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Mammalian trophectoderm gene expression is a predictor of IVF outcome
ORAL PRESENTATIONS Monday, October 19, 2009 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-1 Monday, October 19, 2009 11:15 AM OVARIAN AGING AFTER CHEMOTHERAPY: A GAME OF CHUTES AND LADDERS. H. I. Su, E. W. Freeman, A. DeMichele, M. D. Sammel. Reproductive Endocrinology and Infertility, University of Pennsylvania, Philadelphia, PA; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA; Obstetrics and Psychiatry, University of Pennsylvania, Philadelphia, PA; Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. OBJECTIVE: It is not known if ovarian aging after gonadotoxic chemotherapy is merely an accelerated version of natural ovarian aging. Using anti-Mullerian hormone (AMH) as a measure of ovarian function, this study tests if the long-term rate of AMH decline for breast cancer survivors is similar to that of healthy controls. DESIGN: Prospective cohort MATERIALS AND METHODS: A cohort of 36 breast cancer survivors who were younger than 45, premenopausal at cancer diagnosis and received cyclophosphamide were enrolled 1-4 years from chemotherapy and followed longitudinally. AMH levels were assayed from enrollment and first follow up serum. The annual percentage change in AMH from baseline was calculated for each participant and compared to late reproductive-aged healthy controls. The 20 controls provided 186 repeated measures of AMH over 8 years. The average rate of change in AMH and 95% confidence interval (CI) was determined using random effects regression. The proportion of cancer subjects whose change in AMH fell outside the control 95% CI was compared with a chi-squared test of proportions. RESULTS: 36 breast cancer subjects (mean age 40.9 [29-44]) were enrolled on average 1.8 years from chemotherapy and were followed an average of 3.2 years. Mean AMH (95%CI) was 436 pg/mL (0-1657) at enrollment and 293(0-1780) at follow up. In controls (the chutes), AMH declined by 27% per year (95%CI -59 to þ4%). Compared to controls, a significant number, 15 (42%), of cancer subjects (the ladders) experienced an increase in AMH that was greater than the upper confidence bound of normal (p<0.001). CONCLUSIONS: After chemotherapy, AMH does not always fall as expected from normal aging and in fact increased for a subset of subjects. The pattern and rate of change in AMH in breast cancer survivors is NOT the same as in healthy women. These novel observations suggest the possibility of ovarian recovery after cyclophosphamide exposure and support a different process of ovarian aging in cancer survivors. Supported by: American Cancer Society, WRHR, NIA, Penn CTRC
O-2 Monday, October 19, 2009 11:30 AM MATERNAL OBESITY ADVERSELY AFFECTS ASSISTED REPRODUCTIVE TECHNOLOGY (ART) PREGNANCY RATES AND OBSTETRIC OUTCOMES. B. Luke, M. B. Brown, J. E. Stern, S. A. Missmer, V. Y. Fujimoto, R. Leach. Obstetrics, Gynecology, and Reproductive Biology, Michigan State University, East Lansing, MI; Biostatistics, University of Michigan, Ann Arbor, MI; Obstetrics and Gynecology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; Obstetrics, Gynecology, and Reproductive Biology, Brigham and Womens Hospital, Boston, MA; Obstetrics, Gynecology, and Reproductive Sciences, University of California at San Francisco, San Francisco, CA. OBJECTIVE: To evaluate the effect of maternal obesity on ART pregnancy rates and obstetric outcomes. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: The study population included 48,682 ART cycles from the SART-CORS Online database for 2007 and was limited to women with both height and weight recorded. Women were categorized by their body mass index (BMI) as normal weight (18.5-24.9), overweight (25.0-29.9), or obese (Class I, 30.0-34.9; Class II, 35.0-39.9, Class III,
FERTILITY & STERILITYÒ
R40.0). Logistic regression was used to model the odds of pregnancy (presence of a gestational sac on early ultrasound) as the treatment outcome, live birth or stillbirth as the pregnancy outcome, and early preterm (<32 weeks) or preterm birth (<37 weeks), as adjusted odds ratios and 95% confidence intervals, with normal weight women as the reference group. Models were adjusted for maternal age, race and ethnicity, smoking, number of embryos transferred, and infertility diagnoses. Among live births, models of gestation were additionally adjusted for plurality. RESULTS: The odds of pregnancy were significantly reduced for obese women (0.91, 0.72, and 0.65, respectively for Class I, II, and III), and the odds of a live birth were reduced for overweight and obese women (0.87, 0.80, 0.74, and 0.75, respectively). The odds of stillbirth were increased more than twofold for obese women, significantly for Class I and II. Among live births, the odds of early preterm birth significantly paralleled increasing obesity (1.26, 1.52, and 1.59, respectively for Class I, II, and III), and the odds of preterm birth were significantly increased for all women (1.16, 1.33, 1.38, and 1.34, respectively). CONCLUSIONS: Among obese women, the chance of pregnancy and live birth decreased significantly with increasing BMI; they also had greater chances of a stillbirth, early preterm, or preterm birth. Future research should focus on clarifying the underlying causes of these disparities. Supported by: SART
O-3 Monday, October 19, 2009 11:45 AM MELOXICAM INHIBITS OVULATION IN NON-HUMAN PRIMATES WHEN ADMINISTERED DURING THE FOLLICULAR PHASE OF THE MENSTRUAL CYCLE TO SIMULATE EMERGENCY CONTRACEPTION. D. M. Duffy, K. E. Hester, M. J. K. Harper. Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA; CICCR/CONRAD, Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA. OBJECTIVE: To determine if a 5 day course of oral administration of the cyclooxygenase inhibitor meloxicam can prevent ovulation while maintaining normal menstrual cycles in nonhuman primates. DESIGN: Adult cynomolgus monkeys were studied in each of 4 menstrual cycles. In cycle 1, serum obtained each day was assayed for estradiol, progesterone, and luteinizing hormone (LH); first menses was also noted. In cycle 2, meloxicam was administered orally once each day for 5 days beginning at either mid follicular, late follicular, or periovulatory stage of the menstrual cycle; serum and menses were assessed as in cycle 1. In cycle 3, the ovary bearing the ovulatory follicle was removed 2 days after the expected day of ovulation. In cycle 4, monkeys received 5 days of oral meloxicam beginning at either mid follicular, late follicular, or periovulatory stage; the remaining ovary was removed 2 days after the expected day of ovulation. Ovarian sections were examined for the presence of an oocyte within the follicle. MATERIALS AND METHODS: Estradiol and progesterone were assessed by immunoassay; LH was assayed by RIA. Hormones levels were compared by ANOVA. Menstrual cycle length was compared by paired ttest. Ovaries were fixed, serial sectioned, and stained. RESULTS: All monkeys showed the expected pattern of changing estradiol, progesterone, and LH levels during cycle 1; meloxicam treatment in cycle 2 did not alter hormone levels or luteal phase length. Ovaries removed in cycle 3 did not contain oocytes within follicles, indicating successful ovulation. In contrast, large follicles did contain oocytes, indicating ovulation failure, after treatment with meloxicam during the mid follicular (67%), late follicular (100%), or periovulatory (50%) stage of cycle 4. CONCLUSIONS: A 5 day course of oral meloxicam can reduce the rate of oocyte release without alteration of reproductive hormones or menstrual cycle length. Meloxicam may be an effective emergency contraceptive. Supported by: Supported by CICCR, a program of CONRAD, Eastern Virginia Medical School
O-4 Monday, October 19, 2009 12:00 PM MAMMALIAN TROPHECTODERM GENE EXPRESSION IS A PREDICTOR OF IVF OUTCOME. J. Parks, B. McCallie, A. Janesch, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Foundation for Fertility Research, Lone Tree, CO; Colorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: Despite progressively improving pregnancy rates, the majority of transferred human IVF embryos result in implantation failure. Understanding the blastocyst’s role in the implantation process is vital. The aim
S1
of this study was to investigate mammalian trophectoderm (TE) gene expression relative to IVF outcome. DESIGN: Experimental Study. MATERIALS AND METHODS: Mouse IVF blastocysts were derived from BDF-1 hybrids. Hatching TE cells were biopsied for transcriptome analysis prior to single blastocyst transfer (one per uterine horn) to a recipient female mouse. On day 16 of fetal development, uterine horns were surgically removed to confirm either the presence of a fetus, an implantation site or lack of implantation. Total RNA was isolated from each individual TE biopsy sample (n¼12) and quantitative real-time PCR was performed. RESULTS: Six key developmental genes, Actr3 (TE development), B3gnt5 (cell differentiation and adhesion), Eomes & Cdx2 (TE differentiation), Slc7a5 (cell growth and differentiation) and Wnt5a (JNK/Wnt signaling) were expressed in each individual TE biopsy relative to the housekeeping gene, Gapdh. Statistical analysis revealed blastocysts that resulted in complete implantation failure to have significant downregulation of 3 TE genes: B3gnt5, Slc7a5 & Cdx2 in comparison to blastocysts displaying ongoing healthy fetal development (P<0.05). In addition, blastocysts that implanted but were reabsorbed displayed significant downregulation in biopsied TE cells of the gene Wnt5a when compared with ongoing healthy fetal development (P<0.05). Wnt5a is implicated in several biological processes including preimplantation embryonic development and postimplantation embryonic limb morphogenesis. CONCLUSIONS: An individual TE gene expression profile directly predicted ongoing healthy fetal development in contrast to non-viable implantation or complete implantation failure. TE transcriptome analysis may form the basis of quantifying blastocyst implantation potential.
O-5 Monday, October 19, 2009 12:15 PM THE IMPACT OF GROUP MIND/BODY PARTICIPATION ON PREGNANCY RATES IN IVF PATIENTS: A RANDOMIZED CONTROLLED TRIAL. A. D. Domar, K. Backman, M. M. Alper, B. M. Berger, B. Wiegand, J. Nikolovski. Boston IVF, Waltham, MA; Johnson & Johnson Consumer and Personal Products Worldwide, Johnson & Johnson Consumer Companies, Inc, Skillman, NJ. OBJECTIVE: To determine if participation in a mind/body program was associated with higher pregnancy rates in IVF patients DESIGN: A randomized, controlled study MATERIALS AND METHODS: 148 women scheduled to begin their first IVF cycle were included. Subjects were aged 40 or below, and had day 3 FSH/E2 levels of 12 and 80 or below. Subjects completed baseline questionnaires and were randomized to participate in a ten session mind/body program (MB) or to a control (C) group. C subjects received a spa gift certificate for each three month period. 51 subjects withdrew so a total of 97 subjects underwent at least one IVF cycle. Patients randomized to a MB group attended the next scheduled group; groups began every eight weeks so most subjects began their IVF cycle prior to beginning the group. Clinical pregnancy was defined as a heartbeat and sac on ultrasound. RESULTS: There were no significant differences between the two groups for age, FSH or E2 level. The clinical pregnancy rate for the first IVF cycle was 43% for both groups. 46% of the MB patients had not attended any MB session prior to beginning their first IVF cycle, 43.9% had done 1-5 sessions, and 9.7% had done 6-10. The pregnancy rates for the second cycle were 52% for the MB patients and 20% for the C (p¼ .05). 2.4% of the MB patients had not attended any MB sessions prior to beginning their second IVF cycle, 39% had attended 1-5, and 58.5% had attended 6-10. Subjects who scored at least moderate baseline symptoms of depression were analyzed: pregnancy rates for cycle 1 were 62% for MB and 39% for C, and for cycle 2 were 67% vs 0%. CONCLUSIONS: There is an apparent relationship between participation in a MB program and increased pregnancy rates for the second IVF cycle; the lack of a relationship with the first cycle may be due to the delay in MB exposure. Patients with higher baseline depression scores were the most likely to benefit from the intervention. Supported by: Johnson & Johnson Consumer and Personal Products Worldwide division of Johnson & Johnson Consumer Companies, Inc.
O-6 Monday, October 19, 2009 12:30 PM VALIDATION OF MICROARRAY CGH FOR PGD BY FISH REANALYSIS. S. Munne, C. Gutierrez-Mateo, J. F. Sanchez-Garcia, K. Ketterson, R. Prates, D. Kenigsberg. Reprogenetics, Livingston, NJ; Long Island IVF, Melville, NY.
S2
Abstracts
OBJECTIVE: Comparative genome hybridization (CGH) is successfully applied to trophectoderm biopsy, but it is not compatible with a fresh embryo transfer. Array CGH (aCGH) result turnaround is 24 hrs. We aimed to determine the error rate using aCGH on single cells compared to FISH and what percentage of abnormal embryos would be detected by FISH compared to aCGH. DESIGN: Embryos donated for research (avg. maternal age 32.7) had one cell biopsied on day 3 and analyzed in 24hrs by aCGH. Each remaining embryo was fixed and analyzed by FISH. MATERIALS AND METHODS: Single day 3 cells were biopsied, amplified, fluorescently labeled and hybridized onto BlueGnome 24sure BAC arrays. The remaining embryos were fixed and analyzed by 12 probe FISH and reanalyzed in a fourth hybridization for chromosomes found to be abnormal by aCGH. RESULTS: Six out of 55 biopsied embryos did not yield aCGH results (10.9%). Twenty-nine embryos were classified abnormal by aCGH (59%); 15 had single or double aneuploidy events, 10 were chaotic (3-9 chromosomes affected) and 4 had structural abnormalities. Twenty embryos were classified as normal. One embryo classified by aCGH as normal had a trisomy 22 by FISH (1/20, 5% false negative rate). One embryo diagnosed aneuploid and one as chaotic by aCGH were determined to be normal by FISH (2/29, 7% false positive rate). The total error rate for full chromosome abnormalities was 3/49 (6%). The 9 and 12 FISH probe would have classified 79% and 83%, respectively, of embryos as abnormal. CONCLUSIONS: aCGH seems to detect about 20% more abnormal embryos than FISH with a 6% error for full chromosome abnormalities. This is similar to the lowest error rate reported by FISH. The technique is not yet reliable for detection of small gains and loses of DNA, and is also partially reliable for gender determination. Technical improvements or more cells analyzed should improve these parameters, but we consider the test to be ready for clinical assessment of aneuploidy.
O-7 Monday, October 19, 2009 12:45 PM HUMAN LEIOMYOMA CELL PROLIFERATION AND EXTRACELLULAR MATRIX EXPRESSION IS INHIBITED BY INTEGRIN SIGNALING. M. Malik, D. Jardine, C. Owen, D. McCarthy-Keith, J. Segars, W. H. Catherino. OBG, Uniformed Services University of the Health Sciences, Bethesda, MD; NICHD, National Institutes of Health, Bethesda, MD. OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased b1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production. DESIGN: Experimental intervention MATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to a2, a3, a6 and b1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR. RESULTS: After 72hrs of incubation and at 1mg/ml b1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the a-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following b1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of b1 antibody, leiomyoma cells showed a 3.16þ0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes. CONCLUSIONS: Inhibition of b1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon b1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth. Supported by: NICHD NIH.
Vol. 92., No. 3, Supplement, September 2009
PRIZE PAPER CANDIDATES O-1 Monday, October 19, 2009 11:15 AM OVARIAN AGING AFTER CHEMOTHERAPY: A GAME OF CHUTES AND LADDERS. H. I. Su, E. W. Freeman, A. DeMichele, M. D. Sammel. Reproductive Endocrinology and Infertility, University of Pennsylvania, Philadelphia, PA; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA; Obstetrics and Psychiatry, University of Pennsylvania, Philadelphia, PA; Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. OBJECTIVE: It is not known if ovarian aging after gonadotoxic chemotherapy is merely an accelerated version of natural ovarian aging. Using anti-Mullerian hormone (AMH) as a measure of ovarian function, this study tests if the long-term rate of AMH decline for breast cancer survivors is similar to that of healthy controls. DESIGN: Prospective cohort MATERIALS AND METHODS: A cohort of 36 breast cancer survivors who were younger than 45, premenopausal at cancer diagnosis and received cyclophosphamide were enrolled 1-4 years from chemotherapy and followed longitudinally. AMH levels were assayed from enrollment and first follow up serum. The annual percentage change in AMH from baseline was calculated for each participant and compared to late reproductive-aged healthy controls. The 20 controls provided 186 repeated measures of AMH over 8 years. The average rate of change in AMH and 95% confidence interval (CI) was determined using random effects regression. The proportion of cancer subjects whose change in AMH fell outside the control 95% CI was compared with a chi-squared test of proportions. RESULTS: 36 breast cancer subjects (mean age 40.9 [29-44]) were enrolled on average 1.8 years from chemotherapy and were followed an average of 3.2 years. Mean AMH (95%CI) was 436 pg/mL (0-1657) at enrollment and 293(0-1780) at follow up. In controls (the chutes), AMH declined by 27% per year (95%CI -59 to þ4%). Compared to controls, a significant number, 15 (42%), of cancer subjects (the ladders) experienced an increase in AMH that was greater than the upper confidence bound of normal (p<0.001). CONCLUSIONS: After chemotherapy, AMH does not always fall as expected from normal aging and in fact increased for a subset of subjects. The pattern and rate of change in AMH in breast cancer survivors is NOT the same as in healthy women. These novel observations suggest the possibility of ovarian recovery after cyclophosphamide exposure and support a different process of ovarian aging in cancer survivors. Supported by: American Cancer Society, WRHR, NIA, Penn CTRC
O-2 Monday, October 19, 2009 11:30 AM MATERNAL OBESITY ADVERSELY AFFECTS ASSISTED REPRODUCTIVE TECHNOLOGY (ART) PREGNANCY RATES AND OBSTETRIC OUTCOMES. B. Luke, M. B. Brown, J. E. Stern, S. A. Missmer, V. Y. Fujimoto, R. Leach. Obstetrics, Gynecology, and Reproductive Biology, Michigan State University, East Lansing, MI; Biostatistics, University of Michigan, Ann Arbor, MI; Obstetrics and Gynecology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; Obstetrics, Gynecology, and Reproductive Biology, Brigham and Womens Hospital, Boston, MA; Obstetrics, Gynecology, and Reproductive Sciences, University of California at San Francisco, San Francisco, CA. OBJECTIVE: To evaluate the effect of maternal obesity on ART pregnancy rates and obstetric outcomes. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: The study population included 48,682 ART cycles from the SART-CORS Online database for 2007 and was limited to women with both height and weight recorded. Women were categorized by their body mass index (BMI) as normal weight (18.5-24.9), overweight (25.0-29.9), or obese (Class I, 30.0-34.9; Class II, 35.0-39.9, Class III,
FERTILITY & STERILITYÒ
R40.0). Logistic regression was used to model the odds of pregnancy (presence of a gestational sac on early ultrasound) as the treatment outcome, live birth or stillbirth as the pregnancy outcome, and early preterm (<32 weeks) or preterm birth (<37 weeks), as adjusted odds ratios and 95% confidence intervals, with normal weight women as the reference group. Models were adjusted for maternal age, race and ethnicity, smoking, number of embryos transferred, and infertility diagnoses. Among live births, models of gestation were additionally adjusted for plurality. RESULTS: The odds of pregnancy were significantly reduced for obese women (0.91, 0.72, and 0.65, respectively for Class I, II, and III), and the odds of a live birth were reduced for overweight and obese women (0.87, 0.80, 0.74, and 0.75, respectively). The odds of stillbirth were increased more than twofold for obese women, significantly for Class I and II. Among live births, the odds of early preterm birth significantly paralleled increasing obesity (1.26, 1.52, and 1.59, respectively for Class I, II, and III), and the odds of preterm birth were significantly increased for all women (1.16, 1.33, 1.38, and 1.34, respectively). CONCLUSIONS: Among obese women, the chance of pregnancy and live birth decreased significantly with increasing BMI; they also had greater chances of a stillbirth, early preterm, or preterm birth. Future research should focus on clarifying the underlying causes of these disparities. Supported by: SART
O-3 Monday, October 19, 2009 11:45 AM MELOXICAM INHIBITS OVULATION IN NON-HUMAN PRIMATES WHEN ADMINISTERED DURING THE FOLLICULAR PHASE OF THE MENSTRUAL CYCLE TO SIMULATE EMERGENCY CONTRACEPTION. D. M. Duffy, K. E. Hester, M. J. K. Harper. Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA; CICCR/CONRAD, Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA. OBJECTIVE: To determine if a 5 day course of oral administration of the cyclooxygenase inhibitor meloxicam can prevent ovulation while maintaining normal menstrual cycles in nonhuman primates. DESIGN: Adult cynomolgus monkeys were studied in each of 4 menstrual cycles. In cycle 1, serum obtained each day was assayed for estradiol, progesterone, and luteinizing hormone (LH); first menses was also noted. In cycle 2, meloxicam was administered orally once each day for 5 days beginning at either mid follicular, late follicular, or periovulatory stage of the menstrual cycle; serum and menses were assessed as in cycle 1. In cycle 3, the ovary bearing the ovulatory follicle was removed 2 days after the expected day of ovulation. In cycle 4, monkeys received 5 days of oral meloxicam beginning at either mid follicular, late follicular, or periovulatory stage; the remaining ovary was removed 2 days after the expected day of ovulation. Ovarian sections were examined for the presence of an oocyte within the follicle. MATERIALS AND METHODS: Estradiol and progesterone were assessed by immunoassay; LH was assayed by RIA. Hormones levels were compared by ANOVA. Menstrual cycle length was compared by paired ttest. Ovaries were fixed, serial sectioned, and stained. RESULTS: All monkeys showed the expected pattern of changing estradiol, progesterone, and LH levels during cycle 1; meloxicam treatment in cycle 2 did not alter hormone levels or luteal phase length. Ovaries removed in cycle 3 did not contain oocytes within follicles, indicating successful ovulation. In contrast, large follicles did contain oocytes, indicating ovulation failure, after treatment with meloxicam during the mid follicular (67%), late follicular (100%), or periovulatory (50%) stage of cycle 4. CONCLUSIONS: A 5 day course of oral meloxicam can reduce the rate of oocyte release without alteration of reproductive hormones or menstrual cycle length. Meloxicam may be an effective emergency contraceptive. Supported by: Supported by CICCR, a program of CONRAD, Eastern Virginia Medical School
O-4 Monday, October 19, 2009 12:00 PM MAMMALIAN TROPHECTODERM GENE EXPRESSION IS A PREDICTOR OF IVF OUTCOME. J. Parks, B. McCallie, A. Janesch, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Foundation for Fertility Research, Lone Tree, CO; Colorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: Despite progressively improving pregnancy rates, the majority of transferred human IVF embryos result in implantation failure. Understanding the blastocyst’s role in the implantation process is vital. The aim
S1
of this study was to investigate mammalian trophectoderm (TE) gene expression relative to IVF outcome. DESIGN: Experimental Study. MATERIALS AND METHODS: Mouse IVF blastocysts were derived from BDF-1 hybrids. Hatching TE cells were biopsied for transcriptome analysis prior to single blastocyst transfer (one per uterine horn) to a recipient female mouse. On day 16 of fetal development, uterine horns were surgically removed to confirm either the presence of a fetus, an implantation site or lack of implantation. Total RNA was isolated from each individual TE biopsy sample (n¼12) and quantitative real-time PCR was performed. RESULTS: Six key developmental genes, Actr3 (TE development), B3gnt5 (cell differentiation and adhesion), Eomes & Cdx2 (TE differentiation), Slc7a5 (cell growth and differentiation) and Wnt5a (JNK/Wnt signaling) were expressed in each individual TE biopsy relative to the housekeeping gene, Gapdh. Statistical analysis revealed blastocysts that resulted in complete implantation failure to have significant downregulation of 3 TE genes: B3gnt5, Slc7a5 & Cdx2 in comparison to blastocysts displaying ongoing healthy fetal development (P<0.05). In addition, blastocysts that implanted but were reabsorbed displayed significant downregulation in biopsied TE cells of the gene Wnt5a when compared with ongoing healthy fetal development (P<0.05). Wnt5a is implicated in several biological processes including preimplantation embryonic development and postimplantation embryonic limb morphogenesis. CONCLUSIONS: An individual TE gene expression profile directly predicted ongoing healthy fetal development in contrast to non-viable implantation or complete implantation failure. TE transcriptome analysis may form the basis of quantifying blastocyst implantation potential.
O-5 Monday, October 19, 2009 12:15 PM THE IMPACT OF GROUP MIND/BODY PARTICIPATION ON PREGNANCY RATES IN IVF PATIENTS: A RANDOMIZED CONTROLLED TRIAL. A. D. Domar, K. Backman, M. M. Alper, B. M. Berger, B. Wiegand, J. Nikolovski. Boston IVF, Waltham, MA; Johnson & Johnson Consumer and Personal Products Worldwide, Johnson & Johnson Consumer Companies, Inc, Skillman, NJ. OBJECTIVE: To determine if participation in a mind/body program was associated with higher pregnancy rates in IVF patients DESIGN: A randomized, controlled study MATERIALS AND METHODS: 148 women scheduled to begin their first IVF cycle were included. Subjects were aged 40 or below, and had day 3 FSH/E2 levels of 12 and 80 or below. Subjects completed baseline questionnaires and were randomized to participate in a ten session mind/body program (MB) or to a control (C) group. C subjects received a spa gift certificate for each three month period. 51 subjects withdrew so a total of 97 subjects underwent at least one IVF cycle. Patients randomized to a MB group attended the next scheduled group; groups began every eight weeks so most subjects began their IVF cycle prior to beginning the group. Clinical pregnancy was defined as a heartbeat and sac on ultrasound. RESULTS: There were no significant differences between the two groups for age, FSH or E2 level. The clinical pregnancy rate for the first IVF cycle was 43% for both groups. 46% of the MB patients had not attended any MB session prior to beginning their first IVF cycle, 43.9% had done 1-5 sessions, and 9.7% had done 6-10. The pregnancy rates for the second cycle were 52% for the MB patients and 20% for the C (p¼ .05). 2.4% of the MB patients had not attended any MB sessions prior to beginning their second IVF cycle, 39% had attended 1-5, and 58.5% had attended 6-10. Subjects who scored at least moderate baseline symptoms of depression were analyzed: pregnancy rates for cycle 1 were 62% for MB and 39% for C, and for cycle 2 were 67% vs 0%. CONCLUSIONS: There is an apparent relationship between participation in a MB program and increased pregnancy rates for the second IVF cycle; the lack of a relationship with the first cycle may be due to the delay in MB exposure. Patients with higher baseline depression scores were the most likely to benefit from the intervention. Supported by: Johnson & Johnson Consumer and Personal Products Worldwide division of Johnson & Johnson Consumer Companies, Inc.
O-6 Monday, October 19, 2009 12:30 PM VALIDATION OF MICROARRAY CGH FOR PGD BY FISH REANALYSIS. S. Munne, C. Gutierrez-Mateo, J. F. Sanchez-Garcia, K. Ketterson, R. Prates, D. Kenigsberg. Reprogenetics, Livingston, NJ; Long Island IVF, Melville, NY.
S2
Abstracts
OBJECTIVE: Comparative genome hybridization (CGH) is successfully applied to trophectoderm biopsy, but it is not compatible with a fresh embryo transfer. Array CGH (aCGH) result turnaround is 24 hrs. We aimed to determine the error rate using aCGH on single cells compared to FISH and what percentage of abnormal embryos would be detected by FISH compared to aCGH. DESIGN: Embryos donated for research (avg. maternal age 32.7) had one cell biopsied on day 3 and analyzed in 24hrs by aCGH. Each remaining embryo was fixed and analyzed by FISH. MATERIALS AND METHODS: Single day 3 cells were biopsied, amplified, fluorescently labeled and hybridized onto BlueGnome 24sure BAC arrays. The remaining embryos were fixed and analyzed by 12 probe FISH and reanalyzed in a fourth hybridization for chromosomes found to be abnormal by aCGH. RESULTS: Six out of 55 biopsied embryos did not yield aCGH results (10.9%). Twenty-nine embryos were classified abnormal by aCGH (59%); 15 had single or double aneuploidy events, 10 were chaotic (3-9 chromosomes affected) and 4 had structural abnormalities. Twenty embryos were classified as normal. One embryo classified by aCGH as normal had a trisomy 22 by FISH (1/20, 5% false negative rate). One embryo diagnosed aneuploid and one as chaotic by aCGH were determined to be normal by FISH (2/29, 7% false positive rate). The total error rate for full chromosome abnormalities was 3/49 (6%). The 9 and 12 FISH probe would have classified 79% and 83%, respectively, of embryos as abnormal. CONCLUSIONS: aCGH seems to detect about 20% more abnormal embryos than FISH with a 6% error for full chromosome abnormalities. This is similar to the lowest error rate reported by FISH. The technique is not yet reliable for detection of small gains and loses of DNA, and is also partially reliable for gender determination. Technical improvements or more cells analyzed should improve these parameters, but we consider the test to be ready for clinical assessment of aneuploidy.
O-7 Monday, October 19, 2009 12:45 PM HUMAN LEIOMYOMA CELL PROLIFERATION AND EXTRACELLULAR MATRIX EXPRESSION IS INHIBITED BY INTEGRIN SIGNALING. M. Malik, D. Jardine, C. Owen, D. McCarthy-Keith, J. Segars, W. H. Catherino. OBG, Uniformed Services University of the Health Sciences, Bethesda, MD; NICHD, National Institutes of Health, Bethesda, MD. OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased b1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production. DESIGN: Experimental intervention MATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to a2, a3, a6 and b1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR. RESULTS: After 72hrs of incubation and at 1mg/ml b1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the a-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following b1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of b1 antibody, leiomyoma cells showed a 3.16þ0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes. CONCLUSIONS: Inhibition of b1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon b1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth. Supported by: NICHD NIH.
Vol. 92., No. 3, Supplement, September 2009