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Successful propagation of human prepubertal spermatogonial stem cells
ORAL PRESENTATIONS Monday, October 25, 2010 The following seven papers are candidates for the ASRM Scientific Program Prize Paper Awards. Seven additional candidates will be presented during the Prize Paper Candidates’ Session on Tuesday morning.
PRIZE PAPER CANDIDATES O-01 Monday, October 25, 2010 11:15 AM PREGNANCY RATES ACROSS MULTIPLE TREATMENT CYCLES: DATA FROM THE FAST TRACK AND STANDARD TREATMENT (FASTT) TRIAL. M. B. Goldman, M. M. Regan, M. M. Alper, K. L. Thornton, R. H. Reindollar. Obstetrics & Gynecology, DartmouthHitchcock Medical Center, Lebanon, NH; Biostatistics & Computational Biology, Dana-Farber Cancer Institute, Boston, MA; Boston IVF, Waltham, MA. OBJECTIVE: To assess pregnancy rates by type of treatment and cycle number in women aged 21-39 with unexplained infertility. DESIGN: Data are from a randomized clinical trial to evaluate optimal infertility treatment. MATERIALS AND METHODS: Couples (n¼503) who had a history of at least 12 months of attempted conception, no pelvic pathology or previous treatment, and normal semen analysis and ovarian reserve were enrolled. Treatment course and results were recorded prospectively. Pregnancy was determined by confirmation of a fetal heart beat. RESULTS: 247 couples were randomized to a conventional treatment protocol (3 cycles of clomiphene(CC)/IUI, 3 cycles of FSH/IUI, and up to 6 cycles of IVF); 256 couples were randomized to the accelerated treatment arm (3 cycles of CC/IUI and up to 6 cycles of IVF). Twenty-eight couples either conceived or withdrew before their first treatment cycle.
OBJECTIVE: To achieve in vitro proliferation of prepuberal spermatogonial stem cells (SSCs) in order to obtain a sufficient number of SSCs for successful autotransplantation in sterile childhood cancer survivors. DESIGN: In vitro culture of SSCs from testis of prepuberal boys. MATERIALS AND METHODS: We obtained testicular biopsies from 2 boys of 6 and 8 years old diagnosed with Hodgkin lymphoma. Testicular cells and germ line stem cell (GSC) clusters were cultured using our previously established system for adult SSCs. The presence of spermatogonia in culture was determined by RT-PCR and/or immunohistochemistry using spermatogonial markers ITGA6, ITGB1, GFRA1, CD9, GPR125, UCHL1 and ZBTB16. To determine the presence of SSCs, xenotransplantation of cultured testicular cells to the testes of busulphan treated immunodeficient mice was performed. RESULTS: In testicular cell cultures from both patients, GSC clusters were formed 2.5-3 weeks after initiation of the culture. Testicular cells and subcultured GSC clusters could be cultured for at least 15 and 23 weeks respectively. Expression of spermatogonial markers confirmed the presence of spermatogonia throughout the entire culture period for both patients. Xenotransplantation assays showed successful colonization of mouse testes with cultured human prepuberal SSCs of both patients confirming their stem cell capacity. Transplantation of cells from an early and a later passage of the cultured testicular cells showed a 922-fold increase in the number of human prepuberal SSCs within a time frame of 28 days. CONCLUSION: Our study demonstrated that prepuberal SSCs can be propagated in vitro to the extend that successful autotransplantation of SSCs to infertile childhood cancer survivors should become feasible in the near future. Cryopreservation of testicular biopsies in all prepuberal boys with cancer undergoing gonadotoxic treatment should therefore be considered. Supported by: The Dutch Children Cancer-Free Foundation (KIKA).
O-03 Monday, October 25, 2010 11:45 AM FASTT Trial Pregnancy Rates by Treatment and Cycle Number Randomized Group Conventional
Cycle CC/IUI 1 2 3 FSH/IUI 1 2 3 IVF 1 2 3 4 5 6
No. No. Initiated Conceived
% (95% CI)
233 217 181
15 20 20
6.4 (3.6-10.4) 9.2 (5.7-13.9) 11.0 (6.9-16.5)
169 141 120
19 19 11
11.2 (6.9-17.0) 13.5 (8.3-20.2) 9.2 (4.7-15.8)
111 73 43 20 9 5
45 24 18 6 2 0
40.5 (31.3-50.3) 32.9 (22.3-44.9) 41.9 (27.0-57.9) 30.0 (11.9-54.3) 22.2 (2.8-60.0) 0
Accelerated No. No. Initiated Conceived
% (95% CI)
242 204 189
37 15 15
15.3 (11.0-20.5) 7.4 (4.2-11.8) 7.9 (4.5-12.8)
172 105 54 22 7 1
72 41 22 8 2 0
41.9 (34.4-49.6) 39.0 (29.7-49.1) 40.7 (27.6-55.0) 36.4 (17.2-59.3) 28.6 (3.7-71.0) 0
CONCLUSION: In couples with unexplained infertility undergoing sequential treatment, success rates did not differ significantly over the first three cycles of a given treatment. For each treatment type, couples continued to have similar pregnancy rates, suggesting that unexplained infertility is a lowering of the fecundity set point. Supported by: NIH grant R01 HD38561. O-02 Monday, October 25, 2010 11:30 AM SUCCESSFUL PROPAGATION OF HUMAN PREPUBERTAL SPERMATOGONIAL STEM CELLS. H. Sadri-Ardekani, M. M. Akhondi, F. van der Veen, D. G. de Rooij, A. M. M. van Pelt, S. Repping. Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Islamic Republic of Iran.
FERTILITY & STERILITYÒ
DONORS’ INTERACTIONS WITH NURSING AND PHYSICIANS AFFECTS IMMEDIATE WILLINGNESS TO DONATE AGAIN AND ATTITUDES ONE YEAR POST RETRIEVAL. A. M. Braverman, D. Taylor, R. A. Nicholson, B. Galen, R. T. Scott, Jr. Reproducitve Medicine Associates of New Jersey, Morristown, NJ; Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology and Reproductive Science, Robert Wood Johnson Medical School UMDNJ, New Brunswick, NJ. OBJECTIVE: To explore the influences on ovum donors’ attitudes, reactions and emotional concerns after they donate and a year following their donation. DESIGN: A prospective follow up correlational study with ovum donors. MATERIALS AND METHODS: A post ovum retrieval study utilizing Likert scale questions) was developed to inquiry about donors’ experiences with all aspects of donation. Another fifteen item questionnaire with Likert scale questions about their attitudes and feelings one year post donation. Descriptive statistics were derived on 75 subjects and an ordinal logit regression assessed the significance of correlations between variables. RESULTS: Donors’ satisfaction with nurses correlated with their general happiness with being a donor (p< .003). Nursing interactions were correlated with willingness after retrieval to donate again (p< .005), why they would not donate again (p< .005) and feeling happy with the donor experience (p< .003). Nursing interactions were also positively correlated one year post retrieval with willingness to meet offspring at age 18 (p< .04), willingness to meet recipients (p< .03) and willingness to participate in a registry (p< .03). Similarly, physician interactions were positively correlated with donor’s feelings a year later: happy with being a donor (p< .03) and proud (p< .03). Medical preparation was correlated one year later with pride in being a donor (p< .0008). Psychological education was correlated one year later with willingness to donate again (p< .03) and feeling proud about being a donor (p< .001). CONCLUSION: Donors’ perceptions of nurses, doctors and mental health professionals are strongly correlated with donor experience, attitudes and choices, and this correlation persists a year after donation. Medical professionals should be cognizant that their care may influence a donor’s decision to donate again and the experience of being a donor. Medical interactions may directly influence decisions donors make in the future that may affect both the offspring and recipients.
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PRIZE PAPER CANDIDATES O-01 Monday, October 25, 2010 11:15 AM PREGNANCY RATES ACROSS MULTIPLE TREATMENT CYCLES: DATA FROM THE FAST TRACK AND STANDARD TREATMENT (FASTT) TRIAL. M. B. Goldman, M. M. Regan, M. M. Alper, K. L. Thornton, R. H. Reindollar. Obstetrics & Gynecology, DartmouthHitchcock Medical Center, Lebanon, NH; Biostatistics & Computational Biology, Dana-Farber Cancer Institute, Boston, MA; Boston IVF, Waltham, MA. OBJECTIVE: To assess pregnancy rates by type of treatment and cycle number in women aged 21-39 with unexplained infertility. DESIGN: Data are from a randomized clinical trial to evaluate optimal infertility treatment. MATERIALS AND METHODS: Couples (n¼503) who had a history of at least 12 months of attempted conception, no pelvic pathology or previous treatment, and normal semen analysis and ovarian reserve were enrolled. Treatment course and results were recorded prospectively. Pregnancy was determined by confirmation of a fetal heart beat. RESULTS: 247 couples were randomized to a conventional treatment protocol (3 cycles of clomiphene(CC)/IUI, 3 cycles of FSH/IUI, and up to 6 cycles of IVF); 256 couples were randomized to the accelerated treatment arm (3 cycles of CC/IUI and up to 6 cycles of IVF). Twenty-eight couples either conceived or withdrew before their first treatment cycle.
OBJECTIVE: To achieve in vitro proliferation of prepuberal spermatogonial stem cells (SSCs) in order to obtain a sufficient number of SSCs for successful autotransplantation in sterile childhood cancer survivors. DESIGN: In vitro culture of SSCs from testis of prepuberal boys. MATERIALS AND METHODS: We obtained testicular biopsies from 2 boys of 6 and 8 years old diagnosed with Hodgkin lymphoma. Testicular cells and germ line stem cell (GSC) clusters were cultured using our previously established system for adult SSCs. The presence of spermatogonia in culture was determined by RT-PCR and/or immunohistochemistry using spermatogonial markers ITGA6, ITGB1, GFRA1, CD9, GPR125, UCHL1 and ZBTB16. To determine the presence of SSCs, xenotransplantation of cultured testicular cells to the testes of busulphan treated immunodeficient mice was performed. RESULTS: In testicular cell cultures from both patients, GSC clusters were formed 2.5-3 weeks after initiation of the culture. Testicular cells and subcultured GSC clusters could be cultured for at least 15 and 23 weeks respectively. Expression of spermatogonial markers confirmed the presence of spermatogonia throughout the entire culture period for both patients. Xenotransplantation assays showed successful colonization of mouse testes with cultured human prepuberal SSCs of both patients confirming their stem cell capacity. Transplantation of cells from an early and a later passage of the cultured testicular cells showed a 922-fold increase in the number of human prepuberal SSCs within a time frame of 28 days. CONCLUSION: Our study demonstrated that prepuberal SSCs can be propagated in vitro to the extend that successful autotransplantation of SSCs to infertile childhood cancer survivors should become feasible in the near future. Cryopreservation of testicular biopsies in all prepuberal boys with cancer undergoing gonadotoxic treatment should therefore be considered. Supported by: The Dutch Children Cancer-Free Foundation (KIKA).
O-03 Monday, October 25, 2010 11:45 AM FASTT Trial Pregnancy Rates by Treatment and Cycle Number Randomized Group Conventional
Cycle CC/IUI 1 2 3 FSH/IUI 1 2 3 IVF 1 2 3 4 5 6
No. No. Initiated Conceived
% (95% CI)
233 217 181
15 20 20
6.4 (3.6-10.4) 9.2 (5.7-13.9) 11.0 (6.9-16.5)
169 141 120
19 19 11
11.2 (6.9-17.0) 13.5 (8.3-20.2) 9.2 (4.7-15.8)
111 73 43 20 9 5
45 24 18 6 2 0
40.5 (31.3-50.3) 32.9 (22.3-44.9) 41.9 (27.0-57.9) 30.0 (11.9-54.3) 22.2 (2.8-60.0) 0
Accelerated No. No. Initiated Conceived
% (95% CI)
242 204 189
37 15 15
15.3 (11.0-20.5) 7.4 (4.2-11.8) 7.9 (4.5-12.8)
172 105 54 22 7 1
72 41 22 8 2 0
41.9 (34.4-49.6) 39.0 (29.7-49.1) 40.7 (27.6-55.0) 36.4 (17.2-59.3) 28.6 (3.7-71.0) 0
CONCLUSION: In couples with unexplained infertility undergoing sequential treatment, success rates did not differ significantly over the first three cycles of a given treatment. For each treatment type, couples continued to have similar pregnancy rates, suggesting that unexplained infertility is a lowering of the fecundity set point. Supported by: NIH grant R01 HD38561. O-02 Monday, October 25, 2010 11:30 AM SUCCESSFUL PROPAGATION OF HUMAN PREPUBERTAL SPERMATOGONIAL STEM CELLS. H. Sadri-Ardekani, M. M. Akhondi, F. van der Veen, D. G. de Rooij, A. M. M. van Pelt, S. Repping. Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Islamic Republic of Iran.
FERTILITY & STERILITYÒ
DONORS’ INTERACTIONS WITH NURSING AND PHYSICIANS AFFECTS IMMEDIATE WILLINGNESS TO DONATE AGAIN AND ATTITUDES ONE YEAR POST RETRIEVAL. A. M. Braverman, D. Taylor, R. A. Nicholson, B. Galen, R. T. Scott, Jr. Reproducitve Medicine Associates of New Jersey, Morristown, NJ; Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology and Reproductive Science, Robert Wood Johnson Medical School UMDNJ, New Brunswick, NJ. OBJECTIVE: To explore the influences on ovum donors’ attitudes, reactions and emotional concerns after they donate and a year following their donation. DESIGN: A prospective follow up correlational study with ovum donors. MATERIALS AND METHODS: A post ovum retrieval study utilizing Likert scale questions) was developed to inquiry about donors’ experiences with all aspects of donation. Another fifteen item questionnaire with Likert scale questions about their attitudes and feelings one year post donation. Descriptive statistics were derived on 75 subjects and an ordinal logit regression assessed the significance of correlations between variables. RESULTS: Donors’ satisfaction with nurses correlated with their general happiness with being a donor (p< .003). Nursing interactions were correlated with willingness after retrieval to donate again (p< .005), why they would not donate again (p< .005) and feeling happy with the donor experience (p< .003). Nursing interactions were also positively correlated one year post retrieval with willingness to meet offspring at age 18 (p< .04), willingness to meet recipients (p< .03) and willingness to participate in a registry (p< .03). Similarly, physician interactions were positively correlated with donor’s feelings a year later: happy with being a donor (p< .03) and proud (p< .03). Medical preparation was correlated one year later with pride in being a donor (p< .0008). Psychological education was correlated one year later with willingness to donate again (p< .03) and feeling proud about being a donor (p< .001). CONCLUSION: Donors’ perceptions of nurses, doctors and mental health professionals are strongly correlated with donor experience, attitudes and choices, and this correlation persists a year after donation. Medical professionals should be cognizant that their care may influence a donor’s decision to donate again and the experience of being a donor. Medical interactions may directly influence decisions donors make in the future that may affect both the offspring and recipients.
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