- Email: [email protected]
Suitability of mouse embryos for human IVF QC: zygotes vs. 2-cell embryos.
can be used routinely for long-term storage of human spermatozoa. Rapid freezing of sperm in cryopreservation gives better survival rates compared to gradual freezing, although one may assume that gradual acclimatization to very low temperatures would maintain the functional integrity of the sperm to a greater extent. This as ascertained by morphology is the similar by both protocols thus refuting common belief. Supported by: This project was supported by a grant from the Cleveland Clinic Foundation.
P-357 Suitability of mouse embryos for human IVF QC: zygotes vs. 2-cell embryos. M. Li, K. C. Drury, R. S. Williams. Univ of Florida, Gainesville, FL. Objective: Mouse embryos are widely used for quality control (QC) and media formulation in human IVF laboratories. The present study evaluated two different stages of mouse embryo culture to test the suitability of new media. Design: In-vivo fertilized mouse zygotes and 2 cell stage embryos derived from the same strain (B6C3F-1XB6D2F-1) were purchased frozen from Embryotech. Embryos were cultured in different media and endpoints compared using Chi square (p ⬍ 0.05). Materials/Methods: Frozen–thawed zygotes and 2 cell stage mouse embryos were cultured (avg. ⫽ 20/droplet) in 100 ul droplets of media supplemented with 10% SSS (Irvine) under washed Squibb mineral oil. Two cell embryos were scored for # blastocyst on day 3 (D3) and # hatched on 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 of testing, human IVF was performed using P-1 ⫹ 10% SSS for embryo culture with day 3 (8 cell) transfer. Results: The implantation rate of human embryos transferred on D3 during year 2000 using P-1 ⫹ 10% SSS was 22.4% (N ⫽ 126). In accordance, mouse 2 cell embryo development in the same medium resulted in 91.8% blastocyst (B) on D3 and 51.1% hatched (H) on D4 (N ⫽ 233) with good cell architecture. However, zygote culture in P-1 ⫹ 10% SSS gave only 29.1% B on D4 and just 1.2% H on D5 (N ⫽ 86) (p ⫽ ⬍0.01) with the majority undergoing degeneration at the morula stage. These results are contrasted with the following more complex sequential media using mouse zygote stage: (1) P-1 for 48 hrs then Blastocyst medium (Irvine) 81.8% B, 18.2% H (N ⫽ 22); (2) Quinn’s Sequential (Q-Fert, QCleave, Q-Blast-Biopharma) 78.0% B, 2.6% H (N ⫽ 38); (3) Quinn’s Sequential with Insulin, Transferrin, Selenium (ITS- Sigma) added to QBlast. 95.5% B, 4.5% H (N ⫽ 22); (4) Quinn’s Q-Fert only. 98.4% B, 4.8% H (N ⫽ 63) (p 0.05). Conclusions: It has long been appreciated that although mouse embryos constitute a biological test system for culture conditions, they are notoriously permissive and resistant to levels of toxicity that can negatively impact human embryos. Cell cycle blocks, common in certain strains of mouse embryos, may also not reflect specific conditions found in humans, although they have played a role in the understanding of glucose and phosphates in early human embryo culture. It is with interest then that mouse zygotes did not respond well to P-1 ⫹ 10% SSS under the same conditions where 2 cell embryos progressed to the blastocyst and hatched stage at high rates (91.8% B, 51.1% H). At the same time, P-1 supports reasonable human embryo implantation rates (22.4%) when embryos are transferred at the 8 cell stage. In contrast to P-1, new media from Quinn (Q-Fert) supported blastocyst development from mouse zygotes at high levels (98.4%) but lacked corresponding hatch rates (4.8%). We are continuing to test prospective complex sequential media with a mouse zygote system in order to examine their suitability for human IVF laboratory use. Mouse zygotes are able to distinguish between culture media, but are deficient in their ability to accurately reflect conditions relevant to use in the IVF laboratory. Supported by: Department of Obstetrics and Gynecology, Shands Hospital at University of Florida.
P-358 Coculture with buffalo rat liver (BRL) cells enhanced day-3 embryo development in poor prognosis patients. S. Shen, K. Ho, D. Jaffe, E.
S230
Abstracts
Pritts, M. Cedars, V. Y. Fujimoto. Dept of Obstetrics, Gynecology and Reproductive Science, Univ of CA, San Francisco, San Francisco, CA. Objective: Methods to improve embryo quality are needed to enhance IVF outcome. Coculture systems have been suggested to minimize embryo fragmentation and improve blastomere development. A randomized controlled study showed BRL cell coculture significantly improved implantation 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 cell coculture system for patients who had failed previous cycles with poor embryo quality. For this study we examined day-3 embryo quality and pregnancy rates from coculture compared to regular culture system. Design: A retrospective analysis of patients who had BRL cell coculture at UCSF In Vitro Fertilization program. Materials/Methods: IVF outcome and embryo quality were assessed in cycles from 1999 and 2000 where the patients who had at least one cycle without coculture and one cycle with coculture. A total of 34 patients included 38 cycles without coculture vs. 35 cycles with coculture. Day-3 embryo quality was compared between two culture systems. Embryo quality was based on cleavage on day-3 and morphological “grade” relating to fragmentation. Grade 1 and 2 are considered good or fair embryos; Grade 3 and 4 are poor quality. Clinical pregnancy was defined as the presence of a gestational sac on ultrasound examination. Fisher exact and unpaired t testing were used. Results: Clinical pregnancy rates were 5/38 (13%) in regular culture cycles and 14/35 (40%) in coculture cycles (p ⫽ 0.015). However, ongoing pregnancy rates were not significantly different (8% in regular culture cycles vs. 20% in coculture cycles, p ⫽ 0.179). The effect of BRL cell coculture on embryo development at 72 hour post retrieval.
Culture system Regular Culture Coculture with BRL P value
No. of No. of embryos blastomeres/ cultured embryo 437 342
% of 6-cell
5.29 ⫾ 2.11 44% 6.46 ⫾ 2.05 69% ⬍0.0001 ⬍0.0001
Grade/embryo 2.30 ⫾ 1.03 2.12 ⫾ 0.91 ⫽0.011
Conclusions: These data provide evidence that, with BRL cell coculture, day-3 embryo development is significantly improved. Although randomized studies are needed to assess the impact of BRL cell coculture on pregnancy outcome, these results support the theory of using BRL cell coculture to improve embryo development and subsequent pregnancy rates for patients with poor embryo quality during in vitro fertilization treatment.
P-359 Intracytoplasmic sperm injection using non-motile testicular spermatozoa selected by modified hypo-osmotic swelling test. H. Sallam, A. Rahaman, A. Eid, A. Sallam, A. A. Agameya. Alexandria Univ, Alexandria, Egypt; Alexandria Fertility Ctr, Alexandria, Egypt. Objective: The aim of this work was to explore the fertilizing capacity of totally non-motile testicular spermatozoa selected by a modified hypoosmotic swelling test (consisting of 50% culture medium and 50% milli-Q water), compared to motile testicular spermatozoa in patients with infertility due to non-obstructive azoospermia. Design: A comparison of fertilization, clinical pregnancy and on-going pregnancy rates in azoospermic patients with non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test versus azoospermic patients with motile testicular spermatozoa. Materials/Methods: Fifty five cycles in infertile couples due to nonobstructive azoospermia treated with intracytoplasmic sperm injection (ICSI), consisting of 16 cycles where ICSI was performed using non-motile spermatozoa and 39 cycles where ICSI was performed with motile testicular spermatozoa. In the 16 cycles with total absence of motility, the spermatozoa used for injection were selected by a modified hypo-osmotic swelling test (a solution consisting of 50% culture medium and 50% milli-Q water), while in the 39 cycles with motile spermatozoa, these were randomly selected. Results: The fertilization rate was 32.4% in the non-motile sperm group
Vol. 76, No. 3, Suppl. 1, September 2001
P-357 Suitability of mouse embryos for human IVF QC: zygotes vs. 2-cell embryos. M. Li, K. C. Drury, R. S. Williams. Univ of Florida, Gainesville, FL. Objective: Mouse embryos are widely used for quality control (QC) and media formulation in human IVF laboratories. The present study evaluated two different stages of mouse embryo culture to test the suitability of new media. Design: In-vivo fertilized mouse zygotes and 2 cell stage embryos derived from the same strain (B6C3F-1XB6D2F-1) were purchased frozen from Embryotech. Embryos were cultured in different media and endpoints compared using Chi square (p ⬍ 0.05). Materials/Methods: Frozen–thawed zygotes and 2 cell stage mouse embryos were cultured (avg. ⫽ 20/droplet) in 100 ul droplets of media supplemented with 10% SSS (Irvine) under washed Squibb mineral oil. Two cell embryos were scored for # blastocyst on day 3 (D3) and # hatched on 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 of testing, human IVF was performed using P-1 ⫹ 10% SSS for embryo culture with day 3 (8 cell) transfer. Results: The implantation rate of human embryos transferred on D3 during year 2000 using P-1 ⫹ 10% SSS was 22.4% (N ⫽ 126). In accordance, mouse 2 cell embryo development in the same medium resulted in 91.8% blastocyst (B) on D3 and 51.1% hatched (H) on D4 (N ⫽ 233) with good cell architecture. However, zygote culture in P-1 ⫹ 10% SSS gave only 29.1% B on D4 and just 1.2% H on D5 (N ⫽ 86) (p ⫽ ⬍0.01) with the majority undergoing degeneration at the morula stage. These results are contrasted with the following more complex sequential media using mouse zygote stage: (1) P-1 for 48 hrs then Blastocyst medium (Irvine) 81.8% B, 18.2% H (N ⫽ 22); (2) Quinn’s Sequential (Q-Fert, QCleave, Q-Blast-Biopharma) 78.0% B, 2.6% H (N ⫽ 38); (3) Quinn’s Sequential with Insulin, Transferrin, Selenium (ITS- Sigma) added to QBlast. 95.5% B, 4.5% H (N ⫽ 22); (4) Quinn’s Q-Fert only. 98.4% B, 4.8% H (N ⫽ 63) (p 0.05). Conclusions: It has long been appreciated that although mouse embryos constitute a biological test system for culture conditions, they are notoriously permissive and resistant to levels of toxicity that can negatively impact human embryos. Cell cycle blocks, common in certain strains of mouse embryos, may also not reflect specific conditions found in humans, although they have played a role in the understanding of glucose and phosphates in early human embryo culture. It is with interest then that mouse zygotes did not respond well to P-1 ⫹ 10% SSS under the same conditions where 2 cell embryos progressed to the blastocyst and hatched stage at high rates (91.8% B, 51.1% H). At the same time, P-1 supports reasonable human embryo implantation rates (22.4%) when embryos are transferred at the 8 cell stage. In contrast to P-1, new media from Quinn (Q-Fert) supported blastocyst development from mouse zygotes at high levels (98.4%) but lacked corresponding hatch rates (4.8%). We are continuing to test prospective complex sequential media with a mouse zygote system in order to examine their suitability for human IVF laboratory use. Mouse zygotes are able to distinguish between culture media, but are deficient in their ability to accurately reflect conditions relevant to use in the IVF laboratory. Supported by: Department of Obstetrics and Gynecology, Shands Hospital at University of Florida.
P-358 Coculture with buffalo rat liver (BRL) cells enhanced day-3 embryo development in poor prognosis patients. S. Shen, K. Ho, D. Jaffe, E.
S230
Abstracts
Pritts, M. Cedars, V. Y. Fujimoto. Dept of Obstetrics, Gynecology and Reproductive Science, Univ of CA, San Francisco, San Francisco, CA. Objective: Methods to improve embryo quality are needed to enhance IVF outcome. Coculture systems have been suggested to minimize embryo fragmentation and improve blastomere development. A randomized controlled study showed BRL cell coculture significantly improved implantation 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 cell coculture system for patients who had failed previous cycles with poor embryo quality. For this study we examined day-3 embryo quality and pregnancy rates from coculture compared to regular culture system. Design: A retrospective analysis of patients who had BRL cell coculture at UCSF In Vitro Fertilization program. Materials/Methods: IVF outcome and embryo quality were assessed in cycles from 1999 and 2000 where the patients who had at least one cycle without coculture and one cycle with coculture. A total of 34 patients included 38 cycles without coculture vs. 35 cycles with coculture. Day-3 embryo quality was compared between two culture systems. Embryo quality was based on cleavage on day-3 and morphological “grade” relating to fragmentation. Grade 1 and 2 are considered good or fair embryos; Grade 3 and 4 are poor quality. Clinical pregnancy was defined as the presence of a gestational sac on ultrasound examination. Fisher exact and unpaired t testing were used. Results: Clinical pregnancy rates were 5/38 (13%) in regular culture cycles and 14/35 (40%) in coculture cycles (p ⫽ 0.015). However, ongoing pregnancy rates were not significantly different (8% in regular culture cycles vs. 20% in coculture cycles, p ⫽ 0.179). The effect of BRL cell coculture on embryo development at 72 hour post retrieval.
Culture system Regular Culture Coculture with BRL P value
No. of No. of embryos blastomeres/ cultured embryo 437 342
% of 6-cell
5.29 ⫾ 2.11 44% 6.46 ⫾ 2.05 69% ⬍0.0001 ⬍0.0001
Grade/embryo 2.30 ⫾ 1.03 2.12 ⫾ 0.91 ⫽0.011
Conclusions: These data provide evidence that, with BRL cell coculture, day-3 embryo development is significantly improved. Although randomized studies are needed to assess the impact of BRL cell coculture on pregnancy outcome, these results support the theory of using BRL cell coculture to improve embryo development and subsequent pregnancy rates for patients with poor embryo quality during in vitro fertilization treatment.
P-359 Intracytoplasmic sperm injection using non-motile testicular spermatozoa selected by modified hypo-osmotic swelling test. H. Sallam, A. Rahaman, A. Eid, A. Sallam, A. A. Agameya. Alexandria Univ, Alexandria, Egypt; Alexandria Fertility Ctr, Alexandria, Egypt. Objective: The aim of this work was to explore the fertilizing capacity of totally non-motile testicular spermatozoa selected by a modified hypoosmotic swelling test (consisting of 50% culture medium and 50% milli-Q water), compared to motile testicular spermatozoa in patients with infertility due to non-obstructive azoospermia. Design: A comparison of fertilization, clinical pregnancy and on-going pregnancy rates in azoospermic patients with non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test versus azoospermic patients with motile testicular spermatozoa. Materials/Methods: Fifty five cycles in infertile couples due to nonobstructive azoospermia treated with intracytoplasmic sperm injection (ICSI), consisting of 16 cycles where ICSI was performed using non-motile spermatozoa and 39 cycles where ICSI was performed with motile testicular spermatozoa. In the 16 cycles with total absence of motility, the spermatozoa used for injection were selected by a modified hypo-osmotic swelling test (a solution consisting of 50% culture medium and 50% milli-Q water), while in the 39 cycles with motile spermatozoa, these were randomly selected. Results: The fertilization rate was 32.4% in the non-motile sperm group
Vol. 76, No. 3, Suppl. 1, September 2001