- Email: [email protected]
Use of subcutaneous HCG (Ovidrel) for final follicular maturation does not change reproductive outcome
Table 1: Summary of results
Conclusions: We found no statistically or clinically significant differences in ART outcomes between the r-hCG and the u-hCG groups. Therefore, r-hCG is as effective as u-hCG when administered to egg donors after COS. Given the equivalent clinical outcomes in these two groups, and given the increased patient tolerability and ease of administration of r-hCG compared to u-hCG, egg donors at Boston IVF will be prescribed r-hCG as the preferred agent for ovulation induction.
P-378 Pregnancy outcome from an In Vitro Fertilization-Embryo Transfer (IVF-ET) cycle is not a predictor for frozen embryo transfer success. Lucinda B. White, Dawn A. Kelk, Sandra J. Hahn, Melissa Rosario, Deborah Smith. Univ of Colorado, Aurora, CO. Objective: To determine the significance of pregnancy test results from a fresh IVF-ET cycle on pregnancy outcomes in subsequent frozen embryo transfer (FET) cycles. Design: Retrospective study performed at an academic IVF program. Material and Methods: Charts from women undergoing FET from 7/1/99 until 4/1/03 were retrospectively reviewed. Pregnancy rates during the fresh In vitro fertilization cycle were compared with the pregnancy rates during the FET cycles. Results were stratified by pregnancy outcome. Outcomes examined include intrauterine pregnancy (IUP), spontaneous abortion (SAB), biochemical pregnancy (BIO), ectopic pregnancy (ECT) and negative pregnancy test results (NEG). Results: There were 119 patients undergoing 176 FET cycles at the University of Colorado. The overall pregnancy rate for an FET cycle was 51% per patient. Of patients undergoing an FET cycle 30% of patients had more than one FET cycle performed. Patients having a positive pregnancy test on their fresh IVF-ET cycle (IUP, SAB, BIO, ECT) had a 58% (20/34) chance of having a clinical pregnancy with their FET cycle. Those patients having a negative pregnancy test on their fresh IVF-ET cycle had a 48% (41/85) chance of achieving pregnancy on their subsequent FET attempt. These pregnancy rates were not significantly different. Of the study population (N⫽119) approximately 49% still have frozen embryos for future use. Conclusions: Women who had a negative pregnancy test in their fresh IVF-ET cycle had the same likelihood of achieving pregnancy during a subsequent FET attempt as those patients having a positive pregnancy test during their IVF-ET cycle.
P-379 Follicular development is more dependent on day 3 FSH levels rather than age. Mary R. Vietzke, Tracy Birkenkamp, Jeannine Cavanaugh, Cheryl Costa, Vishvanath C. Karande. Karande and Assoc, S.C., Hoffman Estates, IL. Objective: In a recent article published by Ilse A. J. van Rooij and colleagues in March 2003 (Fertil Steril 2003 79: 482-488), the issue of whether maternal age or basal FSH levels are more important for follicular development and pregnancy outcomes was addressed. The authors examined two groups of women: women ⬎41 years with FSH levels ⬍15 IU/L and women ⬍41 years with FSH levels ⬎15 IU/L. They determined that maternal age is a more important factor in terms of oocyte quality as opposed to FSH levels which play a greater role in determining oocyte quantity. However, a basal FSH value of 10 IU/L has generally been used to determine responder status and whether a patient should strongly consider oocyte donation as a primary option. We wanted to determine if these
S246
Abstracts
conclusions regarding maternal age and FSH levels would be true in our practice using a threshold level of 10 IU/L and using approximately the same overall data analysis strategy. Design: Retrospective study. Materials and Methods: We retrospectively examined the follicular development, oocyte and pregancy data on 149 cycles between October 2002 and March 2003. Cycles examined were categorized into two groups: women aged ⬎41 years with FSH levels ⬍10 IU/L and women aged ⬍41 years with FSH levels ⬎10 IU/L. This practice utilizes an automated immunometric FSH assay system (Bayer, Tarrytown, NY) which is the same as the study published by Rooij et al. Means and standard deviation values were determined for age, basal FSH, follicle number, oocytes retrieved, mature oocytes, fertilized oocytes and embryos transfered. Clinical pregnancy rates per cycle and per embryo transfer were also calculated. Statistical analysis was performed using Student’s t-test and Fisher’s exact as appropriate. Results: Twenty-two cycles were categorized into the two different groups. By design, there were statistically significant differences between the two groups for age and for FSH levels. Surprisingly, even with the small number of cycles examined, there was also a statistcially significant difference in the number of follicles determined at oocyte retrieval favoring a higher number of follicles for women aged ⬎41 years with an FSH level of ⬍10 IU/L. Although not statistically significant, there was a trend for more oocytes and more mature oocytes for this group as well. There was a higher trend in pregnancy rates for women aged ⬍41 years with FSH levels ⬎10 IU/L, but that is most likely due to the higher cancellation rate for this group.
Conclusions: We believe, as also proposed by Rooij and colleagues in March of 2003, that FSH is a more predominant factor in determining follicular development rather than maternal age. It remains to be seen whether this trend toward a higher number of oocytes retrieved and mature oocytes for women ⬎41 years with “normal” (⬍10 IU/L) FSH levels will become statistically significant with more cycles examined. So far, our conclusions are similar to those in Rooij et al 2003 and prove promising for elucidating the role of FSH and maternal age for pregnancy outcomes.
P-380 Use of subcutaneous HCG (Ovidrel) for final follicular maturation does not change reproductive outcome. Melanie Acosta, Tanmoy Mukherjee, Lawrence Grunfeld, Benjamin Sandler, Paul A. Bergh, Alan B. Copperman. Reproductive Medicine Assoc of New York, New York, NY; Reproductive Medicine Assoc of New Jersey, Morristown, NJ; Mount Sinai Sch of Medicine, New York, NY. Objective: Subcutaneous rhCG has been approved for the induction of final follicular maturation and early luteinization in infertile women who have undergone pituitary desensitization and who have been appropriately pretreated with follicle stimulating hormone as part of an Assisted Reproductive Technology (ART) program. rhCG can also be used to induce ovulation in women undergoing controlled ovarian hyperstimulation with or without intrauterine insemination. Its ease of use makes it attractive to patients, but as with any new medication, it is only through experience and data collection that its clinical utility can be validated. This analysis was designed to compare the clinical efficacy of subcutaneous rhCG in a large program as compared to traditional intramuscular injection of urinary hCG. Design: A retrospective analysis of 518 patients undergoing controlled ovarian hyperstimulatino and intrauterine insemination (IUI) and 690 re-
Vol. 80, Suppl. 3, September 2003
cipients of donor eggs using a combined data base of Reproductive Medicine Associates of New York and New Jersey. Materials and Methods: In the first group of patients controlled ovarian hyperstimulation was performed on 518 women under the age of 40 undergoing treatment for unexplained infertility. All patients received between 150 and 300 IU of recombinant FSH or hMG. When two follicles reached ⬎17mm, one of four forms of hCG were prescribed at the treating physician’s discretion to trigger ovulation. An intrauterine insemination (IUI) was performed 36 hours after hCG. An analysis was also undertaken on a second patient group, consisting of ovum donors in our egg donation program. The records of 690 donors were retrospectively analyzed to determine the choice of hCG used to trigger final oocyte maturation. As with our IUI group, hCG was designated by the physician with no criteria for specifying one product over another. Continuous variables were analyzed using a t-test, pregnancy rates were compared using Chi-square where appropriate. Results: The results presented in the table showed no significant difference in the percentage of pregnancy rate for both groups (IUI and Donors) using Novarel, Ovidrel, Pregnyl or Profasi. There was also no change in the rate of spontaneous abortion or multiple gestation in both groups.
Conclusion: Clinical outcomes did not differ significantly between treatment arms. Induction of final follicular maturation using subcutaneous Ovidrel (rhCG) is similar to other treatment options, and is easier to self-administer.
REPRODUCTIVE BIOLOGY: HUMAN STUDIES P-381 Cloning of human and mouse testis and spermatogenesis related gene 4 (TSARG4), a SPAG4 like gene. Xing Xiaowei, Li Luyun, Fu Junjiang, Liu Gang, Liu Shangfeng, Lu Guangxiu. Institute of Human Reproduction & Stem Cell Engineering, Central South University, Changsha, China. Objective: Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. To clone such a gene like Spag4, may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. Design: Using rat and human Spag4 amino acid sequences to hook testis ESTs in mouse and human EST database, which coded amino acid sequences were highly homology with rat and human Spag4. Cloned human and mouse testis and spermatogensis related gene 4 full-length cDNA sequences. RT-PCR and Northern blot were performed to clarify the patters of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Materials and Methods: Using the new program of the National Center for Biotechnology Information (NCBI) and ExPASy Molecular Biology Server to search for proper ESTs. We hooked two ESTs BG101130 and BG100990 which expressed in mouse spermatocytes, and other testis ESTs. By information analysis, we got a hypothetical protein (GenBank accession number AK006225). Meanwhile, we found two EST BG720564 and AI700454 in human testis and the gap between these two ESTs was filled by polymerase chain reaction, and a fragment 1,252 bp which encoded a new gene was obtained. The cDNA sequences were proved in human and mouse testis by RT-PCR. Multi-tissues RT-PCR and Northern blot were performed to clarify the patterns of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Results: A new fragment 1,252 bp which encoded a new gene was obtained in human testis and was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94-1,233 bp. The hypothetical protein AK006225 was named mtsarg4, which full-length 1,136 bp and its opening reading frame was 68-1,114 bp. The homologies of amino acid sequences were 74% between human TSARG4 and mouse mtsarg4 gene and 45% between mtsarg4 and rat Spag4 gene, respectively. Northern blot results showed
FERTILITY & STERILITY威
human TSARG4 gene only expressed in testis, not in spleen, thymus, prostate, overy, small intestine, colon, peripheral blood leukocyte. Multitissues RT-PCR results showed mtsarg4 gene expressed specifically in the testis. In mouse cryptorchidism models, with the days increased after operation, the expression of mtsarg4 decreased. Conclusion: Two new genes, human and mouse TSARG4 were identified, which may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. P-382 Fertilization and developmental potential after Intracytoplasmic Sperm Injection (ICSI) of in vitro matured human Metaphase I oocytes retrieved in stimulated cycles. Hanna Balakier, Agata Sojecki, Gelareh Motamedi, Clifford Librach, Gelareh Motamedi. Create Program Inc., Toronto, ON, Canada; Create Program Inc. and Dept. Obstetrics and Gynecology, Sunnubrook and Women’s Coll Health Science Ctr, Univ of Toronto, Toronto, ON, Canada. Objective: About 5-15% of all oocytes denuded prior to ICSI procedure are in Metaphase I stage (MI) of meiotic division. They have already undergone germinal vesicle breakdown but did not extrude yet the first polar body (PB). Some of these oocytes mature within a few hours in culture and might be suitable for sperm injection. The aim of our study was to evaluate the fertilization rate and developmental potential of in vitro matured MI oocytes compared to their sibling oocytes retrieved at metaphase II (MII) stage. Design: Obtained from stimulated cycles, immature MI oocytes were monitored for the extrusion of the first PB and subjected to ICSI at different times after maturation. Fertilization rates and quality of embryos were compared between the groups of embryos derived from in vitro matured MI and in vivo matured MII oocytes. Materials and Methods: Retrieved oocytes were denuded with hyaluronidase and immature MI oocytes were separated from mature metaphase II oocytes and were cultured in vitro for further maturation. During that time they were examined every half hour for the extrusion of the first polar body. Both, immature MI oocytes that have reached metaphase II stage (MI-MII) and their matured metaphase (MII) siblings were subjected to ICSI. Those matured in vitro oocytes were injected with patient’s sperm at different times after extrusion of the first PB (1-1.5, 2-2.5 and 3-6 hours). Fertilization rates, embryo cleavage and embryo quality were evaluated at 18, 44 and 72 hours after ICSI. Results: Our study involved 326 ICSI cycles in which 3116 MII and 468 in vitro matured MI-MII oocytes were injected. Generally, fertilization rate of in vitro matured oocytes was significantly lower than in vivo matured MII oocytes (42% versus 77% respectively, P⬍0.0001). The time of sperm injection appeared to be important. The majority of MI-MII oocytes injected one hour after PB extrusion remained unfertilized (64%; 98/154 oocytes) and only 25% had formed two pronuclei (PN) and two PBs. About 45% (78/172) of oocytes subjected to ICSI 2-2.5 hours post PB extrusion were unfertilized, 12% exhibited abnormal fertilization (1PN or 3PN) and 43% had normal pronuclei. In contrast, longer incubation before ICSI (3 to 6 hours) resulted in a 63% (86/138 oocytes) fertilization rate while 29% of oocytes remained still unfertilized. Embryos derived from in vitro matured MI oocytes were frequently arrested at the 2- or 4-cell stage of development (39%, 78/198; P⬍0.0001) and had more multinucleated blastomeres (23% embryos, P⬍0.0001) than those originating from MII oocytes (17% and 12% respectively). Otherwise the embryo quality was similar in both groups. Conclusion: The fertilization rate of in vitro matured MI oocytes is significantly lower than that of MII in vivo matured MII oocytes, which may reflect their cytoplasmic immaturity despite their nuclear maturity. The post fertilization cleavage capacity of these oocytes is also decreased and a high proportion of embryos become arrested, exhibiting multinucleated blastomeres, which suggests the presence of chromosomal abnormalities. P-383 Sperm morphology and seminal leukocytes as predictors of increased production of Reactive Oxygen Species (ROS) in infertile men semen. Nabil Aziz, D. Iwan Lewis-Jones, Ashok Agarwal, Ramadan A. Saleh, Rakesh K. Sharma, Anthony J. Thomas Jr. Univ of Liverpool, Liverpool Women’s Hosp, Liverpool, United Kingdom; Cleveland Clin Fdn, Cleveland, OH; Dept of Dermatology and Venereology,, Sohag, Egypt.
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Conclusions: We found no statistically or clinically significant differences in ART outcomes between the r-hCG and the u-hCG groups. Therefore, r-hCG is as effective as u-hCG when administered to egg donors after COS. Given the equivalent clinical outcomes in these two groups, and given the increased patient tolerability and ease of administration of r-hCG compared to u-hCG, egg donors at Boston IVF will be prescribed r-hCG as the preferred agent for ovulation induction.
P-378 Pregnancy outcome from an In Vitro Fertilization-Embryo Transfer (IVF-ET) cycle is not a predictor for frozen embryo transfer success. Lucinda B. White, Dawn A. Kelk, Sandra J. Hahn, Melissa Rosario, Deborah Smith. Univ of Colorado, Aurora, CO. Objective: To determine the significance of pregnancy test results from a fresh IVF-ET cycle on pregnancy outcomes in subsequent frozen embryo transfer (FET) cycles. Design: Retrospective study performed at an academic IVF program. Material and Methods: Charts from women undergoing FET from 7/1/99 until 4/1/03 were retrospectively reviewed. Pregnancy rates during the fresh In vitro fertilization cycle were compared with the pregnancy rates during the FET cycles. Results were stratified by pregnancy outcome. Outcomes examined include intrauterine pregnancy (IUP), spontaneous abortion (SAB), biochemical pregnancy (BIO), ectopic pregnancy (ECT) and negative pregnancy test results (NEG). Results: There were 119 patients undergoing 176 FET cycles at the University of Colorado. The overall pregnancy rate for an FET cycle was 51% per patient. Of patients undergoing an FET cycle 30% of patients had more than one FET cycle performed. Patients having a positive pregnancy test on their fresh IVF-ET cycle (IUP, SAB, BIO, ECT) had a 58% (20/34) chance of having a clinical pregnancy with their FET cycle. Those patients having a negative pregnancy test on their fresh IVF-ET cycle had a 48% (41/85) chance of achieving pregnancy on their subsequent FET attempt. These pregnancy rates were not significantly different. Of the study population (N⫽119) approximately 49% still have frozen embryos for future use. Conclusions: Women who had a negative pregnancy test in their fresh IVF-ET cycle had the same likelihood of achieving pregnancy during a subsequent FET attempt as those patients having a positive pregnancy test during their IVF-ET cycle.
P-379 Follicular development is more dependent on day 3 FSH levels rather than age. Mary R. Vietzke, Tracy Birkenkamp, Jeannine Cavanaugh, Cheryl Costa, Vishvanath C. Karande. Karande and Assoc, S.C., Hoffman Estates, IL. Objective: In a recent article published by Ilse A. J. van Rooij and colleagues in March 2003 (Fertil Steril 2003 79: 482-488), the issue of whether maternal age or basal FSH levels are more important for follicular development and pregnancy outcomes was addressed. The authors examined two groups of women: women ⬎41 years with FSH levels ⬍15 IU/L and women ⬍41 years with FSH levels ⬎15 IU/L. They determined that maternal age is a more important factor in terms of oocyte quality as opposed to FSH levels which play a greater role in determining oocyte quantity. However, a basal FSH value of 10 IU/L has generally been used to determine responder status and whether a patient should strongly consider oocyte donation as a primary option. We wanted to determine if these
S246
Abstracts
conclusions regarding maternal age and FSH levels would be true in our practice using a threshold level of 10 IU/L and using approximately the same overall data analysis strategy. Design: Retrospective study. Materials and Methods: We retrospectively examined the follicular development, oocyte and pregancy data on 149 cycles between October 2002 and March 2003. Cycles examined were categorized into two groups: women aged ⬎41 years with FSH levels ⬍10 IU/L and women aged ⬍41 years with FSH levels ⬎10 IU/L. This practice utilizes an automated immunometric FSH assay system (Bayer, Tarrytown, NY) which is the same as the study published by Rooij et al. Means and standard deviation values were determined for age, basal FSH, follicle number, oocytes retrieved, mature oocytes, fertilized oocytes and embryos transfered. Clinical pregnancy rates per cycle and per embryo transfer were also calculated. Statistical analysis was performed using Student’s t-test and Fisher’s exact as appropriate. Results: Twenty-two cycles were categorized into the two different groups. By design, there were statistically significant differences between the two groups for age and for FSH levels. Surprisingly, even with the small number of cycles examined, there was also a statistcially significant difference in the number of follicles determined at oocyte retrieval favoring a higher number of follicles for women aged ⬎41 years with an FSH level of ⬍10 IU/L. Although not statistically significant, there was a trend for more oocytes and more mature oocytes for this group as well. There was a higher trend in pregnancy rates for women aged ⬍41 years with FSH levels ⬎10 IU/L, but that is most likely due to the higher cancellation rate for this group.
Conclusions: We believe, as also proposed by Rooij and colleagues in March of 2003, that FSH is a more predominant factor in determining follicular development rather than maternal age. It remains to be seen whether this trend toward a higher number of oocytes retrieved and mature oocytes for women ⬎41 years with “normal” (⬍10 IU/L) FSH levels will become statistically significant with more cycles examined. So far, our conclusions are similar to those in Rooij et al 2003 and prove promising for elucidating the role of FSH and maternal age for pregnancy outcomes.
P-380 Use of subcutaneous HCG (Ovidrel) for final follicular maturation does not change reproductive outcome. Melanie Acosta, Tanmoy Mukherjee, Lawrence Grunfeld, Benjamin Sandler, Paul A. Bergh, Alan B. Copperman. Reproductive Medicine Assoc of New York, New York, NY; Reproductive Medicine Assoc of New Jersey, Morristown, NJ; Mount Sinai Sch of Medicine, New York, NY. Objective: Subcutaneous rhCG has been approved for the induction of final follicular maturation and early luteinization in infertile women who have undergone pituitary desensitization and who have been appropriately pretreated with follicle stimulating hormone as part of an Assisted Reproductive Technology (ART) program. rhCG can also be used to induce ovulation in women undergoing controlled ovarian hyperstimulation with or without intrauterine insemination. Its ease of use makes it attractive to patients, but as with any new medication, it is only through experience and data collection that its clinical utility can be validated. This analysis was designed to compare the clinical efficacy of subcutaneous rhCG in a large program as compared to traditional intramuscular injection of urinary hCG. Design: A retrospective analysis of 518 patients undergoing controlled ovarian hyperstimulatino and intrauterine insemination (IUI) and 690 re-
Vol. 80, Suppl. 3, September 2003
cipients of donor eggs using a combined data base of Reproductive Medicine Associates of New York and New Jersey. Materials and Methods: In the first group of patients controlled ovarian hyperstimulation was performed on 518 women under the age of 40 undergoing treatment for unexplained infertility. All patients received between 150 and 300 IU of recombinant FSH or hMG. When two follicles reached ⬎17mm, one of four forms of hCG were prescribed at the treating physician’s discretion to trigger ovulation. An intrauterine insemination (IUI) was performed 36 hours after hCG. An analysis was also undertaken on a second patient group, consisting of ovum donors in our egg donation program. The records of 690 donors were retrospectively analyzed to determine the choice of hCG used to trigger final oocyte maturation. As with our IUI group, hCG was designated by the physician with no criteria for specifying one product over another. Continuous variables were analyzed using a t-test, pregnancy rates were compared using Chi-square where appropriate. Results: The results presented in the table showed no significant difference in the percentage of pregnancy rate for both groups (IUI and Donors) using Novarel, Ovidrel, Pregnyl or Profasi. There was also no change in the rate of spontaneous abortion or multiple gestation in both groups.
Conclusion: Clinical outcomes did not differ significantly between treatment arms. Induction of final follicular maturation using subcutaneous Ovidrel (rhCG) is similar to other treatment options, and is easier to self-administer.
REPRODUCTIVE BIOLOGY: HUMAN STUDIES P-381 Cloning of human and mouse testis and spermatogenesis related gene 4 (TSARG4), a SPAG4 like gene. Xing Xiaowei, Li Luyun, Fu Junjiang, Liu Gang, Liu Shangfeng, Lu Guangxiu. Institute of Human Reproduction & Stem Cell Engineering, Central South University, Changsha, China. Objective: Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. To clone such a gene like Spag4, may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. Design: Using rat and human Spag4 amino acid sequences to hook testis ESTs in mouse and human EST database, which coded amino acid sequences were highly homology with rat and human Spag4. Cloned human and mouse testis and spermatogensis related gene 4 full-length cDNA sequences. RT-PCR and Northern blot were performed to clarify the patters of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Materials and Methods: Using the new program of the National Center for Biotechnology Information (NCBI) and ExPASy Molecular Biology Server to search for proper ESTs. We hooked two ESTs BG101130 and BG100990 which expressed in mouse spermatocytes, and other testis ESTs. By information analysis, we got a hypothetical protein (GenBank accession number AK006225). Meanwhile, we found two EST BG720564 and AI700454 in human testis and the gap between these two ESTs was filled by polymerase chain reaction, and a fragment 1,252 bp which encoded a new gene was obtained. The cDNA sequences were proved in human and mouse testis by RT-PCR. Multi-tissues RT-PCR and Northern blot were performed to clarify the patterns of these new genes. Changes of mtsarg4 expression in mouse cryptorchidism models were observed by semi-quatitive RT-PCR. Results: A new fragment 1,252 bp which encoded a new gene was obtained in human testis and was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94-1,233 bp. The hypothetical protein AK006225 was named mtsarg4, which full-length 1,136 bp and its opening reading frame was 68-1,114 bp. The homologies of amino acid sequences were 74% between human TSARG4 and mouse mtsarg4 gene and 45% between mtsarg4 and rat Spag4 gene, respectively. Northern blot results showed
FERTILITY & STERILITY威
human TSARG4 gene only expressed in testis, not in spleen, thymus, prostate, overy, small intestine, colon, peripheral blood leukocyte. Multitissues RT-PCR results showed mtsarg4 gene expressed specifically in the testis. In mouse cryptorchidism models, with the days increased after operation, the expression of mtsarg4 decreased. Conclusion: Two new genes, human and mouse TSARG4 were identified, which may do help to understand molecular mechanism of spermatogenesis, especially sperm tail development. P-382 Fertilization and developmental potential after Intracytoplasmic Sperm Injection (ICSI) of in vitro matured human Metaphase I oocytes retrieved in stimulated cycles. Hanna Balakier, Agata Sojecki, Gelareh Motamedi, Clifford Librach, Gelareh Motamedi. Create Program Inc., Toronto, ON, Canada; Create Program Inc. and Dept. Obstetrics and Gynecology, Sunnubrook and Women’s Coll Health Science Ctr, Univ of Toronto, Toronto, ON, Canada. Objective: About 5-15% of all oocytes denuded prior to ICSI procedure are in Metaphase I stage (MI) of meiotic division. They have already undergone germinal vesicle breakdown but did not extrude yet the first polar body (PB). Some of these oocytes mature within a few hours in culture and might be suitable for sperm injection. The aim of our study was to evaluate the fertilization rate and developmental potential of in vitro matured MI oocytes compared to their sibling oocytes retrieved at metaphase II (MII) stage. Design: Obtained from stimulated cycles, immature MI oocytes were monitored for the extrusion of the first PB and subjected to ICSI at different times after maturation. Fertilization rates and quality of embryos were compared between the groups of embryos derived from in vitro matured MI and in vivo matured MII oocytes. Materials and Methods: Retrieved oocytes were denuded with hyaluronidase and immature MI oocytes were separated from mature metaphase II oocytes and were cultured in vitro for further maturation. During that time they were examined every half hour for the extrusion of the first polar body. Both, immature MI oocytes that have reached metaphase II stage (MI-MII) and their matured metaphase (MII) siblings were subjected to ICSI. Those matured in vitro oocytes were injected with patient’s sperm at different times after extrusion of the first PB (1-1.5, 2-2.5 and 3-6 hours). Fertilization rates, embryo cleavage and embryo quality were evaluated at 18, 44 and 72 hours after ICSI. Results: Our study involved 326 ICSI cycles in which 3116 MII and 468 in vitro matured MI-MII oocytes were injected. Generally, fertilization rate of in vitro matured oocytes was significantly lower than in vivo matured MII oocytes (42% versus 77% respectively, P⬍0.0001). The time of sperm injection appeared to be important. The majority of MI-MII oocytes injected one hour after PB extrusion remained unfertilized (64%; 98/154 oocytes) and only 25% had formed two pronuclei (PN) and two PBs. About 45% (78/172) of oocytes subjected to ICSI 2-2.5 hours post PB extrusion were unfertilized, 12% exhibited abnormal fertilization (1PN or 3PN) and 43% had normal pronuclei. In contrast, longer incubation before ICSI (3 to 6 hours) resulted in a 63% (86/138 oocytes) fertilization rate while 29% of oocytes remained still unfertilized. Embryos derived from in vitro matured MI oocytes were frequently arrested at the 2- or 4-cell stage of development (39%, 78/198; P⬍0.0001) and had more multinucleated blastomeres (23% embryos, P⬍0.0001) than those originating from MII oocytes (17% and 12% respectively). Otherwise the embryo quality was similar in both groups. Conclusion: The fertilization rate of in vitro matured MI oocytes is significantly lower than that of MII in vivo matured MII oocytes, which may reflect their cytoplasmic immaturity despite their nuclear maturity. The post fertilization cleavage capacity of these oocytes is also decreased and a high proportion of embryos become arrested, exhibiting multinucleated blastomeres, which suggests the presence of chromosomal abnormalities. P-383 Sperm morphology and seminal leukocytes as predictors of increased production of Reactive Oxygen Species (ROS) in infertile men semen. Nabil Aziz, D. Iwan Lewis-Jones, Ashok Agarwal, Ramadan A. Saleh, Rakesh K. Sharma, Anthony J. Thomas Jr. Univ of Liverpool, Liverpool Women’s Hosp, Liverpool, United Kingdom; Cleveland Clin Fdn, Cleveland, OH; Dept of Dermatology and Venereology,, Sohag, Egypt.
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