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Fluorescence-activated cell sorting may allow separation of malignant leukemic cells from a testicular cell suspension
origin subcutaneously or next to muscle or kidney. Four monkeys had transplants to both the arm & abdomen, 2 to the kidney & abdomen, and 1 to only the arm. Serum estradiol (E2) & progesterone (P4) were measured 1-3 x/wk for 1-8 yrs. Previously, we fertilized eggs retrieved from abdominal ovarian transplants, and transferred morulas to a recipient, resulting in a healthy female monkey. This monkey grew to sexual maturity (5 yrs) and then mated in a timed breeding program. E2 & P4 were checked. When E2 was 200 pg/ml, she was paired with a male, and unpaired when P4 rise showed ovulation. RESULTS: Four animals with transplants to the abdomen & arm had cyclic E2 production of 100pg/ml during the first year, while the one with arm transplants had cyclic E2 of 50 pg/ml. One with transplants to the kidney & abdomen was ovulatory for 3 years and had E2s of 150-300 pg/ml. The 5 yr old monkey conceived after her first pairing. She had a normal gestation, but a term, intrapartum fetal demise. At age 6, she again conceived spontaneously in the first attempt, and delivered a healthy female monkey at term via C-section. CONCLUSION: Heterotopic, fresh, ovarian grafts can produce sex steroids for 1-3 years after transplantation; the kidney may be the best site. A non-human primate, born via fertilization of eggs derived from transplanted ovarian tissue, is herself fertile. Her live-born appears to be normal and healthy. Supported by: Bidwell Foundation, NIH.
O-39 Monday, October 25, 2010 06:00 PM FLUORESCENCE-ACTIVATED CELL SORTING MAY ALLOW SEPARATION OF MALIGNANT LEUKEMIC CELLS FROM A TESTICULAR CELL SUSPENSION. S. L. Dovey, H. Valli, B. P. Hermann, K. Orwig. Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh Medical Center/Magee Womens Hospital, Pittsburgh, PA; Magee Womens Research Institute, Pittsburgh, PA. OBJECTIVE: Spermatogonial stem cell (SSCs) may have clinical application for treating male infertility (e.g., secondary to cancer treatment). However, with certain hematologic malignancies, there is concern about reintroducing malignant cells during transplantation. The objective of this study is to examine the feasibility of separating leukemic cells from a suspension of human testicular cells via fluorescence-activated cell sorting (FACS). DESIGN: Basic science research. MATERIALS AND METHODS: The expression of 22 cell surface markers was analyzed via flow cytometry on human testicular cell suspensions as well as on three different human leukemic cell lines. Human testis cells were sorted by FACS based on expression of selected markers and the resulting fractions were stained with SALL-4, a marker of stem and progenitor spermatogonia. In this manner, markers of interest were identified if 1) they did not stain malignant cells but did stain the population of SALL-4 positive cells, or 2) they stained all of the malignant cells but not the SALL-4 positive cells. RESULTS: FACs analyses indicate that SALL-4+ cells were in the EpCAM+ fraction of human testis cells. Ep-CAM was not expressed on the MOLT-4 T cell leukemia cell line. In contrast, HLA-ABC and CD49e were highly expressed on the MOLT-4 cell line, but were not expressed by SALL4+ human testis cells. CONCLUSION: SALL4+ human spermatogonia (Ep-CAM+, HLA-ABC-, CD49e-) and MOLT-4 leukemia cells (Ep-CAM-, HLA-ABC+, CD49e+) have distinct cell surface characteristics that might be exploited to remove malignant contaminants from potentially therapeutic SSCs. FACS experiments utilizing these cell surface markers to fractionate MOLT-4 spiked human testis cell suspensions are ongoing. We are awaiting transplant results to determine the relative SSC activity and malignant potential of each fraction. Supported by: NIH grants R01HD055475 and R21HD061289; and Magee-Womens Research Institute and Foundation.
CLINICAL FEMALE INFERTILITY AND GYNECOLOGY O-40 Monday, October 25, 2010 04:15 PM THE ECONOMIC IMPACT OF SCREENING FOR FETAL KARYOTYPIC ABNORMALITIES IN WOMEN PRESENTING WITH THEIR SECOND PREGNANCY LOSS. N. Foyouzi, M. I. Cedars, M. Rosen, H. G. Huddleston. Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA.
S12
Abstracts
OBJECTIVE: The search to identify a cause for recurrent pregnancy loss (RPL) is often initiated following a second loss. In the majority of all pregnancy losses, fetal aneuploidy is the cause and knowledge of this obviates the need for an extensive workup. The aim of our study was to compare the cost efficiency of obtaining karyotypic analysis on products of conception after a second pregnancy loss compared to proceeding with a full RPL workup at that juncture. DESIGN: Economic Analysis. MATERIALS AND METHODS: The cost of a commonly performed RPL workup, including: saline sonogram, TSH, glucose, lupus anti-coagulant, anti-cardiolipin antibodies, anti-B2glycoprotein antibody, factor V Leiden, prothrombin gene mutation, protein C, protein S, homocysteine, and parental chromosome analyses, was compared to the cost of first performing karyotyping of the products of conception, followed by the full workup for those patients found to have a normal or inconclusive fetal karyotype (35%). We assumed that 50% of patients in the RPL workup group and 100% of the karyotype group would undergo an outpatient D&C. Costs were calculated as the mean from the self pay price of tests obtained from LabCorp, Quest, Mayo Clinic and UCSF. Assumptions regarding the content of the RPL evaluation were derived from current literature. RESULTS: The per patient cost of proceeding with the common RPL workup is $4949. The per patient cost of karyotyping products of conception prior to initiating the workup is $2169. The cost of proceeding with RPL screening for those subsequently found to have a normal or inconclusive fetal karyotype was $1733. Thus, the total cost for beginning with a karyotype was $3902. Globally, the per patient savings of screening products of conception as the first step in managing a patient with recurrent pregnancy loss is $1048. CONCLUSION: Our model suggests obtaining a karyotype on the products of conception in women after a second miscarriage is more cost-effective than proceeding with an RPL evaluation at that juncture.
O-41 Monday, October 25, 2010 04:30 PM ABSORPTION OF VAGINAL PROGESTERONE GEL DURING SEXUAL INTERCOURSE: A PROSPECTIVE, RANDOMIZED, CONTROLLED TRIAL. K. A. Leake, M. Elliot, J. Barker, P. B. Marshburn, R. S. Usadi, B. S. Hurst. Department of Obstetrics & Gynecology, Carolinas Medical Center, Charlotte, NC. OBJECTIVE: To determine if sexual intercourse reduces the absorption of vaginal progesterone gel in women, and to determine if progesterone is absorbed by their male partner. If so, intercourse during luteal support could compromise pregnancy outcome and have adverse effects on the male. DESIGN: Prospective, randomized, placebo-controlled, blinded, crossover study. MATERIALS AND METHODS: 20 reproductive-aged women and their male sexual partners were randomized to receive vaginal progesterone (CrinoneÒ 8% gel) or placebo while taking a monophasic oral contraceptive (MircetteÒ) to suppress ovulation. The progesterone (or placebo) was administered at night 1 hour before intercourse, and serum progesterone was measured for the subject and her partner 10 hours after intercourse. One week later, the subjects were crossed-over to receive the opposite formulation. In the third week, women used progesterone gel at night and abstained from intercourse, and blood was drawn 11 hours later. Groups were compared with the Wilcoxon signed-rank test. RESULTS: Serum progesterone was significantly (p¼0.0075) reduced with Crinone + intercourse (median 2.9 ng/ml) compared to Crinone + abstinence (median 6.9 ng/ml). Serum progesterone with placebo + intercourse was low (median 0.8 ng/ml), significantly less (p%0.0001) than Crinone + intercourse. Men absorbed low but significant (p¼0.0008) progesterone during intercourse with Crinone (0.9 ng/ml) compared to placebo (0.5 ng/ml). CONCLUSION: Intercourse lowers serum progesterone >50% in women using vaginal progesterone. When vaginal progesterone is used for luteal support after fertility treatment, it is possible that intercourse could lower pregnancy rates or increase the risk of miscarriage. Furthermore, although men absorb low levels of progesterone during intercourse, exposure during the typical 8-12 weeks used during early pregnancy could cause adverse effects. Couples should be warned to avoid intercourse while using vaginal progesterone. Supported by: Cannon Education and Research Grants Committee.
Vol. 94., No. 4, Supplement, September 2010
O-39 Monday, October 25, 2010 06:00 PM FLUORESCENCE-ACTIVATED CELL SORTING MAY ALLOW SEPARATION OF MALIGNANT LEUKEMIC CELLS FROM A TESTICULAR CELL SUSPENSION. S. L. Dovey, H. Valli, B. P. Hermann, K. Orwig. Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh Medical Center/Magee Womens Hospital, Pittsburgh, PA; Magee Womens Research Institute, Pittsburgh, PA. OBJECTIVE: Spermatogonial stem cell (SSCs) may have clinical application for treating male infertility (e.g., secondary to cancer treatment). However, with certain hematologic malignancies, there is concern about reintroducing malignant cells during transplantation. The objective of this study is to examine the feasibility of separating leukemic cells from a suspension of human testicular cells via fluorescence-activated cell sorting (FACS). DESIGN: Basic science research. MATERIALS AND METHODS: The expression of 22 cell surface markers was analyzed via flow cytometry on human testicular cell suspensions as well as on three different human leukemic cell lines. Human testis cells were sorted by FACS based on expression of selected markers and the resulting fractions were stained with SALL-4, a marker of stem and progenitor spermatogonia. In this manner, markers of interest were identified if 1) they did not stain malignant cells but did stain the population of SALL-4 positive cells, or 2) they stained all of the malignant cells but not the SALL-4 positive cells. RESULTS: FACs analyses indicate that SALL-4+ cells were in the EpCAM+ fraction of human testis cells. Ep-CAM was not expressed on the MOLT-4 T cell leukemia cell line. In contrast, HLA-ABC and CD49e were highly expressed on the MOLT-4 cell line, but were not expressed by SALL4+ human testis cells. CONCLUSION: SALL4+ human spermatogonia (Ep-CAM+, HLA-ABC-, CD49e-) and MOLT-4 leukemia cells (Ep-CAM-, HLA-ABC+, CD49e+) have distinct cell surface characteristics that might be exploited to remove malignant contaminants from potentially therapeutic SSCs. FACS experiments utilizing these cell surface markers to fractionate MOLT-4 spiked human testis cell suspensions are ongoing. We are awaiting transplant results to determine the relative SSC activity and malignant potential of each fraction. Supported by: NIH grants R01HD055475 and R21HD061289; and Magee-Womens Research Institute and Foundation.
CLINICAL FEMALE INFERTILITY AND GYNECOLOGY O-40 Monday, October 25, 2010 04:15 PM THE ECONOMIC IMPACT OF SCREENING FOR FETAL KARYOTYPIC ABNORMALITIES IN WOMEN PRESENTING WITH THEIR SECOND PREGNANCY LOSS. N. Foyouzi, M. I. Cedars, M. Rosen, H. G. Huddleston. Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA.
S12
Abstracts
OBJECTIVE: The search to identify a cause for recurrent pregnancy loss (RPL) is often initiated following a second loss. In the majority of all pregnancy losses, fetal aneuploidy is the cause and knowledge of this obviates the need for an extensive workup. The aim of our study was to compare the cost efficiency of obtaining karyotypic analysis on products of conception after a second pregnancy loss compared to proceeding with a full RPL workup at that juncture. DESIGN: Economic Analysis. MATERIALS AND METHODS: The cost of a commonly performed RPL workup, including: saline sonogram, TSH, glucose, lupus anti-coagulant, anti-cardiolipin antibodies, anti-B2glycoprotein antibody, factor V Leiden, prothrombin gene mutation, protein C, protein S, homocysteine, and parental chromosome analyses, was compared to the cost of first performing karyotyping of the products of conception, followed by the full workup for those patients found to have a normal or inconclusive fetal karyotype (35%). We assumed that 50% of patients in the RPL workup group and 100% of the karyotype group would undergo an outpatient D&C. Costs were calculated as the mean from the self pay price of tests obtained from LabCorp, Quest, Mayo Clinic and UCSF. Assumptions regarding the content of the RPL evaluation were derived from current literature. RESULTS: The per patient cost of proceeding with the common RPL workup is $4949. The per patient cost of karyotyping products of conception prior to initiating the workup is $2169. The cost of proceeding with RPL screening for those subsequently found to have a normal or inconclusive fetal karyotype was $1733. Thus, the total cost for beginning with a karyotype was $3902. Globally, the per patient savings of screening products of conception as the first step in managing a patient with recurrent pregnancy loss is $1048. CONCLUSION: Our model suggests obtaining a karyotype on the products of conception in women after a second miscarriage is more cost-effective than proceeding with an RPL evaluation at that juncture.
O-41 Monday, October 25, 2010 04:30 PM ABSORPTION OF VAGINAL PROGESTERONE GEL DURING SEXUAL INTERCOURSE: A PROSPECTIVE, RANDOMIZED, CONTROLLED TRIAL. K. A. Leake, M. Elliot, J. Barker, P. B. Marshburn, R. S. Usadi, B. S. Hurst. Department of Obstetrics & Gynecology, Carolinas Medical Center, Charlotte, NC. OBJECTIVE: To determine if sexual intercourse reduces the absorption of vaginal progesterone gel in women, and to determine if progesterone is absorbed by their male partner. If so, intercourse during luteal support could compromise pregnancy outcome and have adverse effects on the male. DESIGN: Prospective, randomized, placebo-controlled, blinded, crossover study. MATERIALS AND METHODS: 20 reproductive-aged women and their male sexual partners were randomized to receive vaginal progesterone (CrinoneÒ 8% gel) or placebo while taking a monophasic oral contraceptive (MircetteÒ) to suppress ovulation. The progesterone (or placebo) was administered at night 1 hour before intercourse, and serum progesterone was measured for the subject and her partner 10 hours after intercourse. One week later, the subjects were crossed-over to receive the opposite formulation. In the third week, women used progesterone gel at night and abstained from intercourse, and blood was drawn 11 hours later. Groups were compared with the Wilcoxon signed-rank test. RESULTS: Serum progesterone was significantly (p¼0.0075) reduced with Crinone + intercourse (median 2.9 ng/ml) compared to Crinone + abstinence (median 6.9 ng/ml). Serum progesterone with placebo + intercourse was low (median 0.8 ng/ml), significantly less (p%0.0001) than Crinone + intercourse. Men absorbed low but significant (p¼0.0008) progesterone during intercourse with Crinone (0.9 ng/ml) compared to placebo (0.5 ng/ml). CONCLUSION: Intercourse lowers serum progesterone >50% in women using vaginal progesterone. When vaginal progesterone is used for luteal support after fertility treatment, it is possible that intercourse could lower pregnancy rates or increase the risk of miscarriage. Furthermore, although men absorb low levels of progesterone during intercourse, exposure during the typical 8-12 weeks used during early pregnancy could cause adverse effects. Couples should be warned to avoid intercourse while using vaginal progesterone. Supported by: Cannon Education and Research Grants Committee.
Vol. 94., No. 4, Supplement, September 2010