaw were available. We analyzed semen analysis and cryopreservation technique variables to determine predictors of total motile sperm yield at test thaw. Variables analyzed included sperm concentration (106/mL), motility (%), sperm motility enhancement (none, pentoxifylline (PX), pentoxifylline + deoxyadenosine (PX/DOA), preparation medium (HTF, HamsF10) and freezing rate (rapid and slow). Iterative multivariate linear regression models were generated using the predictor variables. Test-thaw motility, total motile count (TMC) and the change in these parameters from the raw samples were assessed as outcomes of interest. RESULTS: The strongest predictor of sperm motility and TMC after cryopreservation is raw semen TMC. The percent recovery of TMC and % motility after test-thaw increased significantly by 24% and 4% for each quartile of improvement in raw TMC. When raw semen parameters are controlled for, PX and PX/DOA yielded significant improvements in motile sperm recovery, +76.0% (95%CI 53.3-98.6) and +73.7% (95%CI 44.9-102.5). The use of HamsF10 (compared to HTF) and rapid freezing (compared to slow) demonstrated minor improvements in motile sperm recovery, +7.1% (95%CI 3.0-11.2) and +4.6% (95%CI 1.4-7.8). CONCLUSION: Raw semen TMC is the best predictor of %motility and TMC after cryopreservation. PX and PX/DOA yielded large improvements in % motility with Hams F10 and rapid freezing technique yielding minor improvements. Sperm motility enhancement and choice of optimal buffer and freeze protocol can improve sperm survival post-freeze.
P-507 Wednesday, October 27, 2010 A NOVEL CLOSED SYSTEM VIAL WITH SENTINEL TEST SEGMENT FOR SPERM CRYOPRESERVATION. E. J. Woods, L. Newton, J. K. Critser. General BioTechnology, LLC, Indianapolis, IN; Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN; College of Veterinary Medicine, University Of Missouri, Columbia, MO. OBJECTIVE: This study investigated a novel closed system vial (CellSealÔ, Indianapolis, IN, USA) for cryopreservation of human spermatozoa. DESIGN: This study was designed to determine if the system could maintain its container closure integrity (CCI) under liquid nitrogen (LN2) submersion and still maintain post-thaw motility comparable to controls. An attached segment integral to the vial was also evaluated as a sentinel for representative testing, again evaluating post-thaw motility as compared to the control as well as the vial body.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: First, vials frozen/thawed with saline or bacterial support medium were subjected to CCI testing by dye or bacterial immersion, respectively, under pressure or vacuum. Dye ingress was measured using spectrophotometry and bacterial ingress was measured via CFU assay. Next, a total of 9 semen samples from 3 different donors were processed for long term storage using either a standard 2ml Corning cryovial (control), a 2ml CellSeal cryovial and the CellSeal vial integral test segment. Each sample was then frozen for at least 24 hours at -196 C under liquid nitrogen. After freezing, samples were then evaluated for post-thaw motility using standard methods. Means (normalized to non-frozen control) and standard error of the mean were calculated and compared statistically via student’s t-test. RESULTS: All vials passed the CCI testing with no dye or bacterial ingress. Post-thaw samples were comparable among the control and experimental groups and no difference in motility as per t-test comparing post-thaw control (58 2%) to the CellSeal vials (57 2%; P¼0.70); post-thaw control to the segment (52 2%; P¼0.25); or segment to vial body (57 2%; P¼0.32) was observed. CONCLUSION: The novel closed system vials present a means of assuring sample integrity even under liquid nitrogen and allows measurement of post-thaw motility of a specimen without thawing the primary container. Supported by: Genome Resources, a division of General BioTechnology, LLC.
P-508 Wednesday, October 27, 2010 EFFECT OF SEMEN SAMPLE COLLECTION SITE ON SEMEN ANALYSIS PARAMETERS AND INTRAUTERINE INSEMINATION (IUI) PREGNANCY RATES. J. M. Biggs, K. S. Richter, J. Osheroff, E. A. Widra. Department of Obstetrics and Gynecology, Georgetown University Hospital, Washington, DC; Shady Grove Fertility Reproductive Science Center, Rockville, MD. OBJECTIVE: To determine whether semen analysis parameters and IUI pregnancy outcomes differ between office and home semen sample collection. DESIGN: Retrospective review. MATERIALS AND METHODS: All semen preparations for IUI cycles performed from 2004-09 were reviewed. Semen was prepared for insemination by density gradient centrifugation. Analysis was restricted to the first cycle per couple during the review period, and limited to non-frozen samples collected on the morning of insemination. Men were given the option of collecting semen either on site in a collection room at the office or at home. Age, volume, concentration, motility and total motile sperm (TMS) are reported as means and compared by t-test. Progression and time between collection and processing are reported as medians and compared by Mann-Whitney U test. Clinical pregnancy (gestational sac) was compared among the subset of couples diagnosed with unexplained infertility and stimulated with a standard CC/FSH protocol. RESULTS: See Table.
Patients Collection to analysis time (min) Age (years) Pre-Wash Vol (ml) Pre-Wash Conc Pre-Wash Mot (%) Pre Wash Prog Pre-Wash TMS Post-Wash Conc Post-Wash Mot (%) Post-Wash Prog Post-Wash TMS Pregnancy/cycle
Office
Home
P-value
4100 20
4606 60
<0.0001
36.7 2.7 65.9 62.2 2/2+ 107 78.3 78.4 2+/3 20.2 174/1047 (16.6%)
36.7 2.5 67.3 61.9 2 105 78.6 76.9 2+ 19.1 213/1341 (15.9%)
0.95 <0.0001 0.16 0.40 <0.0001 0.26 0.80 <0.0001 <0.0001 0.0053 0.63
CONCLUSION: There were statistically significant differences in favor of on site collection for pre-wash volume and progression and post-wash motility, progression, and TMS. However, all differences in semen parameters were clinically insignificant despite a 3-fold difference in the median time between collection and processing. There was no difference in pregnancy rates between the groups.
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P-506 Wednesday, October 27, 2010 MODIFIABLE FACTORS THAT MAY IMPROVE MOTILE YIELD IN SPERM CRYOPRESERVATION. J. M. Hotaling, C. H. Muller, E. R. Pagel, H. J. Christianson, T. J. Walsh. Department of Urology, University of Washington, Seattle, WA. OBJECTIVE: To identify modifiable, laboratory factors that may improve the yield of motile sperm following cryopreservation of ejaculated samples. DESIGN: Retrospective review, Academic Medical Center. MATERIALS AND METHODS: We performed retrospective review of laboratory data from the University of Washington Male Infertility Laboratory from 1994-2009. 1000 semen samples from 423 men that underwent cryopreservation and at least one test thaw were available. We analyzed semen analysis and cryopreservation technique variables to determine predictors of total motile sperm yield at test thaw. Variables analyzed included sperm concentration (106/mL), motility (%), sperm motility enhancement (none, pentoxifylline (PX), pentoxifylline + deoxyadenosine (PX/DOA), preparation medium (HTF, HamsF10) and freezing rate (rapid and slow). Iterative multivariate linear regression models were generated using the predictor variables. Test-thaw motility, total motile count (TMC) and the change in these parameters from the raw samples were assessed as outcomes of interest. RESULTS: The strongest predictor of sperm motility and TMC after cryopreservation is raw semen TMC. The percent recovery of TMC and % motility after test-thaw increased significantly by 24% and 4% for each quartile of improvement in raw TMC. When raw semen parameters are controlled for, PX and PX/DOA yielded significant improvements in motile sperm recovery, +76.0% (95%CI 53.3-98.6) and +73.7% (95%CI 44.9-102.5). The use of HamsF10 (compared to HTF) and rapid freezing (compared to slow) demonstrated minor improvements in motile sperm recovery, +7.1% (95%CI 3.0-11.2) and +4.6% (95%CI 1.4-7.8). CONCLUSION: Raw semen TMC is the best predictor of %motility and TMC after cryopreservation. PX and PX/DOA yielded large improvements in % motility with Hams F10 and rapid freezing technique yielding minor improvements. Sperm motility enhancement and choice of optimal buffer and freeze protocol can improve sperm survival post-freeze.
P-507 Wednesday, October 27, 2010 A NOVEL CLOSED SYSTEM VIAL WITH SENTINEL TEST SEGMENT FOR SPERM CRYOPRESERVATION. E. J. Woods, L. Newton, J. K. Critser. General BioTechnology, LLC, Indianapolis, IN; Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN; College of Veterinary Medicine, University Of Missouri, Columbia, MO. OBJECTIVE: This study investigated a novel closed system vial (CellSealÔ, Indianapolis, IN, USA) for cryopreservation of human spermatozoa. DESIGN: This study was designed to determine if the system could maintain its container closure integrity (CCI) under liquid nitrogen (LN2) submersion and still maintain post-thaw motility comparable to controls. An attached segment integral to the vial was also evaluated as a sentinel for representative testing, again evaluating post-thaw motility as compared to the control as well as the vial body.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: First, vials frozen/thawed with saline or bacterial support medium were subjected to CCI testing by dye or bacterial immersion, respectively, under pressure or vacuum. Dye ingress was measured using spectrophotometry and bacterial ingress was measured via CFU assay. Next, a total of 9 semen samples from 3 different donors were processed for long term storage using either a standard 2ml Corning cryovial (control), a 2ml CellSeal cryovial and the CellSeal vial integral test segment. Each sample was then frozen for at least 24 hours at -196 C under liquid nitrogen. After freezing, samples were then evaluated for post-thaw motility using standard methods. Means (normalized to non-frozen control) and standard error of the mean were calculated and compared statistically via student’s t-test. RESULTS: All vials passed the CCI testing with no dye or bacterial ingress. Post-thaw samples were comparable among the control and experimental groups and no difference in motility as per t-test comparing post-thaw control (58 2%) to the CellSeal vials (57 2%; P¼0.70); post-thaw control to the segment (52 2%; P¼0.25); or segment to vial body (57 2%; P¼0.32) was observed. CONCLUSION: The novel closed system vials present a means of assuring sample integrity even under liquid nitrogen and allows measurement of post-thaw motility of a specimen without thawing the primary container. Supported by: Genome Resources, a division of General BioTechnology, LLC.
P-508 Wednesday, October 27, 2010 EFFECT OF SEMEN SAMPLE COLLECTION SITE ON SEMEN ANALYSIS PARAMETERS AND INTRAUTERINE INSEMINATION (IUI) PREGNANCY RATES. J. M. Biggs, K. S. Richter, J. Osheroff, E. A. Widra. Department of Obstetrics and Gynecology, Georgetown University Hospital, Washington, DC; Shady Grove Fertility Reproductive Science Center, Rockville, MD. OBJECTIVE: To determine whether semen analysis parameters and IUI pregnancy outcomes differ between office and home semen sample collection. DESIGN: Retrospective review. MATERIALS AND METHODS: All semen preparations for IUI cycles performed from 2004-09 were reviewed. Semen was prepared for insemination by density gradient centrifugation. Analysis was restricted to the first cycle per couple during the review period, and limited to non-frozen samples collected on the morning of insemination. Men were given the option of collecting semen either on site in a collection room at the office or at home. Age, volume, concentration, motility and total motile sperm (TMS) are reported as means and compared by t-test. Progression and time between collection and processing are reported as medians and compared by Mann-Whitney U test. Clinical pregnancy (gestational sac) was compared among the subset of couples diagnosed with unexplained infertility and stimulated with a standard CC/FSH protocol. RESULTS: See Table.
Patients Collection to analysis time (min) Age (years) Pre-Wash Vol (ml) Pre-Wash Conc Pre-Wash Mot (%) Pre Wash Prog Pre-Wash TMS Post-Wash Conc Post-Wash Mot (%) Post-Wash Prog Post-Wash TMS Pregnancy/cycle
Office
Home
P-value
4100 20
4606 60
<0.0001
36.7 2.7 65.9 62.2 2/2+ 107 78.3 78.4 2+/3 20.2 174/1047 (16.6%)
36.7 2.5 67.3 61.9 2 105 78.6 76.9 2+ 19.1 213/1341 (15.9%)
0.95 <0.0001 0.16 0.40 <0.0001 0.26 0.80 <0.0001 <0.0001 0.0053 0.63
CONCLUSION: There were statistically significant differences in favor of on site collection for pre-wash volume and progression and post-wash motility, progression, and TMS. However, all differences in semen parameters were clinically insignificant despite a 3-fold difference in the median time between collection and processing. There was no difference in pregnancy rates between the groups.
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