p-257 sperm motility as a determinate of the outcome of intrauterine insemination (iui);...

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dium was GEM plus 5 mg/ml BSA, and growth medium was GEM with additional 80#M EDTA and no BSA. Results: Average patient age (35), infertility factors and rates of fertilization and cleavage were not statistically different among the groups in Series A and Series B. Four- teen (31%) of the 45 patients in Series A achieved ongoing clinical pregnancies. Of the total of 57 embryos transferred into the pregnant patients, 18 (32%) developed into appar- ently normal offspring. Six (27%) of the 22 patients in Series B achieved ongoing clinical pregnancies, including one 41 year old undergoing her seventh IVF attempt. Of the total of 20 embryos transferred into the 6 pregnant Series B patients, 9 (45%) developed into apparently nor- mal offspring. The increased surface tension of the pro- tein-free medium necessitated more extensive catheter rinsing prior to embryo transfer. Conclusions: These results demonstrate that protein is not essential in the EDTA-containing culture medium used in these IVF centers. The results from Series A sug- gest that sperm-egg interaction does not require protein, although a beneficial role of protein during sperm purifi- cation is not excluded. Series B embryos cleaved and trans- ferred without protein also underwent high rates of im- plantation and development. The use of fully defined, protein-free culture medium for human IVF will markedly improve the safety of embryos and mothers by avoiding exposure to potential pathogens in serum and albumin preparations. (a) Biol of Repro 41:835; (b)Ibid 43:600; (c)Fertil & Steril. 60:1088. P-256 The Fate of Cryopreserved Human Preembryos. 1R. M. Shelden, 1J. Beloni, 1G. H. Corsan, 1E. Kemmann. ~Department of Obstetrics, Gynecology and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Objective: The fate of abandoned cryopreserved human pre-embryos has received recent attention as the result of a 1991 UK law requiring destruction of 'abandoned' hu- man pre-embryos which had been kept frozen for more than 5 years. We were interested in determining how simi- lar legislation in the US would have impacted our human pre-embryo cryopreservation program. Design: Retrospective analysis of the fate of all human pre-embryos cryopreserved in our program, from January, 1988 through December, 1996. Material and Methods: Since the inception of our pre- embryo cryopreservation program, couples have signed consents detailing the fate of their cryopreserved pre-em- bryos. Options were cryopreservation for future use by the couple, donation to another party, or donation to Institu- tional Review Board approved research protocols. More recently, pre-embryo destruction has been recognized as an option. In addition, pre-embryo fate was defined by the patient in the event of patient debility, divorce, or death. Couples were contacted annually relative to storage fees for continued maintenance of the pre-embryos. Results: Since the start of our cryopreservation pro- gram, 576 patients have had 3,665 pre-embryos cryopre- served for an average of 6.4 pre-embryos per patient. Sixty-two percent of these embryos have been thawed and, if morphologically acceptable, transferred to the patient in a frozen pre-embryo replacement cycle. In addition to pre-embryo replacement, 113 (3.1%) cryopreserved pre- embryos have been transported to other Assisted Repro- ductive Technology programs upon patient request. A lim- ited number of cryopreserved pre-embryos (124, 3.3%) have been donated for research, and 6 patients have re- quested that their 26 (0.7%) pre-embryos be destroyed. Continued cryostorage includes 167 patients (1 to 27 pre- embryos cryopreserved) with 1,149 pre-embryos. Of these, 110 pre-embryos have been in cryostorage for five years or longer. We have lost contact with 5 patients (33 pre- embryos). Conclusion: The majority of cryopreserved pre-embryos in our program have been returned or are expected, at this time, to be returned to the couple of origin. Less than 1% of the cryopreserved pre-embryos in our program would have been affected by a regulation similar to that imposed in the UK relative to continued storage of abandoned pre- embryos. P-257 Sperm Motility as a Determinate of the Outcome of Intrauterine Insemination (IUI); Retrospective and Prospective Analyses. lB. A. Stone, 2j. M. Vargyas, 2G. E. Ringler, 2A. L. Stein, 1R. Faridi, 1E. Ong. 1Inst. for Fertility Research and ~California Fertility Assoc., Santa Monica, CA. Objectives: By retrospective ANOVA of 7,123 consecu- tive IUI cycles in this center, patient age was the main determinant of outcome (F ratio = 23.05), followed by number of follicles (F=9.36) and motility of sperm in the inseminate (F=4.37). When sperm motility in the insemi- nate was <20%, pregnancy occured in 5.5% of IUI cycles, compared with 8.7% conceptions with motility 20-40%, 13.0% with motility 40-60%, and 16.8% pregnancies with motility >60% (P<0.001). With respect to the capacity for pentoxifylline (Pentox) to promote sperm motility, we studied effects of Pentox treatment of ejaculated sperm on CASA properties of the inseminate, and on outcomes of IUI. Design: Prospective analysis (ANOVA) of effects of Pen- tox on properties of processed semen, and of pregnancy outcomes following IUI of specimens processed with or without Pentoxifylline (Chi2). Materials and Methods: Before enrichment of motile sperm fractions through silica gradients, liquefied semen specimens were diluted (1:1, v/v) with HEPES-HTF (Con- trol; 517 cycles) or with HEPES-HTF containing 6.0 mM Pentox (81 cycles). The specimens were immediately over- laid onto dual gradient silica columns, and centrifuged at 200g for 20 minutes. Precipitated sperm pellets were washed and resuspended in HEPES-HTF for IUI. Pre and post-wash semen specimens were analysed by CASA. Results: Prewash semen parameters did not differ be- tween Control and Pentox groups. After processing for IUI, the following average properties of Pentox specimens ex- Abstracts $215

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Page 1: P-257 Sperm motility as a determinate of the outcome of intrauterine insemination (IUI); retrospective and prospective analyses

dium was GEM plus 5 mg/ml BSA, and growth medium was GEM with additional 80#M EDTA and no BSA.

Results: Average patient age (35), infertility factors and rates of fertilization and cleavage were not statistically different among the groups in Series A and Series B. Four- teen (31%) of the 45 patients in Series A achieved ongoing clinical pregnancies. Of the total of 57 embryos transferred into the pregnant patients, 18 (32%) developed into appar- ently normal offspring. Six (27%) of the 22 patients in Series B achieved ongoing clinical pregnancies, including one 41 year old undergoing her seventh IVF attempt. Of the total of 20 embryos transferred into the 6 pregnant Series B patients, 9 (45%) developed into apparently nor- mal offspring. The increased surface tension of the pro- tein-free medium necessitated more extensive catheter rinsing prior to embryo transfer.

Conclusions: These results demonstrate that protein is not essential in the EDTA-containing culture medium used in these IVF centers. The results from Series A sug- gest that sperm-egg interaction does not require protein, although a beneficial role of protein during sperm purifi- cation is not excluded. Series B embryos cleaved and trans- ferred without protein also underwent high rates of im- plantation and development. The use of fully defined, protein-free culture medium for human IVF will markedly improve the safety of embryos and mothers by avoiding exposure to potential pathogens in serum and albumin preparations. (a) Biol of Repro 41:835; (b)Ibid 43:600; (c)Fertil & Steril. 60:1088.

P-256

The Fate of Cryopreserved Human Preembryos. 1R. M. Shelden, 1J. Beloni, 1G. H. Corsan, 1E. Kemmann. ~Department of Obstetrics, Gynecology and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ.

Objective: The fate of abandoned cryopreserved human pre-embryos has received recent attention as the result of a 1991 UK law requiring destruction of 'abandoned' hu- man pre-embryos which had been kept frozen for more than 5 years. We were interested in determining how simi- lar legislation in the US would have impacted our human pre-embryo cryopreservation program.

Design: Retrospective analysis of the fate of all human pre-embryos cryopreserved in our program, from January, 1988 through December, 1996.

Material and Methods: Since the inception of our pre- embryo cryopreservation program, couples have signed consents detailing the fate of their cryopreserved pre-em- bryos. Options were cryopreservation for future use by the couple, donation to another party, or donation to Institu- tional Review Board approved research protocols. More recently, pre-embryo destruction has been recognized as an option. In addition, pre-embryo fate was defined by the patient in the event of patient debility, divorce, or death. Couples were contacted annually relative to storage fees for continued maintenance of the pre-embryos.

Results: Since the s tar t of our cryopreservation pro- gram, 576 patients have had 3,665 pre-embryos cryopre-

served for an average of 6.4 pre-embryos per patient. Sixty-two percent of these embryos have been thawed and, if morphologically acceptable, transferred to the patient in a frozen pre-embryo replacement cycle. In addition to pre-embryo replacement, 113 (3.1%) cryopreserved pre- embryos have been transported to other Assisted Repro- ductive Technology programs upon patient request. A lim- ited number of cryopreserved pre-embryos (124, 3.3%) have been donated for research, and 6 patients have re- quested that their 26 (0.7%) pre-embryos be destroyed. Continued cryostorage includes 167 patients (1 to 27 pre- embryos cryopreserved) with 1,149 pre-embryos. Of these, 110 pre-embryos have been in cryostorage for five years or longer. We have lost contact with 5 patients (33 pre- embryos).

Conclusion: The majority of cryopreserved pre-embryos in our program have been returned or are expected, at this time, to be returned to the couple of origin. Less than 1% of the cryopreserved pre-embryos in our program would have been affected by a regulation similar to that imposed in the UK relative to continued storage of abandoned pre- embryos.

P-257

Sperm Motility as a Determinate of the Outcome of Intrauterine Insemination (IUI); Retrospective and Prospective Analyses. lB. A. Stone, 2j. M. Vargyas, 2G. E. Ringler, 2A. L. Stein, 1R. Faridi, 1E. Ong. 1Inst. for Fertility Research and ~California Fertility Assoc., Santa Monica, CA.

Objectives: By retrospective ANOVA of 7,123 consecu- tive IUI cycles in this center, patient age was the main determinant of outcome (F ratio = 23.05), followed by number of follicles (F=9.36) and motility of sperm in the inseminate (F=4.37). When sperm motility in the insemi- nate was <20%, pregnancy occured in 5.5% of IUI cycles, compared with 8.7% conceptions with motility 20-40%, 13.0% with motility 40-60%, and 16.8% pregnancies with motility >60% (P<0.001). With respect to the capacity for pentoxifylline (Pentox) to promote sperm motility, we studied effects of Pentox t reatment of ejaculated sperm on CASA properties of the inseminate, and on outcomes of IUI.

Design: Prospective analysis (ANOVA) of effects of Pen- tox on properties of processed semen, and of pregnancy outcomes following IUI of specimens processed with or without Pentoxifylline (Chi2).

Materials and Methods: Before enrichment of motile sperm fractions through silica gradients, liquefied semen specimens were diluted (1:1, v/v) with HEPES-HTF (Con- trol; 517 cycles) or with HEPES-HTF containing 6.0 mM Pentox (81 cycles). The specimens were immediately over- laid onto dual gradient silica columns, and centrifuged at 200g for 20 minutes. Precipitated sperm pellets were washed and resuspended in HEPES-HTF for IUI. Pre and post-wash semen specimens were analysed by CASA.

Results: Prewash semen parameters did not differ be- tween Control and Pentox groups. After processing for IUI, the following average properties of Pentox specimens ex-

Abstracts $215

Page 2: P-257 Sperm motility as a determinate of the outcome of intrauterine insemination (IUI); retrospective and prospective analyses

ceeded (P<0.05) respective control values; motility (68% for Pentox vs 57% for Control), percentage of motile sperm >25#m/sec (54 vs 43%), and the percentage of motile sperm >25 #m/sec and >80% linear (46 vs 35%). Neither follicle numbers nor the average total number of motile sperm inseminated differed between groups. Despite a higher average age of patients in the Pentox group (40.0_+0.5 yrs c.f. 38.9_+0.2 for Controls; P<0.05), the preg- nancy rate was higher (16.5%/cycle c.f. 9.6% for Control; P<0.05).

Conclusions: The higher conception rate in patients after IUI with inseminates of higher motility is consistent with reports that sperm necrosis precipitates release of lipid peroxidases which compromize the viability of living cells. Immotile sperm in the inseminate will also obstruct motile sperm. While the higher conception rate following sperm processing with Pentox may result from higher sperm motility, it may also reflect other effects of pentoxi- fylline on sperm function.

P - 2 5 8

Cel lsep: A N a t u r a l D e n s i t y Grad ien t M e d i u m for S p e r m S e p a r a t i o n D e r i v e d F r o m t h e Wood o f Larch Trees . 1I. Khan, ~M. Urich, ~C. Khoury, 1C. H0dges, 2M. H. Fakih. lIVF/Andrology Laboratory and 2FIRST IVF, Sagi- naw General North, Saginaw, MI.

Objective: This study was carried out to determine if Cellsep, a natural polysaccharide density gradient solu- tion, could be utilized in place of Percoll for human sperm selection for IVF and ICSI.

Design: Following parameters were checked: 1) Toxicity against fresh mouse and triploid human embryos, 2) effec- tiveness of various density gradients for optimum sperm selection, 3) rates of fertilization, cleavage and pregnan- cies in humans.

Materials and Methods: Cellsep universal solution (Larex, Inc.) is made of highly purified arabinogalactan, a water soluble polymer extracted from the wood of Larch trees. I t has a density of 1.12g/ml. Various iso-osmotic gradients were obtained by diluting Cellsep solution with Hepes buffered HTF medium. The pH and osmolality ranged between 7.3-7.4 and 280-293 morns, respectively. Toxicity test was performed by injecting 10 #l of 95% Cell- sep solution to 100 #l droplets of culture medium con- taining fresh mouse zygotes. Development of mouse and triploid human embryos was assessed by blastocyst forma- tion. Human sperm survival was also checked. Several density gradients were tested to obtain optimum yield of sperm. Best results were obtained with a single gradient of 80% when sperm values were normal. For such cases, 1.2 ml well liquefied semen was loaded on 0.8 ml of 80% Cellsep solution, centrifuged for 20 minutes at 300 g, pellet and 0.5 ml of 80% solution was washed 2×. Semen with lower than 5 mill/ml sperm gave best results with two gradients, i.e., 47% and 95%. For IVF and ICSI, standard procedures were used.

Results: No deleterious effect of Cellsep solution was noticed when 10#1 of Cellsep solution was introduced in culture medium with developing embryos. Forty seven out

of 58 mouse zygotes reached blastocyst stage (81%). Simi- larly, six out of ten triploid human embryos reached the blastocyst stage. Cellsep selected sperm were used for con- secutive eleven IVF and 9 ICSI cycles. Results are pre- sented in following tables:

Sperm values for IVF cycles

Cycles Conc. % Mot

Pre-wash 11 159 mill/ml 61% Post-wash 64 mill/ml 91%

Conc. Mot Velocity

Pre-wash 100 mill/ml 31 microns/sec Post-wash 59 mill/ml 61 microns/sec

Fertilization, cleavage and pregnancy rates after using Cellsep selected sperm with IVF and ICSI

Cycles Occ Ins 2PN 3PN

IVF 11 164 141 (86%) 11 (7%) ICSI 9 90 70 (78%) 3 (3%)

Cleaved Good emb Pregnancy

IVF 118 (92%)* 99 (84%) 7 (64%) ICSI 54 (98%)* 42 (78%) 4 (44%)

* = 2/11 (IVF), 3/9 (ICSI) were ZIFT.

Conclusions: Our results show that Cellsep is safe and sperm recovered from single and double density gradients were instrumental in excellent fertilization, cleavage and pregnancy rates. For IVF and ICSI, it should be preferred over synthetic coated particles.

P - 2 5 9

Cl in ica l Ef f icacy a n d Cost E f f e c t i v e n e s s o f a Micro- d o s e GnRH-a P r o t o c o l C o m p a r e d to a Lutea l P h a s e GnRH-a P r o t o c o l for C o n t r o l l e d O v a r i a n Hyper- s t i m u l a t i o n . 1M. P. Leondires, 3B. T. Miller, 1M. Escalpes, 2B. J. Rogers, 1j. H. Segars, 2D. L. Gehlbach, eR. T. Scott. 1National Institutes of Health, eWalter Reed Reproductive Science Center, ~National Naval Medical Center, Bethesda, MD.

Objective: To examine the effectiveness of a follicular phase microdose GnRH agonist (GnRH-a; leuprolide ace- tate) protocol for the stimulation of IVF patients as com- pared to a common luteal phase GnRH-a protocol.

Design: Analysis of 132 IVF patients entered serially in 1996. The leuprolide acetate group (LA) consisted of sixty- one women (23-39 yrs) treated with luteal phase GnRH- a. The microdose leuprolide acetate group (MICRO) con- sisted of seventy-one women (25-39 yrs) treated with a follicular phase microdose GnRH-a protocol. Mean Ages: LA=31.13 _ 3.77 (_+SD); MICRO=32.3 _+ 2.74 (p=0.051). All diagnoses of infertility were included.

Interventions: The LA group was treated with GnRH-a (1 mg/day) until pituitary desensitization was docu-

8216 Abstracts