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The efficacy of selected single embryo transfer (SET) with vitrification
Choi, J. S. Yoon, K. R. Hwang, S. H. Kim, W. D. Lee, S. Y. Moon. Seoul National University College of Medicine, Seoul, Republic of Korea; Maria Infertility Hospital, Seoul, Republic of Korea; Asan Medical Center, Seoul, Republic of Korea. OBJECTIVE: To investigate the association of FSH receptor (FSHR) polymorphism at position 680 with outcomes of controlled ovarian hyperstimulation for IVF-ET in Korean women DESIGN: Genetic polymorphism analysis. MATERIALS AND METHODS: The FSHR polymorphism was analyzed by PCR-RFLP in 172 ovulatory women below the age of 40 year. Patients with polycystic ovary syndrome, endometriosis, or previous history of ovarian surgery were excluded. RESULTS: Genotype distribution was 41.9% for the Asn/Asn, 47.7% for the Asn/Ser, and 10.5% for the Ser/Ser FSHR genotype group. There was no difference in age of subjects and infertility diagnosis between genotype groups. When the patients were grouped according to their FSHR genotype, the basal levels of FSH (day 3) were significantly different among the three groups (6.0 ⫾ 0.3 IU/L (mean ⫾ SEM), 5.8 ⫾ 0.3 IU/L, and 8.6 ⫾ 1.2 IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P ⫽0.002). The Ser/Ser group showed a higher total doses of gonadotropins required to achieve ovulation induction, and a lower serum estradiol levels at the time of hCG administration compared with other two groups, but the differences were of no statistical significance. The numbers of oocytes retrieved were significantly different among the three groups (8.6 ⫾ 0.8, 9.9 ⫾ 0.6, and 6.3 ⫾ 0.9, for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P ⫽0.049). Clinical pregnancy rates were 39.7%, 25.6%, and 25.0% for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively. CONCLUSION: Homozygous Ser/Ser genotype of FSHR polymorphism at position 680 was associated with decreased ovarian response to gonadotropin stimulation for IVF-ET. Supported by: supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (01-PJ10-PG6 – 01GN13– 0002)
P-205 Prediction of outcomes of assisted reproductive technology treatment by calcium ionophore-induced acrosome reaction. T. Hara, T. Katsuki, T. Kusuda, K. Ohama, J. Tanaka, K. Ueda. Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; Department of Obstetrics and Gynecology, Hiroshima Prefectural Hospital, Hiroshima, Japan. OBJECTIVE: Conventional semen analysis is not a good predictor of successful outcome of in vivo insemination, intrauterine insemination (IUI) or hysteroscopic intra-tubal insemination (HIT), or a predictor of complete fertilization failure (CFF) in conventional IVF (IVF). We investigated whether calcium ionophore induced acrosome reaction provides an additional indicator of pregnancy among various assisted reproduction modalities as well as an indicator of complete fertilization failure in IVF, independent of conventional semen parameters, sperm concentration and motility. DESIGN: prospective study MATERIALS AND METHODS: Subjects included infertile couples with no female infertility factors (n⫽478) and women with tubal obstruction (n⫽32). Procedures included conventional IVF (IVF): n⫽148, ICSI: n⫽72, HIT: n⫽245, IUI: n⫽61. They all underwent a standard protocol of historytaking, physical examination and investigations including conventional semen analysis as well as a test of the sperm acrosome reaction following ionophore challenge. Every patient gave an informed consent prior to participating in this study, which was approved by the local Institutional Review Board, Faculty of Medicine. Calcium ionophore induced acrosomal reactivity was assessed in spermatozoa selected by swim-up. Spermatozoa on the slide were subjected to staining with fluorescent isothiocyanateconjugated pisum sativum agglutinin (FITC-PSA) and with Hoescht 33258. Spermatozoa were considered viable when their heads were unstained or stained only lightly with Hoechst 33258. When the equatorial segment strongly stained with FITC-PSA, the spermatozoon was considered to have reacted. The percentage of acrosome-reacted spermatozoa to viable spermatozoa (%AR) was calculated. At least 300 spermatozoa per sample were
FERTILITY & STERILITY威
examined to determine the %AR. We excluded abnormal or deformed spermatozoa, as well as unclassifiable fluorescence patterns. Multiple logistic regression analysis and receiver-operator characteristic (ROC) curve analysis were performed to determine the best predictive variables and cut-off values in the prediction of fertilization rate and pregnancy in each modality. -square analysis was used to compare the different fertilization and pregnancy rates. P⬍0.05 was considered significant. RESULTS: Cut-off value of %AR for IVF was 21% and the value was independent from the parameters of conventional semen analysis in predicting fertilization rate and pregnancy. Fertilization rate on IVF was higher for those with %AR over 21% compared to that of those with a lower percentage (63.6% vs. 35.4%; p⬍0.01). The pregnancy rate was also higher (43.1% vs. 18.9%; p⬍0.01). CFF was lower for those with %AR over 21% (24.5% vs. 4.2%,p⬍0.01). Fertilization rate, pregnancy rate, and CFF on ICSI did not differ for those with %AR over 21%. Pregnancy rates were higher for those with %AR over 21% than for those with a lower percentage on IUI (28.0% vs. 5.6%, p⬍0.05) and on HIT (23.9% vs. 6.2%, p⬍0.01). CONCLUSION: These data shows that %AR may be a novel parameter independent of conventional semen parameters in choosing assisted reproduction modalities. Thus, patients with acrosomal reactivity below 21% could not achieve pregnancy by IUI, HIT, or IVF with high rates. With reactivity below 21%, a low fertilization rate for initial IVF, ICSI or split fertilization is indicated. Supported by: None
P-206 The efficacy of selected single embryo transfer (SET) with vitrification. S. Teramoto, K. Uchiyama, F. Aono, M. Kuwayama, H. Osada, O. Kato. Kato Ladies Clinic, Tokyo, Japan; Nihon Univ Sch of Medicine, Tokyo, Japan. OBJECTIVE: To evaluate the efficacy of the repeating SET with cryopreserved surplus embryos by vitrification to prevent multiple pregnancies. DESIGN: Prospective clinical study. MATERIALS AND METHODS: From August in 2003 to February in 2004, 108 patients (33.7⫾3.3 years old; mean⫾SD, ⬍37 years old). They had no tubal infertile factors and no chramidial antibodies. The Ovarian stimulation was done by the Clomiphene cycle with hMG. Clomiphene was initiated on d3 by 50mg/day and continued until the flare up of LH, caused by spraying 300mg of GnRHa. hMG was initiated on d8 by 150IU and continued every other day until the day before the flare up of LH. The mean dosage of total hMG was 375⫾141 IU. SET was applied for the patients who’s past histories of four-cell stage ET were less than two times, and had more than three good four-cell stage embryos. The best was transferred first. The surplus embryos were cultured into blastocysts by Blastocyst-medium (Irvine Scientific). When they grew into expanded blastocyts, they were immediately cryopreserved by vitrification using the Cryotop method (Kuwayama et al, 2000). If the first transfer resulted in failure, single vitifiedthawed blastocyst was transferred in a following natural cycle or a hormonereplacement cycle. We repeated this SET until finishing the stock of the cryopreserved surplus blastocysts. RESULTS: The number of retrieved oocytes was 5.3⫾1.9 and the number of 4-cell stage embryos were 4.2⫾1.5. The pregnancy rate in the first SET was 44.0%. The surplus 287 four-cell stage embryos were cultured into blastocysts. The blastocyst formation rate per 4-cell stage embryos were 68.6% (197 expanded blastocysts), and per patient was 95.0%, respectively. All the blastocysts were successfully cryopreserved by vitrification (100% post-thaw survival). Twenty-nine patients, who resulted in failure in the first SET, underwent single blastocyst transfer. The pregnancy rate per transfer was 57.7% and per cycle was 91.4%, respectively. CONCLUSION: As long as more than three good four-cell stage embryos are obtained in the Clomiphene cycle, increasing the number of transferred embryos does not increase pregnancy rate more than expected, but rather increases multiple pregnancy rate. In such cases, our SET protocol is very effective in increasing pregnancy rate per cycle with preventing multiple pregnancies.
P-207 Liberal performing of office hysteroscopy in the presence of normal HSG does not improve ICSI and ET outcome. H. Yarali, E. Demirtas, G.
S207
P-205 Prediction of outcomes of assisted reproductive technology treatment by calcium ionophore-induced acrosome reaction. T. Hara, T. Katsuki, T. Kusuda, K. Ohama, J. Tanaka, K. Ueda. Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; Department of Obstetrics and Gynecology, Hiroshima Prefectural Hospital, Hiroshima, Japan. OBJECTIVE: Conventional semen analysis is not a good predictor of successful outcome of in vivo insemination, intrauterine insemination (IUI) or hysteroscopic intra-tubal insemination (HIT), or a predictor of complete fertilization failure (CFF) in conventional IVF (IVF). We investigated whether calcium ionophore induced acrosome reaction provides an additional indicator of pregnancy among various assisted reproduction modalities as well as an indicator of complete fertilization failure in IVF, independent of conventional semen parameters, sperm concentration and motility. DESIGN: prospective study MATERIALS AND METHODS: Subjects included infertile couples with no female infertility factors (n⫽478) and women with tubal obstruction (n⫽32). Procedures included conventional IVF (IVF): n⫽148, ICSI: n⫽72, HIT: n⫽245, IUI: n⫽61. They all underwent a standard protocol of historytaking, physical examination and investigations including conventional semen analysis as well as a test of the sperm acrosome reaction following ionophore challenge. Every patient gave an informed consent prior to participating in this study, which was approved by the local Institutional Review Board, Faculty of Medicine. Calcium ionophore induced acrosomal reactivity was assessed in spermatozoa selected by swim-up. Spermatozoa on the slide were subjected to staining with fluorescent isothiocyanateconjugated pisum sativum agglutinin (FITC-PSA) and with Hoescht 33258. Spermatozoa were considered viable when their heads were unstained or stained only lightly with Hoechst 33258. When the equatorial segment strongly stained with FITC-PSA, the spermatozoon was considered to have reacted. The percentage of acrosome-reacted spermatozoa to viable spermatozoa (%AR) was calculated. At least 300 spermatozoa per sample were
FERTILITY & STERILITY威
examined to determine the %AR. We excluded abnormal or deformed spermatozoa, as well as unclassifiable fluorescence patterns. Multiple logistic regression analysis and receiver-operator characteristic (ROC) curve analysis were performed to determine the best predictive variables and cut-off values in the prediction of fertilization rate and pregnancy in each modality. -square analysis was used to compare the different fertilization and pregnancy rates. P⬍0.05 was considered significant. RESULTS: Cut-off value of %AR for IVF was 21% and the value was independent from the parameters of conventional semen analysis in predicting fertilization rate and pregnancy. Fertilization rate on IVF was higher for those with %AR over 21% compared to that of those with a lower percentage (63.6% vs. 35.4%; p⬍0.01). The pregnancy rate was also higher (43.1% vs. 18.9%; p⬍0.01). CFF was lower for those with %AR over 21% (24.5% vs. 4.2%,p⬍0.01). Fertilization rate, pregnancy rate, and CFF on ICSI did not differ for those with %AR over 21%. Pregnancy rates were higher for those with %AR over 21% than for those with a lower percentage on IUI (28.0% vs. 5.6%, p⬍0.05) and on HIT (23.9% vs. 6.2%, p⬍0.01). CONCLUSION: These data shows that %AR may be a novel parameter independent of conventional semen parameters in choosing assisted reproduction modalities. Thus, patients with acrosomal reactivity below 21% could not achieve pregnancy by IUI, HIT, or IVF with high rates. With reactivity below 21%, a low fertilization rate for initial IVF, ICSI or split fertilization is indicated. Supported by: None
P-206 The efficacy of selected single embryo transfer (SET) with vitrification. S. Teramoto, K. Uchiyama, F. Aono, M. Kuwayama, H. Osada, O. Kato. Kato Ladies Clinic, Tokyo, Japan; Nihon Univ Sch of Medicine, Tokyo, Japan. OBJECTIVE: To evaluate the efficacy of the repeating SET with cryopreserved surplus embryos by vitrification to prevent multiple pregnancies. DESIGN: Prospective clinical study. MATERIALS AND METHODS: From August in 2003 to February in 2004, 108 patients (33.7⫾3.3 years old; mean⫾SD, ⬍37 years old). They had no tubal infertile factors and no chramidial antibodies. The Ovarian stimulation was done by the Clomiphene cycle with hMG. Clomiphene was initiated on d3 by 50mg/day and continued until the flare up of LH, caused by spraying 300mg of GnRHa. hMG was initiated on d8 by 150IU and continued every other day until the day before the flare up of LH. The mean dosage of total hMG was 375⫾141 IU. SET was applied for the patients who’s past histories of four-cell stage ET were less than two times, and had more than three good four-cell stage embryos. The best was transferred first. The surplus embryos were cultured into blastocysts by Blastocyst-medium (Irvine Scientific). When they grew into expanded blastocyts, they were immediately cryopreserved by vitrification using the Cryotop method (Kuwayama et al, 2000). If the first transfer resulted in failure, single vitifiedthawed blastocyst was transferred in a following natural cycle or a hormonereplacement cycle. We repeated this SET until finishing the stock of the cryopreserved surplus blastocysts. RESULTS: The number of retrieved oocytes was 5.3⫾1.9 and the number of 4-cell stage embryos were 4.2⫾1.5. The pregnancy rate in the first SET was 44.0%. The surplus 287 four-cell stage embryos were cultured into blastocysts. The blastocyst formation rate per 4-cell stage embryos were 68.6% (197 expanded blastocysts), and per patient was 95.0%, respectively. All the blastocysts were successfully cryopreserved by vitrification (100% post-thaw survival). Twenty-nine patients, who resulted in failure in the first SET, underwent single blastocyst transfer. The pregnancy rate per transfer was 57.7% and per cycle was 91.4%, respectively. CONCLUSION: As long as more than three good four-cell stage embryos are obtained in the Clomiphene cycle, increasing the number of transferred embryos does not increase pregnancy rate more than expected, but rather increases multiple pregnancy rate. In such cases, our SET protocol is very effective in increasing pregnancy rate per cycle with preventing multiple pregnancies.
P-207 Liberal performing of office hysteroscopy in the presence of normal HSG does not improve ICSI and ET outcome. H. Yarali, E. Demirtas, G.
S207