hole. However, an aluminum block with a 51 mm inspection hole did notaffect the rate of cooling. Increasing the temperature of the surface to 45°Cdid not change the rate of cooling over 5 minutes. Since the polystreneplastic dish and the air pocket underneath the dish are poor thermal con-ductors, it is not surprising that the heated surface is inefficient in preventingheat loss.

P-354

The time interval between oocyte retrieval and ICSI can affect fertili-zation rate. D. Harris, R. D. Powers. Boston IVF, Waltham, MA.

Objective: To determine if there are optimal time intervals betweenoocyte retrieval, removal of cumulus cells and ICSI with respect to fertil-ization rates and pregnancy rates.

Design: Fertilization rates and pregnancy rates of ICSI cycles in whichthere were random time intervals between oocyte retrieval, cumulus cellremoval and insemination were compared by chi square analysis to deter-mine the optimal window of time for these procedures.

Materials/Methods: We compared fertilization and pregnancy rates of269 contiguous ICSI cycles at Boston IVF over a three month period in2000 with respect to time intervals between oocyte retrieval, denudation ofoocytes and ICSI.

Results: The difference in fertilization rates when oocytes were injected,2 h after retrieval (64.7%) and when oocytes were injected.5 2 h postretrieval (65.6%) was not statistically significant (p5 .7996). However, thefertilization rates were statistically different for oocytes injected,3 h postretrieval (63.2%) and.5 3 h post retrieval (67.3%) (p5 .0020). Thedifference in fertilization rates when oocytes were injected,3 h afterdenudation (65.7% ) and.5 3 h post denudation (69.8%) was not statis-tically significant (p 5 .0577). The difference in fertilization rates foroocytes injected 2 h post denudation (63.8%) and.5 2 h post denudation(68.7%) was significant (p5 .0370). In the group of cycles where the ICSItime minus the retrieval time (IT-Ret. T) was.5 3 and the ICSI time minusthe denuding time (IT-DT) was.5 2, the fertilization rate (69.7%) wasstatistically significantly higher than in the group where (IT – Ret. T),3and (IT-DT) 2 (63.3%) (p5 .0107). When the pregnancy rates in these twogroups were compared, there was a difference in the rates (58.5% vs.32.8%) but it was not statistically significant due to the limited number oftransfers in the smaller group.

Conclusions: Previously published reports have differed on the effect ofthe time of denudation and insemination on the success of ICSI cycles. Inour laboratory, it appears that the optimal time to perform ICSI is at least 3hours post retrieval and at least 2 hours post denudation of the oocytes.

Supported by: Boston IVF.

P-355

A randomized prospective comparison of sibling human oocytes/em-bryos cultured in two different media. J. F. Mayer, E. L. Jones, F.Nehchiri, S. J. Muasher, W. E. Gibbons, S. C. Oehninger. The JonesInstitute for Reproductive Medicine, Eastern Virginia Medical Sch, Nor-folk, VA.

Objective: To determine if culture medium without glucose or phosphateproduces superior clinical outcomes compared to a more complex culturemedium.

Design: Prospective randomized study of 126 IVF cycles from January2000 to Feb.2001 at an academic, tertiary care institution.

Materials/Methods: Mature oocytes were collected following standardcontrolled ovarian stimulation protocols. During retrieval, sibling oocyteswere randomly assigned to one of two groups. Group 1 oocytes werecultured and inseminated in “P1” media1 10% SSS (no glucose; phos-phate—Irvine Scientific, CA). All resulting embryos in Group 1 werecultured in “P1” media until day 3. If culture was extended beyond Day 3,embryos were subsequently cultured in “Blastocyst” media (Irvine Scien-tific). In Group 2, gametes were cultured and inseminated in Hams F10 with7.5% SSS and the resulting embryos were cultured in G1.2 media (IVFScience Scandinavia). If culture was extended beyond Day 3, embryos inGroup 2 were subsequently cultured in “G2.2” media (IVF Science). Allother culture conditions were identical between the two groups. On day 3 or5, embryos with the best morphology were chosen for fresh transfer.

Embryo morphology was graded on a scale of 1–5 with 1 being best. Alltransfers were to the uterine cavity on Day 3 or Day 5 and were performedidentically for the two groups.

Results: The results (summarized below in Table 1) were analyzed byChi-square.

Table 1. Comparison of outcomes in two culture media.

Media G1.2/G2.2 P1/Blastocyst Statistics

# oocytes 645 628% 2pn fertilization 74% (480) 74% (464) NS% 0pn no fertilization 12% (80) 12% (80) NS% 8-cell embryos

(day 3)47% (186/399) 34% (138/408) S (p5 0.0003)

Avg. # blastomeres(day 3)

6.66 0.11 5.96 0.12 S

Avg. embryo grade(day 3)

2.56 0.06 2.56 0.06 NS

% Blastocyst 40% (39/97) 38% (42/110) NS% Clinical Pregnancy*45% (15/33) 68% (13/19) NS (p. 0.15)

* All embryos transferred were from the same media,6 5 SEM; S 5significant; NS5 not significant.

Conclusions: These results demonstrate a statistically significant increasein the cleavage rate of embryos in the presence of glucose and phosphate(G1.2 medium). A higher percentage of the embryos are 8-cell on day 3 withculture in the G1.2 media. Interestingly, although not statistically significantwith the current number of cycles, there is a trend towards a higherpregnancy when the embryos in the P1 media (no glucose or phosphate)were selected for transfer. This study continues in order to resolve thequestion about the pregnancy potential of these media.

P-356

Comparison of two cryopreservation protocols for freezing humanspermatozoa. H. Kobayashi, P. Ranganathan, A. M. Mahran, R. K.Sharma, A. J. Thomas, A. Agarwal. The Cleveland Clinic Foundation,Cleveland, OH.

Objective: Sperm quality decreases significantly following freezing andresearch on improving cryosurvival rates is crucial. We compared theeffects of two cryopreservation protocols to determine which method allowsbetter preservation of sperm characteristics.

Design: Prospective study in an Andrology Laboratory.Materials/Methods: Each sample was divided into two aliquots after

liquefaction. Each aliquot was cryopreserved using two freezing protocols[Cleveland Clinic Foundation method (gradual freezing) and the IrvineScientific (rapid freezing) method] using TEST-yolk buffer as the freezingmedium. In the Cleveland Clinic Foundation method (CCF method) a 5 mLvial of freezing medium was thawed and an aliquot equal to 25% of theoriginal specimen volume was added to the specimen. This process wasrepeated four times to give a final ratio of 1:1 (v/v) of freezing medium toejaculate. These aliquots were placed in cryovials at220°C for 8 minutesfollowed by nitrogen vapors for 2 hours before being immersed in liquidnitrogen. In the Irvine Scientific method (IS method), the entire volume offreezing medium was added at one time and the specimens were immersedin liquid nitrogen. The step that included placing of cryovials at220°C for8 minutes was omitted. Prefreeze and post-thaw total sperm count, percent-age motility, sperm motion characteristics and morphology (Kruger andWHO) were evaluated. Motility was analyzed at 0, 60, 120, 180 minutesafter thawing.

Results: Percentage motility was significantly lower in postthaw samplescompared to prefreeze values in samples cryopreserved by the two methods.Postthaw sperm motility was greater in specimens processed by IS methodcompared to the CCF method (15.946 9.14 vs. 12.076 7.31; P, 0.006).In addition, percent cryosurvival was also greater in IS method compared tothe CCF method (47.426 17.44 vs. 35.766 17.56; P, 0.008). Morphol-ogy was similar with both methods.

Conclusions: Specimens cryopreserved by the IS method had a smallerdecrease in sperm motility over time when compared to the CCF method.The IS method for sperm cryopreservation is easy and gives good results. It

FERTILITY & STERILITY t S229