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High endometrial aromatase P450 messenger RNA expression is associated with poor IVF outcome
Wednesday, October 15, 2003 4:15 P.M. O-267 Early cleavage assessment in embryos from Intracytoplasmic Sperm Injection (ICSI) cycles. Patrı´cia Guilherme, Lia M. Rossi, Assumpto Iaconelli Jr, Claudia C. Rocha, Christiany V. Locambo, Edson Borges Jr. Fertility - Ctr de Fertilizac¸ ao Assistida, Sao Paulo, Brazil. Objective: More recently, assessment of the time of cleavage to the 2-cell stage has proven to be a reliable parameter for the selection of embryos with the highest capability of implantation and successful pregnancy after transfer. The purpose of this work was to compare the relevance of early cleavage (EC) under embryo development and pregnancy occurrence. Design: Propsective in vitro study. Materials and Methods: This prospective study included 147 ICSI cycles (122 patients). Several classifications were done in the same embryos since fertilization diagnosis until time to transfer. All zygotes that were normally fertilized (two pronuclei) were included. Seventeen hours after ICSI, the pronuclear (PN) and nucleolar precursor body (NPB) arrangement were evaluated. Normal embryos (S0) showed aligned or aligning PN, with no discrepant number of NPB compared between male and female PN. Embryos with different conditions were classified as abnormal (S1). Early cleavage assessment was done, as a rule, 26 hours after ICSI. Three groups were formed: (A) embryos with visible PN; (B) embryos non-cleaved without visible PN and (C) EC-embryos. Embryo selection was performed on day 3 according to number, size, shape of cells and fragmentation. “Good quality embryos” was classified when had regular blastomeres, with at least 6 cells and without fragmentation on day 3. “Top quality embryos” were named when good quality embryos showed also S0 classification. It should be noted that embryos were not selected for transfer on the basis of early cleavage occurrence. Statistical significance was determined using Student’s t test, chi-square and Fisher’s exact tests as appropriate with statistically significant differences considered at p ⬍ 0.05. Results: The mean maternal age (SD) was 36.4 (5.5) and did not differ among three groups (34.5 ⫾ 4.6; 34.1 ⫾ 5; 34.2 ⫾ 4.2; groups A, B and C; respectively; p ⬎ .05). A total, 1203 viable oocytes were obtained after ICSI and of these, 74.3% had shown normal fertilization (n ⫽ 894) and were analyzed. The presence of EC embryos in ICSI cycles seemed to predict the occurrence of pregnancy in cycles that provided 1 or 2 EC embryos (40%) and more than 3 EC embryos (56.1%). Irrespective of other classifications (PN or day 3 morphology), when at least one EC embryo was transferred, pregnancy occurred in 60% of patients, two times higher than that obtained without the transfer of these kind of embryos (33%; p ⫽ 0.025). Complementary results are shown in the table.
surgically retrieved spermatozoa. The aim of this study was to analyze the effect of sub-optimal normal fertilization in ICSI outcomes, according to the source of sperm. Design: Retrospective in vitro study. Materials and Methods: This work included 918 patients with classical indications for ICSI performing 1061 cycles. In 737 couples ICSI was done with ejaculated spermatozoa and in 181 patients the spermatozoa were obtained by aspiration technique. The criterion to exclude female factor was maternal age ⱕ 35 years and more than 4 oocytes retrieved. Patients were ranked based on the fertilization rate namely: group A (normal fertilization rate equal or less than 50%) and group B (normal fertilization rate more than 50%). Statistical significance was determined using Student’s t test, chisquare and Fisher’s exact tests as appropriate with statistically significant differences considered at p ⬍ 0.05. Results: The mean age (SD) of female patients was 30.1 (3.7) with no statistical difference among the groups. The incidence of sub-optimal fertilization was 11.8% (126 of 1061 ICSI cycles). Complementary results are shown in table.
(a and b : p ⬍ 0.05; a group A compared with group B in non-ejaculated sperm group; b group A compared with group B in ejaculated sperm group). Conclusion: Irrespective of source of sperm, cycles that exhibit suboptimal fertilization rates are likely to provide embryos with implantation potential three times lower. Our data suggest that the fertilization rate may be considered a new, additional and valuable parameter to predict successful ICSI outcomes.
Wednesday, October 15, 2003 4:45 P.M. O-269 (a - b; b - c: p ⬍ 0.05; a - c: p ⬍ 0.001). Conclusion: Our data suggest that early cleavage parameter is a powerful indicator of embryo potential, working as a reliable prognostic tool for patients undergoing to ICSI technique. It should be incorporated in embryo classification grading, resulting in overall significantly improved embryo selection.
Wednesday, October 15, 2003 4:30 P.M. O-268 Sub-optimal fertilization: A new parameter to predict successful outcomes in assisted reproduction. Assumpto Iaconelli Jr, Lia M. Rossi, Patrı´cia Guilherme, Claudia C. Rocha, Christiany Locambo, Edson Borges Jr. Fertility—Ctr de Fertilizac¸ ao Assistida, Sao Paulo, Brazil. Objective: Intracytoplasmic sperm injection (ICSI) has become the premier treatment modality for severe male factor infertility. This technique yields good pregnancy and implantation rates using both ejaculated or
FERTILITY & STERILITY威
High endometrial aromatase P450 messenger RNA expression is associated with poor IVF outcome. Jan J. Brosens, European Fertility Association (EFA). Imperial Coll London, London, United Kingdom. Objective: The success of IVF treatment is thought to be dependent upon embryo quality and endometrial receptivity. Previous reports indicated that expression of aromatase P450 in the endometrium is restricted to women with proliferative reproductive tract disorders such as endometriosis, leiomyomas, and adenomyosis. The aim of this study was to determine if the level of endometrial aromatase P450 mRNA expression is a determinant of IVF outcome in infertile women. Design: A prospective multicenter case-control study. Setting: Eight European IVF units and a university center for reproductive research. Patients: A hundred twenty patients awaiting IVF treatment. Intervention(s): Endometrial biopsy prior to starting IVF treatment. Main Outcome Measures: Endometrial aromatase P450 and GAPDH transcripts were quantified by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System. Results: Aromatase P450 transcripts were detected in all endometria examined, although the levels varied considerably between samples ranging
S103
from 0.22 (arbitrary units, a.u.) to 486.6 a.u. (⬎2000 fold difference). The mean aromatase P450 mRNA level (⫾S.D.) in women who subsequently became pregnant after IVF treatment was 3.7 (⫾3.7) compared to 14.3 (⫾52.5) in patients with IVF failure (p ⬎ 0.05). The clinical pregnancy rate in women with high endometrial aromatase P450 mRNA levels (ⱖ 8.2 a.u.; n ⫽ 19) was 5.5% versus 22.8% in those patients with low expression levels (⬍8.2 a.u.; n ⫽ 101) (p ⬍ 0.05). The cycle day of the endometrial biopsy, cause of infertility, age, parity, number of oocytes collected, or number of embryos transferred did not differ between patients with high versus low endometrial aromatase P450 mRNA levels (p ⬎ 0.1). Conclusion: Quantitation of endometrial P450 mRNA levels is a potential diagnostic tool to identify women with high likelihood of IVF treatment failure.
Wednesday, October 15, 2003 5:00 P.M. O-270 Transfer of Embryos with Even a Single Multinucleated Blastomere Significantly Reduces Pregnancy and Implantation Rates. Marlena Duke, Natalia Basile, Melissa Johnson, Alan Copperman, Benjamin Sandler, Jason Barritt. Reproductive Medicine Assoc of New York, New York, NY. Objective: Although the transfer of multinucleated embryos can result in live births, these embryos demonstrate a slower developmental rate and higher incidence of chromosomal abnormalities. The purpose of this study is to determine whether the transfer of embryos with even limited multinucleation, a single binucleated blastomere, impacts pregnancy and implantation. Design: Retrospective review of patient outcomes for cycles between August 2002 and April 2003. Materials and Methods: The presence of multinucleated blastomeres (MNB) was carefully evaluated on days 2 and 3 of culture in 269 fresh day 3 embryo transfers, including 55 oocyte recipients. No multinucleated embryos were replaced in 237 embryo transfers. Only when mononucleated embryos were unavailable, were embryos with limited multinucleation (a single binucleated blastomere) transferred. This occurred in 32 embryo transfers, in which a single embryo with limited multinucleation was transferred in addition to any available mononucleated embryos. Mean patient age, mean number of embryos transferred, pregnancy rates and implantation rates were evaluated between the groups. Data was analyzed using chisquare or t-test as appropriate. Results: The clinical pregnancy and implantation rates were significantly reduced when transfers included even a single embryo with limited multinucleation. There were no significant differences for mean patient age and number of embryos transferred between the groups. A separate evaluation (data not shown) demonstrated that the results reported in this study were not affected by the day the single binucleated blastomere was observed.
ART: OVARIAN STIMULATION STANDARD PROTOCOLS II Wednesday, October 15, 2003 2:00 P.M. O-271 Alternate day triptorelin: A cost effective method for controlled ovar¨ zo¨ rnek, Elif Ergin, Berna ian hyperstimulation. Emre Karatekeli, Hakan O Ongun. EURO FERTIL, ISTANBUL, Turkey. Objective: Long gonadotropin releasing hormone (GnRH) agonist protocol has been the mostly used protocol in controlled ovarian hyperstimulation for IVF cycles. The aim of this study was to compare the effect of the every day protocol and the alternate day protocol triptorelin administration after one week pituitary desensitization period on the IVF results. Design: Prospective randomized study. Materials and Methods: Total of 60 IVF patients who have normal day 3 FSH levels (⬍10IU/L), under 36 of age, first IVF cycle, no previous ovarian surgery or history of endometriosis and polycystic ovarian disease were enrolled in the study. All patients received a daily sc. injection of 100 g triptorelin for 7 days starting from day 21 of the pretreatment cycle. After that patients were randomized in two groups: Group A (every day protocol, 30 patients) received daily 100 g triptorelin and Group B (alternate day protocol, 30 patients) received every other day 100 g triptorelin until the day of the hCG administration. Gonadotropin stimulation was started on cyle day 3. Variables which analyzed and compared were as follows: avarage age of patients, total dose of gonadotrophins used, length of gonadotropin stimulation, number of mature oocytes, number of embriyos transfered, clinical pregnancy rate and total agonist dose. Independent samples t test and chi square analysis used to determine significance. Results: Total 60 IVF cycles were analyzed and the results were summarized in Table I. Every and alternate day triptorelin protocols resulted in similar clinical pregnancy rates. Both protocols had similar numbers of mature oocytes and transferable embriyos. Length of ovarian stimulation and total dose of gonadotrophins were also same. Only totaly used GnRH agonist doses was statistically significant between two groups. Table I
*P ⬍ 0.05 Mean⫾SD Conclusion: Alternate day triptorelin administration is a cost effective stimulation protocol and has comparable IVF outcomes as compared with daily triptorelin protocol.
Wednesday, October 15, 2003 2:15 P.M. O-272
a
P⬍.05; bP⬍.008; cP⬍.02
Conclusion: The presence of more than 1 nucleus in a blastomere is an important morphological marker indicative of possible chromosomal abnormalities in the embryo. Although it may be possible for the embryo to repair, reject or direct a multinucleated blastomere to the trophectoderm, the limited multinucleation of even a single blastomere observed in this study had a significant impact on patient outcomes.
S104
Abstracts
A randomized prospective trial comparing GnRH antagonist/rFSH versus GnRH Agonist/rFSH in women pretreated with oral contraceptives prior to IVF. Larry I. Barmat, Samuel J. Chantilis, Bradley S. Hurst, Richard P. Dickey. Abington Reproductive Medicine, Abington, PA; Presbyterian Hosp of Dallas, Dallas, TX; Carolinas Medical Ctr, Charlotte, NC; Fertility Institute of New Orleans, New Orleans, LA. Objective: To compare the clinical and endocrine outcomes in IVF patients treated with rFSH/GnRH antagonist (ANT) or rFSH/GnRH agonist (AG) stimulation after OCP pretreatment. Design: Prospective, randomized, controlled, multicenter clinical trial.
Vol. 80, Suppl. 3, September 2003
surgically retrieved spermatozoa. The aim of this study was to analyze the effect of sub-optimal normal fertilization in ICSI outcomes, according to the source of sperm. Design: Retrospective in vitro study. Materials and Methods: This work included 918 patients with classical indications for ICSI performing 1061 cycles. In 737 couples ICSI was done with ejaculated spermatozoa and in 181 patients the spermatozoa were obtained by aspiration technique. The criterion to exclude female factor was maternal age ⱕ 35 years and more than 4 oocytes retrieved. Patients were ranked based on the fertilization rate namely: group A (normal fertilization rate equal or less than 50%) and group B (normal fertilization rate more than 50%). Statistical significance was determined using Student’s t test, chisquare and Fisher’s exact tests as appropriate with statistically significant differences considered at p ⬍ 0.05. Results: The mean age (SD) of female patients was 30.1 (3.7) with no statistical difference among the groups. The incidence of sub-optimal fertilization was 11.8% (126 of 1061 ICSI cycles). Complementary results are shown in table.
(a and b : p ⬍ 0.05; a group A compared with group B in non-ejaculated sperm group; b group A compared with group B in ejaculated sperm group). Conclusion: Irrespective of source of sperm, cycles that exhibit suboptimal fertilization rates are likely to provide embryos with implantation potential three times lower. Our data suggest that the fertilization rate may be considered a new, additional and valuable parameter to predict successful ICSI outcomes.
Wednesday, October 15, 2003 4:45 P.M. O-269 (a - b; b - c: p ⬍ 0.05; a - c: p ⬍ 0.001). Conclusion: Our data suggest that early cleavage parameter is a powerful indicator of embryo potential, working as a reliable prognostic tool for patients undergoing to ICSI technique. It should be incorporated in embryo classification grading, resulting in overall significantly improved embryo selection.
Wednesday, October 15, 2003 4:30 P.M. O-268 Sub-optimal fertilization: A new parameter to predict successful outcomes in assisted reproduction. Assumpto Iaconelli Jr, Lia M. Rossi, Patrı´cia Guilherme, Claudia C. Rocha, Christiany Locambo, Edson Borges Jr. Fertility—Ctr de Fertilizac¸ ao Assistida, Sao Paulo, Brazil. Objective: Intracytoplasmic sperm injection (ICSI) has become the premier treatment modality for severe male factor infertility. This technique yields good pregnancy and implantation rates using both ejaculated or
FERTILITY & STERILITY威
High endometrial aromatase P450 messenger RNA expression is associated with poor IVF outcome. Jan J. Brosens, European Fertility Association (EFA). Imperial Coll London, London, United Kingdom. Objective: The success of IVF treatment is thought to be dependent upon embryo quality and endometrial receptivity. Previous reports indicated that expression of aromatase P450 in the endometrium is restricted to women with proliferative reproductive tract disorders such as endometriosis, leiomyomas, and adenomyosis. The aim of this study was to determine if the level of endometrial aromatase P450 mRNA expression is a determinant of IVF outcome in infertile women. Design: A prospective multicenter case-control study. Setting: Eight European IVF units and a university center for reproductive research. Patients: A hundred twenty patients awaiting IVF treatment. Intervention(s): Endometrial biopsy prior to starting IVF treatment. Main Outcome Measures: Endometrial aromatase P450 and GAPDH transcripts were quantified by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System. Results: Aromatase P450 transcripts were detected in all endometria examined, although the levels varied considerably between samples ranging
S103
from 0.22 (arbitrary units, a.u.) to 486.6 a.u. (⬎2000 fold difference). The mean aromatase P450 mRNA level (⫾S.D.) in women who subsequently became pregnant after IVF treatment was 3.7 (⫾3.7) compared to 14.3 (⫾52.5) in patients with IVF failure (p ⬎ 0.05). The clinical pregnancy rate in women with high endometrial aromatase P450 mRNA levels (ⱖ 8.2 a.u.; n ⫽ 19) was 5.5% versus 22.8% in those patients with low expression levels (⬍8.2 a.u.; n ⫽ 101) (p ⬍ 0.05). The cycle day of the endometrial biopsy, cause of infertility, age, parity, number of oocytes collected, or number of embryos transferred did not differ between patients with high versus low endometrial aromatase P450 mRNA levels (p ⬎ 0.1). Conclusion: Quantitation of endometrial P450 mRNA levels is a potential diagnostic tool to identify women with high likelihood of IVF treatment failure.
Wednesday, October 15, 2003 5:00 P.M. O-270 Transfer of Embryos with Even a Single Multinucleated Blastomere Significantly Reduces Pregnancy and Implantation Rates. Marlena Duke, Natalia Basile, Melissa Johnson, Alan Copperman, Benjamin Sandler, Jason Barritt. Reproductive Medicine Assoc of New York, New York, NY. Objective: Although the transfer of multinucleated embryos can result in live births, these embryos demonstrate a slower developmental rate and higher incidence of chromosomal abnormalities. The purpose of this study is to determine whether the transfer of embryos with even limited multinucleation, a single binucleated blastomere, impacts pregnancy and implantation. Design: Retrospective review of patient outcomes for cycles between August 2002 and April 2003. Materials and Methods: The presence of multinucleated blastomeres (MNB) was carefully evaluated on days 2 and 3 of culture in 269 fresh day 3 embryo transfers, including 55 oocyte recipients. No multinucleated embryos were replaced in 237 embryo transfers. Only when mononucleated embryos were unavailable, were embryos with limited multinucleation (a single binucleated blastomere) transferred. This occurred in 32 embryo transfers, in which a single embryo with limited multinucleation was transferred in addition to any available mononucleated embryos. Mean patient age, mean number of embryos transferred, pregnancy rates and implantation rates were evaluated between the groups. Data was analyzed using chisquare or t-test as appropriate. Results: The clinical pregnancy and implantation rates were significantly reduced when transfers included even a single embryo with limited multinucleation. There were no significant differences for mean patient age and number of embryos transferred between the groups. A separate evaluation (data not shown) demonstrated that the results reported in this study were not affected by the day the single binucleated blastomere was observed.
ART: OVARIAN STIMULATION STANDARD PROTOCOLS II Wednesday, October 15, 2003 2:00 P.M. O-271 Alternate day triptorelin: A cost effective method for controlled ovar¨ zo¨ rnek, Elif Ergin, Berna ian hyperstimulation. Emre Karatekeli, Hakan O Ongun. EURO FERTIL, ISTANBUL, Turkey. Objective: Long gonadotropin releasing hormone (GnRH) agonist protocol has been the mostly used protocol in controlled ovarian hyperstimulation for IVF cycles. The aim of this study was to compare the effect of the every day protocol and the alternate day protocol triptorelin administration after one week pituitary desensitization period on the IVF results. Design: Prospective randomized study. Materials and Methods: Total of 60 IVF patients who have normal day 3 FSH levels (⬍10IU/L), under 36 of age, first IVF cycle, no previous ovarian surgery or history of endometriosis and polycystic ovarian disease were enrolled in the study. All patients received a daily sc. injection of 100 g triptorelin for 7 days starting from day 21 of the pretreatment cycle. After that patients were randomized in two groups: Group A (every day protocol, 30 patients) received daily 100 g triptorelin and Group B (alternate day protocol, 30 patients) received every other day 100 g triptorelin until the day of the hCG administration. Gonadotropin stimulation was started on cyle day 3. Variables which analyzed and compared were as follows: avarage age of patients, total dose of gonadotrophins used, length of gonadotropin stimulation, number of mature oocytes, number of embriyos transfered, clinical pregnancy rate and total agonist dose. Independent samples t test and chi square analysis used to determine significance. Results: Total 60 IVF cycles were analyzed and the results were summarized in Table I. Every and alternate day triptorelin protocols resulted in similar clinical pregnancy rates. Both protocols had similar numbers of mature oocytes and transferable embriyos. Length of ovarian stimulation and total dose of gonadotrophins were also same. Only totaly used GnRH agonist doses was statistically significant between two groups. Table I
*P ⬍ 0.05 Mean⫾SD Conclusion: Alternate day triptorelin administration is a cost effective stimulation protocol and has comparable IVF outcomes as compared with daily triptorelin protocol.
Wednesday, October 15, 2003 2:15 P.M. O-272
a
P⬍.05; bP⬍.008; cP⬍.02
Conclusion: The presence of more than 1 nucleus in a blastomere is an important morphological marker indicative of possible chromosomal abnormalities in the embryo. Although it may be possible for the embryo to repair, reject or direct a multinucleated blastomere to the trophectoderm, the limited multinucleation of even a single blastomere observed in this study had a significant impact on patient outcomes.
S104
Abstracts
A randomized prospective trial comparing GnRH antagonist/rFSH versus GnRH Agonist/rFSH in women pretreated with oral contraceptives prior to IVF. Larry I. Barmat, Samuel J. Chantilis, Bradley S. Hurst, Richard P. Dickey. Abington Reproductive Medicine, Abington, PA; Presbyterian Hosp of Dallas, Dallas, TX; Carolinas Medical Ctr, Charlotte, NC; Fertility Institute of New Orleans, New Orleans, LA. Objective: To compare the clinical and endocrine outcomes in IVF patients treated with rFSH/GnRH antagonist (ANT) or rFSH/GnRH agonist (AG) stimulation after OCP pretreatment. Design: Prospective, randomized, controlled, multicenter clinical trial.
Vol. 80, Suppl. 3, September 2003