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Gonadotropin and Modulation of Ovarian Neoplastic Cell Proliferation In Vitro.
menstrual cycle with FSH (50 –225 IU/day) starting on day 7. HCG (10,000 IU) was given when leading follicles were ⬎2 cm followed by IUI. The mean ⫹ SD age and duration of infertility were 36.5 ⫾ 2.96 and 4.6 ⫾ 1.2 years, respectively. Results: FSH only (16 cycles) FSH/cycle (IU) 1,469 ⫾ 498 Stimulation days/cycle 9.1 ⫾ 3 Day of HCG administration 12.2 ⫾ 2.9 Number of mature follicles 1.88 ⫾ 0.7 E2 on HCG day (pmol/L) 1,893 ⫾ 1,066 E2/mature follicle on 1,097 ⫾ 557 HCG day (pmol/L) Endometrial thickness on 0.92 ⫾ 0.15 HCG day (cm) LH on HCG day (IU/L) 14.7 ⫾ 13.2 a
Letrozole ⫹ FSH (8 cycles) P value 603 ⫾ 510 6.8 ⫾ 2.4 13.3 ⫾ 2.1 3.5 ⫾ 1.3 1,689 ⫾ 1,044 483 ⫾ 259
⬍.05 NSa NS ⬍.05 NS ⬍.05
0.93 ⫾ 0.14
NS
10.4 ⫾ 8.5
NS
Background: The putative link between ovulation induction and ovarian neoplasia (ON) has been addressed by several epidemiologic studies with conflicting results. Additionally, it is known that pregnancy protects against ovarian cancer. However, the mechanisms explaining such relationships have yet to be elucidated. The purpose of this study was to evaluate the effects of FSH and hCG on benign, borderline, and malignant ovarian cell lines with known FSH receptors. Materials and Methods: Benign cystadenoma (ML5), borderline cystadenoma (ML46), and cystadenocarcinoma (AO) cell lines were maintained in MEM and RPMI media with 10% FBS, respectively. For cell growth experiments, the cells were plated at a cell density of 5 ⫻ 104 cells/ml and grown as monolayer cultures. At 50 – 60% confluence, treatment was initiated with either placebo (growth media alone), FSH 40 mIU/ml, hCG 200 mIU/ml, or FSH 40 mIU/ml ⫹ hCG 200 mIU/ml. Each treatment was evaluated 6 times per run, and the experiment was run 5 separate times. After 72 hours of treatment, cell proliferation was quantified using a colorimetric MTT assay at an optical density (OD) of 630 nm. After adjusting for the block effect, ANOVA was used for statistical analyses. Results:
NS ⫽ not significant.
During letrozole ⫹ FSH cycles two pregnancies were achieved (25%). Conclusion: In this preliminary report, we demonstrate a potential benefit of letrozole for improving ovarian response to FSH in poor responders. PII S0015-0282(01)01717-4
O-10 How Accurate Are Day Three Criteria for the Selection of the Best Two Embryos? A. A. Milki, J. Gebhardt, M. D. Hinckley, D. Dasig, L. M. Westphal, B. Behr. Stanford University Medical Center, Stanford, CA. Objective: Recent guidelines encourage transferring only two embryos in patients at risk for high-order multiples. Since 1998, we routinely perform blastocyst transfer (BT) in patients with more than three 8-cell embryos to facilitate the selection process. However, even in this population, the additional effort needed for blastocyst culture, compounded by concerns about the occasional sub-optimal performance of sequential media, have led many programs to maintain their day-3 ET practice. In this study we assess the accuracy of the embryologist’s choice of the best two embryos on day 3 to evaluate whether culture to day 5 is warranted for better embryo selection. Material and Methods: This is an ongoing, prospective study of BT since September 2000. On day 3, the embryologist chose the two embryos that would have been selected for transfer that day and cultured them apart from the remaining embryos. On day 5, all embryos were examined and the two most advanced blastocysts were transferred with additional blastocysts frozen on day 5 or 6. All embryos were cultured under oil in P1 with 10% SSS and moved on day 3 to blastocyst medium with 10% SSS. Results: Thirty-four patients had BT. The mean age was 35 (29 – 41 years). The mean number of ⱖ8-cell embryos was 6.3. Picks were ⱖ8-cells and typically grade I (91%). All patients made blastocysts and 31 had cryopreservation. The mean number of blastocysts was 3.2 on day 5 and 2.2 on day 6. Neither pick was chosen in 15 patients. In 11 patients one pick was transferred while both picks were transferred in 8 patients only (23%). Of 41 nontransferred picks, 26 were frozen and 15 arrested, but no patient had both arrest. Conclusions: The value of morphologic criteria for cleavage-stage embryo selection may fall short when the transfer is limited to two embryos. Although a reasonable number of day 3 picks did end up forming blastocysts by day 6, surprisingly, in 44% of our patients the chosen blastocysts were not from the day 3 picks selected by experienced embryologists. Therefore, we believe that BT is the method of choice in this population. PII S0015-0282(01)01718-6
O-11 Gonadotropin and Modulation of Ovarian Neoplastic Cell Proliferation In Vitro. D. E. Tourgeman, J. J. Lu, R. Boostanfar, W. Zheng, F. Z. Stanczyk, J. C. Felix, R. J. Paulson. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Southern California Keck School of Medicine, Los Angeles, CA.
FERTILITY & STERILITY威
Placebo FSH hCG FSH ⫹ hCG
ML5
ML46
AO
0.103 ⫾ 0.005 0.113 ⫾ 0.005 0.087 ⫾ 0.005a,b 0.089 ⫾ 0.005b
0.157 ⫾ 0.006 0.143 ⫾ 0.006 0.123 ⫾ 0.006a,b 0.117 ⫾ 0.006a,b
0.167 ⫾ 0.005 0.145 ⫾ 0.005a 0.113 ⫾ 0.005a,b 0.098 ⫾ 0.005a,b
Results reported as mean OD values ⫾ SEM. a P⬍.05 compared to placebo. b P⬍.05 compared to FSH. In the ML5 and ML46 cell lines, FSH stimulation resulted in cell growth which was similar to placebo treatment. When treated with hCG alone, all cell lines showed significantly decreased cellular proliferation when compared to placebo or FSH alone (P⬍.05). When hCG was given in combination with FSH, there was significantly decreased growth of all cell lines when compared to FSH alone (P⬍.05). Additionally, the combination of FSH and hCG resulted in decreased cell growth when compared to placebo. This difference was statistically significant in the ML46 and AO cell lines (P⬍.05), and approached statistical significance in the ML5 line (P ⫽ .07). Conclusions: These data show that the growth of benign, borderline, and malignant ovarian epithelial cell lines with FSH receptors may be inhibited by hCG at levels which are commonly achieved with hCG administration during ovulation induction. This suppression is evident even in the presence of FSH, suggesting that an ovulation induction regimen consisting of FSH and hCG would be unlikely to stimulate ON cell proliferation. These data also provide a possible mechanism by which the high levels of hCG during pregnancy may protect against ovarian cancer. PII S0015-0272(01)01719-8
O-12 Transvaginal Ultrasound Guided Embryo Transfer Improves Outcome in Patients With Failed IVF Cycles. R. E. Anderson, N. L. Nugent, A. T. Gregg, S. L. Nunn, B. R. Behr. Southern California Center for Reproductive Medicine, Newport Beach, CA. The use of ultrasound guidance during embryo transfer has been reported to increase pregnancy and implantation rates. All reports, however, have utilized transabdominal ultrasonography. We have developed a novel method of embryo transfer using transvaginal ultrasound guidance (TUG), which provides better resolution than transabdominal ultrasonography, allowing very accurate placement of embryos. Eighty day-3 transfers of fresh embryos have been performed during a 5-month period. Thirty-nine pregnancies have resulted (49%). Of these, 11 patients who became pregnant had previously completed 21 failed cycles of IVF without TUG. Seven of these pregnancies were multiple pregnancies. Using these patients as their own controls, there was no significant difference in age (35.7 vs. 36.2 years), ampules of gonadotrophins used (41.5 vs. 49.3), or estradiol level on day of hCG (2268 vs. 2727 pg/ml) in failed vs. successful cycles. There was also no difference in the number of oocytes retrieved (12.2 vs. 13.4), two pronuclear embryos obtained (8.4 vs. 10), or embryos transferred
9S
Letrozole ⫹ FSH (8 cycles) P value 603 ⫾ 510 6.8 ⫾ 2.4 13.3 ⫾ 2.1 3.5 ⫾ 1.3 1,689 ⫾ 1,044 483 ⫾ 259
⬍.05 NSa NS ⬍.05 NS ⬍.05
0.93 ⫾ 0.14
NS
10.4 ⫾ 8.5
NS
Background: The putative link between ovulation induction and ovarian neoplasia (ON) has been addressed by several epidemiologic studies with conflicting results. Additionally, it is known that pregnancy protects against ovarian cancer. However, the mechanisms explaining such relationships have yet to be elucidated. The purpose of this study was to evaluate the effects of FSH and hCG on benign, borderline, and malignant ovarian cell lines with known FSH receptors. Materials and Methods: Benign cystadenoma (ML5), borderline cystadenoma (ML46), and cystadenocarcinoma (AO) cell lines were maintained in MEM and RPMI media with 10% FBS, respectively. For cell growth experiments, the cells were plated at a cell density of 5 ⫻ 104 cells/ml and grown as monolayer cultures. At 50 – 60% confluence, treatment was initiated with either placebo (growth media alone), FSH 40 mIU/ml, hCG 200 mIU/ml, or FSH 40 mIU/ml ⫹ hCG 200 mIU/ml. Each treatment was evaluated 6 times per run, and the experiment was run 5 separate times. After 72 hours of treatment, cell proliferation was quantified using a colorimetric MTT assay at an optical density (OD) of 630 nm. After adjusting for the block effect, ANOVA was used for statistical analyses. Results:
NS ⫽ not significant.
During letrozole ⫹ FSH cycles two pregnancies were achieved (25%). Conclusion: In this preliminary report, we demonstrate a potential benefit of letrozole for improving ovarian response to FSH in poor responders. PII S0015-0282(01)01717-4
O-10 How Accurate Are Day Three Criteria for the Selection of the Best Two Embryos? A. A. Milki, J. Gebhardt, M. D. Hinckley, D. Dasig, L. M. Westphal, B. Behr. Stanford University Medical Center, Stanford, CA. Objective: Recent guidelines encourage transferring only two embryos in patients at risk for high-order multiples. Since 1998, we routinely perform blastocyst transfer (BT) in patients with more than three 8-cell embryos to facilitate the selection process. However, even in this population, the additional effort needed for blastocyst culture, compounded by concerns about the occasional sub-optimal performance of sequential media, have led many programs to maintain their day-3 ET practice. In this study we assess the accuracy of the embryologist’s choice of the best two embryos on day 3 to evaluate whether culture to day 5 is warranted for better embryo selection. Material and Methods: This is an ongoing, prospective study of BT since September 2000. On day 3, the embryologist chose the two embryos that would have been selected for transfer that day and cultured them apart from the remaining embryos. On day 5, all embryos were examined and the two most advanced blastocysts were transferred with additional blastocysts frozen on day 5 or 6. All embryos were cultured under oil in P1 with 10% SSS and moved on day 3 to blastocyst medium with 10% SSS. Results: Thirty-four patients had BT. The mean age was 35 (29 – 41 years). The mean number of ⱖ8-cell embryos was 6.3. Picks were ⱖ8-cells and typically grade I (91%). All patients made blastocysts and 31 had cryopreservation. The mean number of blastocysts was 3.2 on day 5 and 2.2 on day 6. Neither pick was chosen in 15 patients. In 11 patients one pick was transferred while both picks were transferred in 8 patients only (23%). Of 41 nontransferred picks, 26 were frozen and 15 arrested, but no patient had both arrest. Conclusions: The value of morphologic criteria for cleavage-stage embryo selection may fall short when the transfer is limited to two embryos. Although a reasonable number of day 3 picks did end up forming blastocysts by day 6, surprisingly, in 44% of our patients the chosen blastocysts were not from the day 3 picks selected by experienced embryologists. Therefore, we believe that BT is the method of choice in this population. PII S0015-0282(01)01718-6
O-11 Gonadotropin and Modulation of Ovarian Neoplastic Cell Proliferation In Vitro. D. E. Tourgeman, J. J. Lu, R. Boostanfar, W. Zheng, F. Z. Stanczyk, J. C. Felix, R. J. Paulson. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Southern California Keck School of Medicine, Los Angeles, CA.
FERTILITY & STERILITY威
Placebo FSH hCG FSH ⫹ hCG
ML5
ML46
AO
0.103 ⫾ 0.005 0.113 ⫾ 0.005 0.087 ⫾ 0.005a,b 0.089 ⫾ 0.005b
0.157 ⫾ 0.006 0.143 ⫾ 0.006 0.123 ⫾ 0.006a,b 0.117 ⫾ 0.006a,b
0.167 ⫾ 0.005 0.145 ⫾ 0.005a 0.113 ⫾ 0.005a,b 0.098 ⫾ 0.005a,b
Results reported as mean OD values ⫾ SEM. a P⬍.05 compared to placebo. b P⬍.05 compared to FSH. In the ML5 and ML46 cell lines, FSH stimulation resulted in cell growth which was similar to placebo treatment. When treated with hCG alone, all cell lines showed significantly decreased cellular proliferation when compared to placebo or FSH alone (P⬍.05). When hCG was given in combination with FSH, there was significantly decreased growth of all cell lines when compared to FSH alone (P⬍.05). Additionally, the combination of FSH and hCG resulted in decreased cell growth when compared to placebo. This difference was statistically significant in the ML46 and AO cell lines (P⬍.05), and approached statistical significance in the ML5 line (P ⫽ .07). Conclusions: These data show that the growth of benign, borderline, and malignant ovarian epithelial cell lines with FSH receptors may be inhibited by hCG at levels which are commonly achieved with hCG administration during ovulation induction. This suppression is evident even in the presence of FSH, suggesting that an ovulation induction regimen consisting of FSH and hCG would be unlikely to stimulate ON cell proliferation. These data also provide a possible mechanism by which the high levels of hCG during pregnancy may protect against ovarian cancer. PII S0015-0272(01)01719-8
O-12 Transvaginal Ultrasound Guided Embryo Transfer Improves Outcome in Patients With Failed IVF Cycles. R. E. Anderson, N. L. Nugent, A. T. Gregg, S. L. Nunn, B. R. Behr. Southern California Center for Reproductive Medicine, Newport Beach, CA. The use of ultrasound guidance during embryo transfer has been reported to increase pregnancy and implantation rates. All reports, however, have utilized transabdominal ultrasonography. We have developed a novel method of embryo transfer using transvaginal ultrasound guidance (TUG), which provides better resolution than transabdominal ultrasonography, allowing very accurate placement of embryos. Eighty day-3 transfers of fresh embryos have been performed during a 5-month period. Thirty-nine pregnancies have resulted (49%). Of these, 11 patients who became pregnant had previously completed 21 failed cycles of IVF without TUG. Seven of these pregnancies were multiple pregnancies. Using these patients as their own controls, there was no significant difference in age (35.7 vs. 36.2 years), ampules of gonadotrophins used (41.5 vs. 49.3), or estradiol level on day of hCG (2268 vs. 2727 pg/ml) in failed vs. successful cycles. There was also no difference in the number of oocytes retrieved (12.2 vs. 13.4), two pronuclear embryos obtained (8.4 vs. 10), or embryos transferred
9S