- Email: [email protected]
Autologous endometrial coculture biopsy: is timing really everything?
miscarriage. The hypothesized mechanism of action in these indications is promotion of maternal-fetal tolerance. Because maternal-fetal tolerance encompasses a series of systemic changes affecting many cell types, analyses of individual proteins or cell types may not provide a complete picture. We therefore undertook a comprehensive flow cytometric analysis of more than 50 immune cell subsets to gain a broader view of NT100’s systemic effects. DESIGN: Peripheral blood was obtained from a Phase 1 randomized, double-blind, placebo-controlled trial of NT100 in 48 healthy non-pregnant women between the ages of 18 and 40. The study included both single- and multi-dose cohorts across three escalating dose levels. NT100 or placebo was administered subcutaneously as a single dose or once daily for 10 days. MATERIALS AND METHODS: PBMC were obtained at multiple time points and analyzed by ten-color flow cytometry. Antibody combinations were designed to identify various cellular sub-types within the following compartments: antigen-presenting cells, helper T cells, effector T cells, B cells, innate lymphocytes, and myeloid cells. RESULTS: NT100 induced broad and complex immunomodulatory changes within each compartment consistent with a net toleragenic profile. The effects were apparent after multiple-dose administration of NT100, and were not observed after a single dose. These effects were persistent during the period of drug administration and rapidly reversed on cessation of drug treatment. CONCLUSION: NT100 induces a complex and temporary set of changes consistent with a state of enhanced maternal-fetal tolerance. Two randomized, placebo-controlled Phase 2 clinical trials of NT100 are ongoing in unexplained recurrent pregnancy loss (RESPONSE trial) and in repeated implantation failure patients undergoing IVF (ThriveIVF trial). These will provide the opportunity to correlate the observed pharmacodynamics effects of NT100 treatment with changes in pregnancy outcomes. Supported by: This study was Supported by Nora Therapeutics, Inc.
P-285 Tuesday, October 21, 2014 EFFECT OF LAZER-ASSISTED FOUR-POINTS ZONA THINNING ON HATCHING AND PREGNANCY OUTCOME. B. S. Joo, S. H. Park, S. E. Moon, J. S. Koo, H. S. Youm, H. S. Moon. IVF-Busan, Center for Reprodutive Medicine, Good Moonhwa Hospital, Busan, Korea. OBJECTIVE: Hatching process may have polarity and the effect of assisted hatching (AH) may depends on the AH site. In human IVF-ET, AH is usually performed in day 2 or 3 embryos. However, the correct hatching site at these stages is not clear. Therefore, the first objective of this study was to preliminarily examine the effect of laser-assisted one-point zona thinning (LAZT1) or four-points zona thinning (LAZT4) on blastocyst formation and hatching in the mouse. Second, this study examined the effect of LAZT4 on pregnancy outcome in human IVF-ET. DESIGN: Prospective pilot study. MATERIALS AND METHODS: C57BL inbred female mice of two age groups (6-11, and 28-31 weeks) were superovulated with 5 IU pregnant mare’s serum gonadotropin, followed by 5 IU human chorionic gonadotropin injection, and immediately mated with an individual male of the same strain. After 15-16 hours, zygotes were obtained and cultured in vitro. At 8-cell stage, embryos were treated with LAZT1 or LAZT4. One point was randomly chosen at any site of zona pellucid and the sites of four points were chosen randomly at 90 degree intervals. After laser-assisted zona thinning, the embryos were further cultured and the rates of blastocyst formation and hatching were examined 96 hours and 120 hours after zygote collection, respectively. In human IVF-ET, fresh or frozen thawed 8-cell embryos were treated with LAZT4 (n¼28). The control group (n¼71) was embryos without LAZT. RESULTS: In young mice, LAZT had no effect on blastocyst formation rate, but resulted in a significant increase in early hatching and hatching rates compared to the control group (P<0.05). However, in old mice, LAZT significantly increases the blastocyst formation rate as well as early hatching and hatching rates compared to the control group (P<0.05). These effects of AH were more remarkable in LAZT4 than LAZT1. In human IVF-ET, there is no significant effect of LAZT4 on clinical pregnancy outcome according to the female age. However,
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ASRM Abstracts
LAZT4 for frozen-thawed embryos significantly increased clinical pregnancy rate (P<0.05). CONCLUSION: This study shows that the effect of LAZT4 was more noticeable in the old than the young in mice, whereas it is more effective in the frozen-thawed embryos rather than older patients in human IVF-ET. Although the effect of LAZT4 differs with the species, this result suggests that LAZT4 for 8-cell embryos can improve formation and hatching of blastocyst, and in turn, clinical pregnancy outcome.
P-286 Tuesday, October 21, 2014 BLASTOMERE-DERIVED HUMAN TROPHOBLAST STEM CELLS AS A NOVEL SURROGATE FOR BLASTOCYSTS IN MODELING T. Zdravkovic,b M. Gormley,b IMPLANTATION. M. Noel,a S. Fisher.b aCenter for Reproductive Health, University of California, San Francisco, San Francisco, CA; bCenter for Reproductive Sciences, University of California, San Francisco, San Francisco, CA. OBJECTIVE: In vitro studies of human implantation are limited by ethical restrictions on obtaining and working with human embryos, which has necessitated using mouse embryos or carcinoma-derived cell lines as substitutes for blastocysts1. Our objective was to explore a blastomere-derived trophoblast stem cell line as a novel surrogate for human blastocyst trophectoderm in modeling implantation. DESIGN: In vitro experimental study. MATERIALS AND METHODS: Previous work in our lab generated nine embryonic stem cell lines from single blastomeres of day 3 human embryos. These stem cell lines differentiate into trophoblast stem cells (TSCs) that undergo continuous self-renewal in vitro and differentiate into mature descendants2. They express transcription factors that specify trophoblast fate as well as markers of human blastocyst trophectoderm at the time of implantation. In this study, we utilized the Aggrewell system (StemCell Technologies) to create TSC spheroids that mimic the physical properties of the human blastocyst. Protein expression in TSC spheroids was characterized by immunolocalization, and compared to that of expanded blastocyst trophectoderm. Finally, TSC spheroids were cocultured with the endometrial cell line HEC-1-A in order to characterize the rate and extent of of attachment. RESULTS: TSCs were able to form spheroids of 150 cells, measuring 100-150mm in diameter, which remained stable in size and conformation after removal from Aggrewells. Immunolocalization showed that these TSC spheroids expressed transcription factors that specify trophoblast fate including CDX2, TEAD4 and GATA3, as well as cell surface proteins implicated in implantation, including LIF-R, EGFR, and several integrins. Immunolocalization of expanded human blastocysts showed a similar expression pattern of both transcription factors and cell surface markers. In attachment assays with HEC-1-A uterine epithelial cells, 60% of TPC spheroids stably attached over 20 hours. CONCLUSION: TSC spheroids are a novel surrogate for hatched human blastocysts that can be used to model human implantation. Future experiments will involve culturing TSC spheroids with primary endometrial tissue function-perturbing antibodies or siRNAs to elucidate the function of candidate trophoblast mediators in implantation.
P-287 Tuesday, October 21, 2014 AUTOLOGOUS ENDOMETRIAL COCULTURE BIOPSY: IS TIMING REALLY EVERYTHING?. A. P. Melnick,a A. K. Masbou,a E. M. Murphy,a K. J. Sapra,b Z. Rosenwaks,a S. D. Spandorfer.a aThe Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY; bDepartment of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY. OBJECTIVE: Autologous endometrial coculture (AECC) has been shown to increase embryo quality and implantation in patients with a history of IVF failure. Mechanical endometrial injury in the cycle prior to IVF has also been proposed to increase implantation rates. Therefore, the biopsy itself may contribute to AECC success rates. Our objective was to determine whether
Vol. 102, No. 3, Supplement, September 2014
endometrial biopsy timing affects implantation rates and pregnancy outcomes in patients undergoing IVF with AECC. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Patients with a history of at least one failed IVF cycle undergoing IVF with AECC at our center were included. All patients underwent luteal phase endometrial biopsy in preparation for IVF. Biopsy samples were either utilized for IVF in a consecutive menstrual cycle or were frozen for use in a future cycle. Embryos were cultured in AECC media and transferred on day 3. Outcomes included embryo grade, implantation rate, clinical pregnancy rate, and live birth rate. Statistical analysis included Mann Whitney U and c2 tests. P<0.05 was deemed statistically significant. RESULTS: 2568 cycles of 1764 patients who underwent an AECC/IVF cycle between May 2004 and November 2013 were analyzed. 1435 biopsies were performed in the cycle prior to IVF and 1133 were performed more than one cycle prior to IVF. The two groups were similar in age, BMI, number of mature oocytes retrieved, number of embryos transferred, and best embryo grade. There were no significant differences in implantation, clinical pregnancy, or live birth rates with adjusted relative risks of 1.0 (95%CI 0.90-1.11), 1.00 (95%CI 0.89-1.11), and 0.98 (95%CI 0.85-1.14) respectively.
N Age BMI #Oocytes #MII Best Embryo Grade #Transferred Implantation Rate Clinical Pregnancies Live Births
The females were then followed to delivery and their litter sizes compared. RESULTS: An average of 13.4 million (5-32 million)Tregs were transferred back to recipient mice. Adoptive transfer of Tregs performed closer (2 days) to native Treg knockdown resulted in higher whole blood (1.3%) Treg levels compared to transfer further (4 days) from knockdown (0.4%). Adoptive transfer of Tregs significantly increased liter sizes in mice (n¼7) with Treg knockdown compared to mice (n¼7) with Treg knockdown but no adoptive transfer (6.8 pups/litter vs. 4.5 pups/litter, P<0.05). CONCLUSION: Herein we provide further evidence, in a murine model, for the importance of Tregs in embryo implantation and pregnancy establishment. Implantation failure can be corrected using transfusions of Tregs. Altered Treg levels could be a potential cause of infertility due to recurrent implantation failure, which may be amenable to cellular therapy approaches. Supported by: This research was supported, in part, by the intramural research program of the Program in Reproductive and Adult Endocrinology, NICHD and NHLBI, NIH.
EMB<1 cycle
EMB>1 cycle
P-289 Tuesday, October 21, 2014
1435 38(35-41) 22.7(20.8-25.5) *10(7-14) 7(5-11) 2(2-2.5) 3(2-4) 0.160.26 639(44.5%) 331(22.8%)
1133 38(35-41) 22.5(20.6-25.3) 9(6-13) 7(5-11) 2(2-2.5) 3(2-4) 0.160.27 489(43.2%) 269(23.2%)
IMPLANTATION FAILURE OF TRANSLOCATED EMBRYOS CAN BE EXPLAINED BY IMPAIRED TROPHOBLASTIC A. Shpiz,a,b T. Frumkin,a DIFFERENTIATION. Y. Kalma,a M. Telias,a,b T. Shwartz,a T. Cohen,a A. Amit,a D. Ben-Yosef.a,b aWolfe PGD-Embryonic Stem Cell Laboratory, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel; bDepartment of Cell and Developmental Biology, Sackler Medical School, Tel-Aviv University, Israel, Tel Aviv, Israel.
*P<0.05 CONCLUSION: Coculture biopsy in the cycle preceding IVF does not increase implantation, clinical pregnancy, or live birth rates when compared to biopsies performed more than one cycle prior to IVF. Previously demonstrated improvements in embryo quality and pregnancy outcomes in patients undergoing IVF with AECC are not attributable to biopsy-induced endometrial injury.
P-288 Tuesday, October 21, 2014 ADOPTIVE TRANSFER OF TREGS AFTER NATIVE TREG KNOCKDOWN IMPROVES LITTER SIZES BACK TO BASELINE LEVELS IN A MURINE MODEL. R. J. Heitmann,a R. P. Weitzel,b J. F. Tisdale,b E. F. Wolff.a,b aNICHD, National Institutes of Health, Bethesda, MD; bNHLBI, National Institutes of Health, Bethesda, MD. OBJECTIVE: Maternal immune tolerance to the fetal semi-allograft is important in the establishment of pregnancy. T regulatory cells (Tregs); defined by CD4+CD25+FoxP3+ surface antigen expression, appear to play an important role in embryo implantation. We have previously demonstrated significantly smaller litter sizes, as a result of defective implantation, in a murine model after conditional native Treg knockdown (1). We hypothesized with add-back of Tregs, using adoptive transfer, would increase litter sizes to baseline levels. DESIGN: Animal research laboratory. MATERIALS AND METHODS: Using a transgenic (DEREG) mouse, conditional knockdown of Tregs was induced with application of intraperitoneal (IP) injections of diphtheria toxin (DT). Pilot studies were performed to analyze the optimal timing of adoptive Tregs transfer. Tregs from sex and litter matched wild-type mice were separated and purified using magnetic antibody cell sorting technology. Purified Tregs were then injected back into mating females two days after Treg knockdown.
FERTILITY & STERILITYÒ
OBJECTIVE: Carriers of translocation (11;22), the most common recurrent reciprocal translocation in humans, have high risk of repeated miscarriages. Current research models for studying the biological basic for implantation failure in translocated embryos are limited. The aim of this work is to study the reason for repeated miscarriages of unbalanced translocated embryos using a human embryonic stem cell (hESC) line carrying the unbalanced translocation t(11;22). DESIGN: The novel hESC line we have recently derived from an embryo with unbalanced t(11;22) following PGD, was induced to differentiate into trophoblast, and the results were compared to those obtained from control hESCs with normal karyotype. Gene expression of trophoblastic markers and bhCG secretion were analyzed and compared in the translocated and the control hESCs at different time points of in vitro differentiation into trophoblasts. MATERIALS AND METHODS: Fluorescent in-situ hybridization and karyotype analysis were performed to analyze t(11;22). The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Differentiation potential was assessed by spontaneous differentiation into teratomas, as well as by in-vitro differentiation into trophoblasts. Trophoblast development was assessed by measuring bhCG secretion, bhCG immunostaining and by gene expression of trophoblastic markers. RESULTS: We derived the first hESC line carrying an unbalanced t(11;22) which we labeled Lis05_t(11;22). This hESC line showed the typical morphological and molecular characteristics of a hESC line. It was functionally pluripotent, giving rise to derivatives of all three germ layers. Interestigly, Lis05_t(11;22) hESCs failed to differentiate into trophoblasts as evidenced by their failure to secrete b hCG. Gene expression analysis demonstrated reduced and delayed expression of the trophoblastic genes (CDX2, KRT7, GCM1, PPARG, KLF4, TP63 and CGA), concomitant with their failure to secrete b hCG. CONCLUSION: The t(11;22)-hESC line is here presented as a valuable human in-vitro model for studying the mechanisms underlying implantation failure. The inactivation of the trophoblastic genes are probably responsible for the failure in trophoblastic differentiation in t(11;22), correlating with implantation failure in unbalanced translocated embryos. Supported by: This study is Supported by a grant from the Israeli Ministery of Health, and from Tel Aviv University.
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P-285 Tuesday, October 21, 2014 EFFECT OF LAZER-ASSISTED FOUR-POINTS ZONA THINNING ON HATCHING AND PREGNANCY OUTCOME. B. S. Joo, S. H. Park, S. E. Moon, J. S. Koo, H. S. Youm, H. S. Moon. IVF-Busan, Center for Reprodutive Medicine, Good Moonhwa Hospital, Busan, Korea. OBJECTIVE: Hatching process may have polarity and the effect of assisted hatching (AH) may depends on the AH site. In human IVF-ET, AH is usually performed in day 2 or 3 embryos. However, the correct hatching site at these stages is not clear. Therefore, the first objective of this study was to preliminarily examine the effect of laser-assisted one-point zona thinning (LAZT1) or four-points zona thinning (LAZT4) on blastocyst formation and hatching in the mouse. Second, this study examined the effect of LAZT4 on pregnancy outcome in human IVF-ET. DESIGN: Prospective pilot study. MATERIALS AND METHODS: C57BL inbred female mice of two age groups (6-11, and 28-31 weeks) were superovulated with 5 IU pregnant mare’s serum gonadotropin, followed by 5 IU human chorionic gonadotropin injection, and immediately mated with an individual male of the same strain. After 15-16 hours, zygotes were obtained and cultured in vitro. At 8-cell stage, embryos were treated with LAZT1 or LAZT4. One point was randomly chosen at any site of zona pellucid and the sites of four points were chosen randomly at 90 degree intervals. After laser-assisted zona thinning, the embryos were further cultured and the rates of blastocyst formation and hatching were examined 96 hours and 120 hours after zygote collection, respectively. In human IVF-ET, fresh or frozen thawed 8-cell embryos were treated with LAZT4 (n¼28). The control group (n¼71) was embryos without LAZT. RESULTS: In young mice, LAZT had no effect on blastocyst formation rate, but resulted in a significant increase in early hatching and hatching rates compared to the control group (P<0.05). However, in old mice, LAZT significantly increases the blastocyst formation rate as well as early hatching and hatching rates compared to the control group (P<0.05). These effects of AH were more remarkable in LAZT4 than LAZT1. In human IVF-ET, there is no significant effect of LAZT4 on clinical pregnancy outcome according to the female age. However,
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ASRM Abstracts
LAZT4 for frozen-thawed embryos significantly increased clinical pregnancy rate (P<0.05). CONCLUSION: This study shows that the effect of LAZT4 was more noticeable in the old than the young in mice, whereas it is more effective in the frozen-thawed embryos rather than older patients in human IVF-ET. Although the effect of LAZT4 differs with the species, this result suggests that LAZT4 for 8-cell embryos can improve formation and hatching of blastocyst, and in turn, clinical pregnancy outcome.
P-286 Tuesday, October 21, 2014 BLASTOMERE-DERIVED HUMAN TROPHOBLAST STEM CELLS AS A NOVEL SURROGATE FOR BLASTOCYSTS IN MODELING T. Zdravkovic,b M. Gormley,b IMPLANTATION. M. Noel,a S. Fisher.b aCenter for Reproductive Health, University of California, San Francisco, San Francisco, CA; bCenter for Reproductive Sciences, University of California, San Francisco, San Francisco, CA. OBJECTIVE: In vitro studies of human implantation are limited by ethical restrictions on obtaining and working with human embryos, which has necessitated using mouse embryos or carcinoma-derived cell lines as substitutes for blastocysts1. Our objective was to explore a blastomere-derived trophoblast stem cell line as a novel surrogate for human blastocyst trophectoderm in modeling implantation. DESIGN: In vitro experimental study. MATERIALS AND METHODS: Previous work in our lab generated nine embryonic stem cell lines from single blastomeres of day 3 human embryos. These stem cell lines differentiate into trophoblast stem cells (TSCs) that undergo continuous self-renewal in vitro and differentiate into mature descendants2. They express transcription factors that specify trophoblast fate as well as markers of human blastocyst trophectoderm at the time of implantation. In this study, we utilized the Aggrewell system (StemCell Technologies) to create TSC spheroids that mimic the physical properties of the human blastocyst. Protein expression in TSC spheroids was characterized by immunolocalization, and compared to that of expanded blastocyst trophectoderm. Finally, TSC spheroids were cocultured with the endometrial cell line HEC-1-A in order to characterize the rate and extent of of attachment. RESULTS: TSCs were able to form spheroids of 150 cells, measuring 100-150mm in diameter, which remained stable in size and conformation after removal from Aggrewells. Immunolocalization showed that these TSC spheroids expressed transcription factors that specify trophoblast fate including CDX2, TEAD4 and GATA3, as well as cell surface proteins implicated in implantation, including LIF-R, EGFR, and several integrins. Immunolocalization of expanded human blastocysts showed a similar expression pattern of both transcription factors and cell surface markers. In attachment assays with HEC-1-A uterine epithelial cells, 60% of TPC spheroids stably attached over 20 hours. CONCLUSION: TSC spheroids are a novel surrogate for hatched human blastocysts that can be used to model human implantation. Future experiments will involve culturing TSC spheroids with primary endometrial tissue function-perturbing antibodies or siRNAs to elucidate the function of candidate trophoblast mediators in implantation.
P-287 Tuesday, October 21, 2014 AUTOLOGOUS ENDOMETRIAL COCULTURE BIOPSY: IS TIMING REALLY EVERYTHING?. A. P. Melnick,a A. K. Masbou,a E. M. Murphy,a K. J. Sapra,b Z. Rosenwaks,a S. D. Spandorfer.a aThe Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY; bDepartment of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY. OBJECTIVE: Autologous endometrial coculture (AECC) has been shown to increase embryo quality and implantation in patients with a history of IVF failure. Mechanical endometrial injury in the cycle prior to IVF has also been proposed to increase implantation rates. Therefore, the biopsy itself may contribute to AECC success rates. Our objective was to determine whether
Vol. 102, No. 3, Supplement, September 2014
endometrial biopsy timing affects implantation rates and pregnancy outcomes in patients undergoing IVF with AECC. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Patients with a history of at least one failed IVF cycle undergoing IVF with AECC at our center were included. All patients underwent luteal phase endometrial biopsy in preparation for IVF. Biopsy samples were either utilized for IVF in a consecutive menstrual cycle or were frozen for use in a future cycle. Embryos were cultured in AECC media and transferred on day 3. Outcomes included embryo grade, implantation rate, clinical pregnancy rate, and live birth rate. Statistical analysis included Mann Whitney U and c2 tests. P<0.05 was deemed statistically significant. RESULTS: 2568 cycles of 1764 patients who underwent an AECC/IVF cycle between May 2004 and November 2013 were analyzed. 1435 biopsies were performed in the cycle prior to IVF and 1133 were performed more than one cycle prior to IVF. The two groups were similar in age, BMI, number of mature oocytes retrieved, number of embryos transferred, and best embryo grade. There were no significant differences in implantation, clinical pregnancy, or live birth rates with adjusted relative risks of 1.0 (95%CI 0.90-1.11), 1.00 (95%CI 0.89-1.11), and 0.98 (95%CI 0.85-1.14) respectively.
N Age BMI #Oocytes #MII Best Embryo Grade #Transferred Implantation Rate Clinical Pregnancies Live Births
The females were then followed to delivery and their litter sizes compared. RESULTS: An average of 13.4 million (5-32 million)Tregs were transferred back to recipient mice. Adoptive transfer of Tregs performed closer (2 days) to native Treg knockdown resulted in higher whole blood (1.3%) Treg levels compared to transfer further (4 days) from knockdown (0.4%). Adoptive transfer of Tregs significantly increased liter sizes in mice (n¼7) with Treg knockdown compared to mice (n¼7) with Treg knockdown but no adoptive transfer (6.8 pups/litter vs. 4.5 pups/litter, P<0.05). CONCLUSION: Herein we provide further evidence, in a murine model, for the importance of Tregs in embryo implantation and pregnancy establishment. Implantation failure can be corrected using transfusions of Tregs. Altered Treg levels could be a potential cause of infertility due to recurrent implantation failure, which may be amenable to cellular therapy approaches. Supported by: This research was supported, in part, by the intramural research program of the Program in Reproductive and Adult Endocrinology, NICHD and NHLBI, NIH.
EMB<1 cycle
EMB>1 cycle
P-289 Tuesday, October 21, 2014
1435 38(35-41) 22.7(20.8-25.5) *10(7-14) 7(5-11) 2(2-2.5) 3(2-4) 0.160.26 639(44.5%) 331(22.8%)
1133 38(35-41) 22.5(20.6-25.3) 9(6-13) 7(5-11) 2(2-2.5) 3(2-4) 0.160.27 489(43.2%) 269(23.2%)
IMPLANTATION FAILURE OF TRANSLOCATED EMBRYOS CAN BE EXPLAINED BY IMPAIRED TROPHOBLASTIC A. Shpiz,a,b T. Frumkin,a DIFFERENTIATION. Y. Kalma,a M. Telias,a,b T. Shwartz,a T. Cohen,a A. Amit,a D. Ben-Yosef.a,b aWolfe PGD-Embryonic Stem Cell Laboratory, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel; bDepartment of Cell and Developmental Biology, Sackler Medical School, Tel-Aviv University, Israel, Tel Aviv, Israel.
*P<0.05 CONCLUSION: Coculture biopsy in the cycle preceding IVF does not increase implantation, clinical pregnancy, or live birth rates when compared to biopsies performed more than one cycle prior to IVF. Previously demonstrated improvements in embryo quality and pregnancy outcomes in patients undergoing IVF with AECC are not attributable to biopsy-induced endometrial injury.
P-288 Tuesday, October 21, 2014 ADOPTIVE TRANSFER OF TREGS AFTER NATIVE TREG KNOCKDOWN IMPROVES LITTER SIZES BACK TO BASELINE LEVELS IN A MURINE MODEL. R. J. Heitmann,a R. P. Weitzel,b J. F. Tisdale,b E. F. Wolff.a,b aNICHD, National Institutes of Health, Bethesda, MD; bNHLBI, National Institutes of Health, Bethesda, MD. OBJECTIVE: Maternal immune tolerance to the fetal semi-allograft is important in the establishment of pregnancy. T regulatory cells (Tregs); defined by CD4+CD25+FoxP3+ surface antigen expression, appear to play an important role in embryo implantation. We have previously demonstrated significantly smaller litter sizes, as a result of defective implantation, in a murine model after conditional native Treg knockdown (1). We hypothesized with add-back of Tregs, using adoptive transfer, would increase litter sizes to baseline levels. DESIGN: Animal research laboratory. MATERIALS AND METHODS: Using a transgenic (DEREG) mouse, conditional knockdown of Tregs was induced with application of intraperitoneal (IP) injections of diphtheria toxin (DT). Pilot studies were performed to analyze the optimal timing of adoptive Tregs transfer. Tregs from sex and litter matched wild-type mice were separated and purified using magnetic antibody cell sorting technology. Purified Tregs were then injected back into mating females two days after Treg knockdown.
FERTILITY & STERILITYÒ
OBJECTIVE: Carriers of translocation (11;22), the most common recurrent reciprocal translocation in humans, have high risk of repeated miscarriages. Current research models for studying the biological basic for implantation failure in translocated embryos are limited. The aim of this work is to study the reason for repeated miscarriages of unbalanced translocated embryos using a human embryonic stem cell (hESC) line carrying the unbalanced translocation t(11;22). DESIGN: The novel hESC line we have recently derived from an embryo with unbalanced t(11;22) following PGD, was induced to differentiate into trophoblast, and the results were compared to those obtained from control hESCs with normal karyotype. Gene expression of trophoblastic markers and bhCG secretion were analyzed and compared in the translocated and the control hESCs at different time points of in vitro differentiation into trophoblasts. MATERIALS AND METHODS: Fluorescent in-situ hybridization and karyotype analysis were performed to analyze t(11;22). The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Differentiation potential was assessed by spontaneous differentiation into teratomas, as well as by in-vitro differentiation into trophoblasts. Trophoblast development was assessed by measuring bhCG secretion, bhCG immunostaining and by gene expression of trophoblastic markers. RESULTS: We derived the first hESC line carrying an unbalanced t(11;22) which we labeled Lis05_t(11;22). This hESC line showed the typical morphological and molecular characteristics of a hESC line. It was functionally pluripotent, giving rise to derivatives of all three germ layers. Interestigly, Lis05_t(11;22) hESCs failed to differentiate into trophoblasts as evidenced by their failure to secrete b hCG. Gene expression analysis demonstrated reduced and delayed expression of the trophoblastic genes (CDX2, KRT7, GCM1, PPARG, KLF4, TP63 and CGA), concomitant with their failure to secrete b hCG. CONCLUSION: The t(11;22)-hESC line is here presented as a valuable human in-vitro model for studying the mechanisms underlying implantation failure. The inactivation of the trophoblastic genes are probably responsible for the failure in trophoblastic differentiation in t(11;22), correlating with implantation failure in unbalanced translocated embryos. Supported by: This study is Supported by a grant from the Israeli Ministery of Health, and from Tel Aviv University.
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