suitability of mouse embryos for human ivf qc: zygotes vs. 2-cell embryos

can be used routinely for long-term storage of human spermatozoa. Rapidfreezing of sperm in cryopreservation gives better survival rates comparedto gradual freezing, although one may assume that gradual acclimatizationto very low temperatures would maintain the functional integrity of thesperm to a greater extent. This as ascertained by morphology is the similarby both protocols thus refuting common belief.

Supported by: This project was supported by a grant from the ClevelandClinic Foundation.

P-357

Suitability of mouse embryos for human IVF QC: zygotes vs. 2-cellembryos. M. Li, K. C. Drury, R. S. Williams. Univ of Florida, Gainesville,FL.

Objective: Mouse embryos are widely used for quality control (QC) andmedia formulation in human IVF laboratories. The present study evaluatedtwo different stages of mouse embryo culture to test the suitability of newmedia.

Design: In-vivo fertilized mouse zygotes and 2 cell stage embryos de-rived from the same strain (B6C3F-1XB6D2F-1) were purchased frozenfrom Embryotech. Embryos were cultured in different media and endpointscompared using Chi square (p, 0.05).

Materials/Methods: Frozen–thawed zygotes and 2 cell stage mouse em-bryos were cultured (avg.5 20/droplet) in 100 ul droplets of mediasupplemented with 10% SSS (Irvine) under washed Squibb mineral oil.Two cell embryos were scored for # blastocyst on day 3 (D3) and # hatchedon day 4 (D4) of culture. Zygotes were scored for # blastocyst on D4 and# hatched on day 5 (D5) of culture. When sequential media were employed,embryos were washed in 3 ml prior to next step culture. During period oftesting, human IVF was performed using P-11 10% SSS for embryoculture with day 3 (8 cell) transfer.

Results: The implantation rate of human embryos transferred on D3during year 2000 using P-11 10% SSS was 22.4% (N5 126). Inaccordance, mouse 2 cell embryo development in the same medium resultedin 91.8% blastocyst (B) on D3 and 51.1% hatched (H) on D4 (N5 233)with good cell architecture. However, zygote culture in P-11 10% SSSgave only 29.1% B on D4 and just 1.2% H on D5 (N5 86) (p 5 ,0.01)with the majority undergoing degeneration at the morula stage. Theseresults are contrasted with the following more complex sequential mediausing mouse zygote stage: (1) P-1 for 48 hrs then Blastocyst medium(Irvine) 81.8% B, 18.2% H (N5 22); (2) Quinn’s Sequential (Q-Fert, Q-Cleave, Q-Blast-Biopharma) 78.0% B, 2.6% H (N5 38); (3) Quinn’sSequential with Insulin, Transferrin, Selenium (ITS- Sigma) added to Q-Blast. 95.5% B, 4.5% H (N5 22); (4) Quinn’s Q-Fert only. 98.4% B, 4.8%H (N 5 63) (p 0.05).

Conclusions: It has long been appreciated that although mouse embryosconstitute a biological test system for culture conditions, they are notori-ously permissive and resistant to levels of toxicity that can negativelyimpact human embryos. Cell cycle blocks, common in certain strains ofmouse embryos, may also not reflect specific conditions found in humans,although they have played a role in the understanding of glucose andphosphates in early human embryo culture. It is with interest then thatmouse zygotes did not respond well to P-11 10% SSS under the sameconditions where 2 cell embryos progressed to the blastocyst and hatchedstage at high rates (91.8% B, 51.1% H). At the same time, P-1 supportsreasonable human embryo implantation rates (22.4%) when embryos aretransferred at the 8 cell stage. In contrast to P-1, new media from Quinn(Q-Fert) supported blastocyst development from mouse zygotes at highlevels (98.4%) but lacked corresponding hatch rates (4.8%). We are con-tinuing to test prospective complex sequential media with a mouse zygotesystem in order to examine their suitability for human IVF laboratory use.Mouse zygotes are able to distinguish between culture media, but aredeficient in their ability to accurately reflect conditions relevant to use in theIVF laboratory.

Supported by: Department of Obstetrics and Gynecology, Shands Hos-pital at University of Florida.

P-358

Coculture with buffalo rat liver (BRL) cells enhanced day-3 embryodevelopment in poor prognosis patients.S. Shen, K. Ho, D. Jaffe, E.

Pritts, M. Cedars, V. Y. Fujimoto. Dept of Obstetrics, Gynecology andReproductive Science, Univ of CA, San Francisco, San Francisco, CA.

Objective: Methods to improve embryo quality are needed to enhanceIVF outcome. Coculture systems have been suggested to minimize embryofragmentation and improve blastomere development. A randomized con-trolled study showed BRL cell coculture significantly improved implanta-tion rates in patients undergoing conventional IVF (Hum. Reprod, Vol. 13,no.1, pp. 165–168, 1998). In our lab, we have been using a BRL cellcoculture system for patients who had failed previous cycles with poorembryo quality. For this study we examined day-3 embryo quality andpregnancy rates from coculture compared to regular culture system.

Design: A retrospective analysis of patients who had BRL cell cocultureat UCSF In Vitro Fertilization program.

Materials/Methods: IVF outcome and embryo quality were assessed incycles from 1999 and 2000 where the patients who had at least one cyclewithout coculture and one cycle with coculture. A total of 34 patientsincluded 38 cycles without coculture vs. 35 cycles with coculture. Day-3embryo quality was compared between two culture systems. Embryo qualitywas based on cleavage on day-3 and morphological “grade” relating tofragmentation. Grade 1 and 2 are considered good or fair embryos; Grade 3and 4 are poor quality. Clinical pregnancy was defined as the presence of agestational sac on ultrasound examination. Fisher exact and unpaired ttesting were used.

Results: Clinical pregnancy rates were 5/38 (13%) in regular culturecycles and 14/35 (40%) in coculture cycles (p5 0.015). However, ongoingpregnancy rates were not significantly different (8% in regular culturecycles vs. 20% in coculture cycles, p5 0.179).

The effect of BRL cell coculture on embryo development at 72 hour postretrieval.

Culture system

No. ofembryoscultured

No. ofblastomeres/

embryo% of6-cell Grade/embryo

Regular Culture 437 5.296 2.11 44% 2.306 1.03Coculture with BRL 342 6.466 2.05 69% 2.126 0.91P value ,0.0001 ,0.0001 50.011

Conclusions: These data provide evidence that, with BRL cell coculture,day-3 embryo development is significantly improved. Although randomizedstudies are needed to assess the impact of BRL cell coculture on pregnancyoutcome, these results support the theory of using BRL cell coculture toimprove embryo development and subsequent pregnancy rates for patientswith poor embryo quality during in vitro fertilization treatment.

P-359

Intracytoplasmic sperm injection using non-motile testicular sperma-tozoa selected by modified hypo-osmotic swelling test.H. Sallam, A.Rahaman, A. Eid, A. Sallam, A. A. Agameya. Alexandria Univ, Alexan-dria, Egypt; Alexandria Fertility Ctr, Alexandria, Egypt.

Objective: The aim of this work was to explore the fertilizing capacity oftotally non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test (consisting of 50% culture medium and 50% milli-Qwater), compared to motile testicular spermatozoa in patients with infertilitydue to non-obstructive azoospermia.

Design: A comparison of fertilization, clinical pregnancy and on-goingpregnancy rates in azoospermic patients with non-motile testicular sperma-tozoa selected by a modified hypo-osmotic swelling test versus azoospermicpatients with motile testicular spermatozoa.

Materials/Methods: Fifty five cycles in infertile couples due to non-obstructive azoospermia treated with intracytoplasmic sperm injection(ICSI), consisting of 16 cycles where ICSI was performed using non-motilespermatozoa and 39 cycles where ICSI was performed with motile testicularspermatozoa. In the 16 cycles with total absence of motility, the spermato-zoa used for injection were selected by a modified hypo-osmotic swellingtest (a solution consisting of 50% culture medium and 50% milli-Q water),while in the 39 cycles with motile spermatozoa, these were randomlyselected.

Results: The fertilization rate was 32.4% in the non-motile sperm group

S230 Abstracts Vol. 76, No. 3, Suppl. 1, September 2001