- Email: [email protected]
Homeobox transcription factor msx1 is reduced in human endometrial biopsies of women from infertile couples
DOR group (0.0%). Pregnancy outcomes were not independently predicted by endometrial thickness for any group. Adjusting for lead follicle diameter, women in the unexplained group who became pregnant tended to have thinner endometrial measurements than non-pregnant women from the other groups (p<0.05). CONCLUSION: Thin endometrium is more common among women with unexplained infertility undergoing letrozole/IUI therapy than women with other primary diagnoses. However, endometrial thickness is not a significant predictor of pregnancy outcomes, and women with unexplained infertility and thin endometrial measurements may show improved outcomes compared to other women with similar follicular profiles. P-468 Wednesday, October 22, 2014 IS ENDOMETRIAL THICKNESS ON DAY OF TRANSFER A PREDICTOR OF PREGNANCY RATE IN IVF?. M. Irani,a D. B. Seifer,b K. Melzer,b J. Makarov,b D. Chavkin,b R. V. Grazi.b aOB/Gyn, Maimonides Medical Center, Brooklyn, NY; bOB/Gyn, Division of Reproductive Endocrinology and Infertility, Maimonides Medical Center, Brooklyn, NY. OBJECTIVE: Multiple factors can affect IVF success rate including endometrial receptivity, quality of the embryo and the transfer technique. Some studies have shown that endometrial thickness is a good indicator of endometrial receptivity. The majority of studies have examined the sonographic appearance of the endometrium on the day of hCG administration. The objective of this study was to test the hypothesis that endometrial thickness measured on the day of embryo transfer (EMT-ET) may be more informative of IVF success rates than EMT evaluated on the day of hCG administration (EMT-hCG). DESIGN: A prospective cohort study. MATERIALS AND METHODS: EMT-hCG and EMT-ET were measured by transvaginal sonogram in 101 women (22-48 years old) undergoing IVF between September 2013 and February 2014. Chemical pregnancy, clinical pregnancy, and miscarriage rates were evaluated. T-test and Pearson correlation were used as appropriate. RESULTS: Age was a significant predictor of IVF success rate (p¼0.037). After adjusting for age, fresh cycles were associated with significantly higher clinical pregnancy rates compared to frozen cycles (64.2% versus 41.7%; p¼0.024). The overall rate of miscarriage was 24.5%. There was a trend toward a negative correlation between the rate of miscarriage and EMT-ET (r¼-0.41; p¼0.08) but not with EMT-hCG (p¼0.17). For women younger than 35 years, there was a trend toward a positive correlation between EMT-ET and clinical pregnancy rate (r¼0.71; p¼0.055). In the same age group, EMT-hCG was significantly higher in women who achieved clinical pregnancy (11.1mm+/-0.4 [SEM]) when compared to women who did not (9.5mm+/-0.4; p¼0.02). CONCLUSION: EMT-hCG is a better predictor of clinical pregnancy rate than EMT-ET; however, EMT-ET may be more informative of potential miscarriage than EMT-hCG. P-469 Wednesday, October 22, 2014 BENEFICIAL EFFECT OF LOCAL INJURY TO THE ENDOMETRIUM IN INTRACYTOPLASMIC SPERM INJECTION (ICSI) PATIENTS WITH RECURRENT IMPLANTATION FAILURE. P. J. Buzzi, M. P. Zappacosta, L. Auge, L. Isa, E. Young Obejero, A. Bello. IFER Instituto de Ginecologıa y Fertilidad, Buenos Aires, Argentina. OBJECTIVE: Successful embryo implantation depends on a well-functioning endometrium as well as a normal healthy embryo. Several investigators have suggested that patients with implantation failure (IF) may benefit from mechanical endometrial stimulation performed in the cycle preceding the actual treatment cycle. The aim of this study is to assess the influence of local injury to the endometrium in a selected group of ICSI patients with prior IF. DESIGN: Prospective cohort study. MATERIALS AND METHODS: Patients % 36 years old undergoing IVF between October 2012 –December 2013 with R2 IVF failures and with at least 2 good quality embryos transferred were considered for the analysis. A study group (n¼21) included women who underwent hysteroscopy+biopsy in the cycle preceding the current IVF treatment. Control group (n¼20) underwent a repeat cycle with no intervention. Baseline characteristics of both groups confirmed no different history of IVF-ET failures and a similar performance in the present IVF-ET treatment: mean number of IVF trials 2.2 vs. 2.1 cycles, age 31.8 vs. 31.9 yrs Number of oocytes retrieved: 11.0 6.0 vs 10.4 5.3 Number of good quality Day 3 embryos
FERTILITY & STERILITYÒ
obtained 5.7 3.9 (51.8%) 5.8 4.1 (56.3%).A similar number of good quality Day 3 embryos were transferred into the uterus in the control and in the experimental groups (2.8 1.3 and 2.6 1.3, respectively)T-test and chi square test were used. RESULTS: Hystologycal analysis of the endometrium biopsies showed chronic endometritis in 9.5% (2 /21) of the study population. No other pathologycal findings were reported. . Transfer of embryos in the IVF-ET cycle that immediately followed the endometrium treatment (STUDY GROUP) resulted in a clinical pregnancy rate of 63.2% and a 47.4% ongoing /live birth rate, significantly higher than those obtained in the control group (40% and 25%). CONCLUSION: Local injury to the endometrium prior to controlled ovarian stimulation may considerably improve implantation rates and pregnancy outcomes in patients with implantation failure. P-470 Wednesday, October 22, 2014 THE MIR-16 FAMILY IS HORMONALLY REGULATED IN ENDOMETRIAL STROMAL CELLS AND ALTERED BY SUPEROVULATION IN A MURINE MODEL. S. White P. T. Jimenez. Obstetrics & Gynecology, University of Texas Southwestern Medical Center, Dallas, TX. OBJECTIVE: The purpose of this study was to determine the expression and hormonal regulation of the microRNA (miRNA, miR) -16 family in endometrial stromal cells during embryo implantation. DESIGN: Experimental laboratory study in CD1/ICR mice. MATERIALS AND METHODS: To determine the differential expression of miRNAs before and at the time of implantation, eight week old CD1/ICR female mice were mated with males of known fertility and sacrificed at 0.5 dpc (morning of vaginal plug) or 4.5 dpc. RNA from endometrial stromal cells isolated from the whole uterus at 0.5 dpc and from implantation sites at 4.5 dpc of three mice at each time point underwent microRNA microarray analysis in triplicate. A separate cohort of mice was sacrificed at 0.5, 2.5, 4.5, and 8.5 dpc to confirm the microarray results. To analyze hormonal regulation of the miRNAs, ovariectomized mice were subcutaneously injected with vehicle (sesame oil) or 1 mg estradiol-17b for 48 hr. Another cohort of mice was either injected with 10 IU PMSG followed by 10 IU hCG 48 hrs later to induce superovulation or allowed to ovulate spontaneously. Endometrial stromal cells were isolated. After RNA isolation, RT-qPCR was performed for the three members of the miR-16 family, miR-15a, -15b, and -16, as well as for their predicted targets, Indian Hedgehog (IHH), HOXA10, and PTCH1. GraphPad Prism 5 was used for statistical analysis. P value < 0.05 was considered significant. RESULTS: In the miRNA array, miR-16 was significantly downregulated at 4.5 dpc versus 0.5 dpc; this was confirmed by RT-qPCR. The miR-16 family members, miR-15a and -15b were also decreased at 4.5 dpc. The putative targets of the miR-16 family, IHH, HOXA10 and PTCH1, were significantly, but transiently upregulated at 2.5 dpc. miR-16 family members were increased with estradiol treatment in the ovariectomized mice. On the other hand, IHH and PTCH1 were significantly downregulated following 48 hr of estradiol treatment. Superovulation also increased miR-16 family expression and downregulated the predicted target IHH. CONCLUSION: The miR-16 family is gradually downregulated following ovulation with a maximal decrease at implantation (4.5 dpc). The coordinate increase in the miR-16 targets may serve an important role in embryo implantation. By contrast, superovulation or estradiol treatment caused rapid and profound upregulation of miR-16 family expression. Our findings suggest that aberrant induction of the miR-16 family by estradiol treatment or superovulation may alter the expression of its targets necessary for successful implantation. Supported by: K12 HD000849-25 (PTJ). P-471 Wednesday, October 22, 2014 HOMEOBOX TRANSCRIPTION FACTOR MSX1 IS REDUCED IN HUMAN ENDOMETRIAL BIOPSIES OF WOMEN FROM INFERTILE COUPLES. A. D. Bolnick,a J. M. Bolnick,a B. A. Kilburn,a J. Oakes,a J. Dai,a M. P. Diamond,b S. K. Dey,c D. R. Armant.a aObstetrics and Gynecology, Wayne State University, Detroit, MI; bObstetrics and Gynecology, Georgia Regents University, Augusta, GA; cDivision of Reproductive Sciences, Cincinnati Children’s Hospital, Cincinnati, OH. OBJECTIVE: To evaluate MSX1 protein expression in human endometrial biopsies from fertile and infertile patients across the secretory phase of the menstrual cycle.
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DESIGN: Control Case Study. MATERIALS AND METHODS: MSX1 protein expression was evaluated in endometrial biopsies obtained throughout the secretory phase from women of fertile (N¼89) and infertile (N¼89) couples using high-throughput immunohistochemistry. Samples were grouped into early (ESP; cycle days 14-19), mid (MSP; cycle days 20-24), and late (LSP; cycle days 25-28) secretory phases of the menstrual cycle. Stain intensity was evaluated separately in the luminal epithelium, glands and stroma by quantifying MSX1 protein expression using image analysis. Comparisons were made by ANOVA with Tukey post-hoc analysis. A Student’s t-test was used to determine the significance between fertile and infertile groups for each cell type throughout the secretory phase. RESULTS: Significant cyclic and tissue-specific regulation of MSX1 protein was found, as well as its dysregulation in women of infertile couples. MSX1 was expressed in all cell types across the secretory phase and was highest during the MSP for fertile patients. There was a significant decrease in stain intensity from 32.8 2.33 to 24.7 2.07 in the glands between the MSP and LSP of fertile patients (p<0.05). Infertile patients demonstrated a significant reduction in MSX1 expression throughout the endometrium during the secretory phase compared to fertile couples (p<0.05), with the greatest reductions (3-fold) in glands and stroma. CONCLUSION: The MSX genes encode homeobox transcription factors that are crucial during embryonic development. In fertile patients, MSX1 protein accumulated during the window of receptivity at MSP. MSX1 protein expression was significantly reduced in infertile patients, suggesting that its target genes might be critical for blastocyst implantation and decidualization similar to that found in mice. Supported by: NICHD National Cooperative Reproductive Medicine Network grant HD039005 (MPD) and NIH grant HD068524 (SKD). P-472 Wednesday, October 22, 2014 MICRORNAS AND THEIR TARGET GENES RELATED TO ENDOMETRIAL DECIDUALIZATION. H. Tochigi,a,b T. Kajihara,a Y. Mizuno,b S. Tamaru,a,b Y. Kamei,a Y. Okazaki,b,c O. Ishihara.a aObstetrics and Gynecology, Saitama Medical University, Moroyama-machi Irumagun, Saitama, Japan; bDivision of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Hidaka City, Saitama, Japan; cDivision of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka City, Saitama, Japan. OBJECTIVE: Endometrial decidualization is the essential step for successful implantation of embryo, however, molecular mechanism of the change is still under debate. The aim of this study is to investigate the role of microRNAs and their target genes at decidualization in human endmetrial stromal cells (HESCs). DESIGN: In vitro experiment on primary culture of HESCs. MATERIALS AND METHODS: Human endometrial tissues were obtained at the time of hysterectomy for uterine fibroids from normally cycling premenopausal women. The patients were not on hormonal treatment at the time of surgery. All the samples were collected during the proliferative phase of the cycle. Written informed consent for the study was obtained before the operation and the study protocol was approved by Institutional Review Board of Saitama Medical University Hospital. Human endometrial stromal cells (HESCs) were isolated and cultured as previously described. The HESCs was treated with or without 0.5 mM 8-bromo-cAMP (cAMP) and 10-6 M medroxyprogesterone acetate (MPA) for 6 days. The significantly altered expression of miRNAs target genes were identified with using miRNA microarray, mRNA microarray and RT-qPCR assay. We then investigated the roles of the miRNAs and the predicted target gene IGFBP1 known as a decidualization marker using gene overexpression experiments. Luciferase reporter assay was conducted to confirm whether the miRNA directly regulates the expression of IGFBP1. RESULTS: Nine miRNAs significantly altered at decidualization were identified by microarray experiments. Among them, one miRNA was up-regulated and eight were down-regulated including 0.27 times expression of miR-542-3p that predicted target gene as IGFBP1. The level of IGFBP1 mRNA expression was significantly reduced in decidualized HESCs by overexpression of the miR-542-3p. In addition, overexpression of miR-542-3p in HESCs inhibited morphorogical decidualization by the stimulation with cAMP and MPA. Luciferase reporter assay confirmed that the 3’-UTR of IGFBP-1 mRNA is directly targeted by the miR542-3p.
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ASRM Abstracts
CONCLUSION: These results suggest that miR-542-3p play an important role in endometrial decidualization through regulating IGFBP1 expression. P-473 Wednesday, October 22, 2014 EFFECTS OF LEVONORGESTREL-RELEASING INTRAUTERINE SYSTEM ON THE EXPRESSION OF STEROID RECEPTOR COREGULATORS TIF-2, AIB-1 AND NCOR IN ADENOMYOSIS. M. K. Kim, B. H. Yun, Y. S. Choi, S. K. Seo. Obstetrics and Gynecology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. OBJECTIVE: In the present study, the expressions of steroid receptor coregulators, transcriptional intermediary factor-2 (TIF-2), amplified in breast cancer-1 (AIB-1) and nuclear receptor corepressor (NCoR) in endometrial tissues from control group, untreated adenomyosis group and levonorgestrel-releasing intrauterine system (LNG-IUS)-treated adenomyosis group are immunohistochemically evaluated. DESIGN: Clinical study. MATERIALS AND METHODS: Tissue samples of endometrium were obtained from 38 premenopausal women with symptomatic adenomyosis, and 23 normal ovulatory women who had carcinoma in situ of uterine cervix without any other significant uterine abnormalities after hysterectomy. Seventeen women with adenomyosis were treated with LNG-IUS. Immunostaining for TIF-2, AIB-1 and NCoR was performed and assessed semiquantitatively. RESULTS: TIF-2 was distributed diffusely in endometrial glandular cytoplasm and stromal cell irrespective of menstrual phase. The expression of TIF-2 in glandular and stromal cell showed strong intensity in the control group and untreated adenomyosis group. The endometrium from LNGIUS-treated adenomyosis group demonstrates significantly decreased expression of TIF-2 compared with those from control group and untreated adenomyosis group. The expression of AIB-1 was observed diffusely in endometrial stromal cells and negligible in glandular cells. AIB-1 expression in stromal cell was greatest in untreated adenomyosis group. The endometrium from untreated adenomyosis group demonstrated significantly higher expression of AIB-1 compared with those from control group and LNG-IUS treated adenomyosis. The immunoreactivity of NCoR was observed in both endometrial glandular and stromal cell throughout the menstrual cycle. NCoR expressions in endometrial glandular nucleus were significantly decreased in untreated adenomyosis group compared with normal control group, and significantly increased in LNG-IUS-treated group compared with untreated adenomyosis group during the proliferative phase. CONCLUSION: The alteration of expression of TIF-2, AIB-1 and NCoR in adenomyosis may be a possible underlying pathogenetic mechanism of adenomyosis and these coregulators may also be associated with the treatment mechanism of LNG-IUS in adenomyosis. P-474 Wednesday, October 22, 2014 LIPID PROFILE AS A NON-INVASIVE TOOL TO PREDICT ENDOMETRIAL RECEPTIVITY – A PILOT STUDY. D. P. A. F. Braga,a,b A. S. Setti,a,b M. N. Eberlin,c E. Cabral,c E. Lo Turco,d A. Iaconelli, Jr.,a,b E. Borges, Jr.,a,b aFertility - Centro de Fertilizac¸~ao Assistida, S~ao Paulo, SP, Brazil; bInstituto Sapientiae, S~ao Paulo, SP, Brazil; cThoMSon Mass Spectrometry Laboratory, Bar~ao Geraldo, SP, Brazil; dUniversidade Federal de S~ao Paulo - UNIFESP, S~ao Paulo, SP, Brazil. OBJECTIVE: To make use of the analytical power of mass spectrometry (MS) to characterize the lipid profile of the receptive endometrial fluid of patients undergoing frozen–thawed embryo transfer cycles. DESIGN: Prospective study. MATERIALS AND METHODS: For this study 22 samples of endometrial fluid were collected from patients undergoing hormonal replacement therapy for frozen-thawed embryo transfers. Samples were split into groups according to the pregnancy result: Positive, the Receptive-EndometriumGroup (RE-Group, n¼13) and negative, the Non-Receptive-EndometriumGroup (NRE-Group, n-9). To avoid the bias of the embryo quality to the implantation success, the study included exclusively frozen-thawed cycles with blastocyst embryo transfer in which one or two top quality embryos were transferred. The lipid profiles of samples from the RE-Group were compared with the lipid profiles of samples from the NRE-Group, aiming to identify a possible biomarker of the receptive endometrium. Mass spectra fingerprinting were acquired using a 7.2T LTQ FT Ultra-MS, equipped with
Vol. 102, No. 3, Supplement, September 2014
FERTILITY & STERILITYÒ
obtained 5.7 3.9 (51.8%) 5.8 4.1 (56.3%).A similar number of good quality Day 3 embryos were transferred into the uterus in the control and in the experimental groups (2.8 1.3 and 2.6 1.3, respectively)T-test and chi square test were used. RESULTS: Hystologycal analysis of the endometrium biopsies showed chronic endometritis in 9.5% (2 /21) of the study population. No other pathologycal findings were reported. . Transfer of embryos in the IVF-ET cycle that immediately followed the endometrium treatment (STUDY GROUP) resulted in a clinical pregnancy rate of 63.2% and a 47.4% ongoing /live birth rate, significantly higher than those obtained in the control group (40% and 25%). CONCLUSION: Local injury to the endometrium prior to controlled ovarian stimulation may considerably improve implantation rates and pregnancy outcomes in patients with implantation failure. P-470 Wednesday, October 22, 2014 THE MIR-16 FAMILY IS HORMONALLY REGULATED IN ENDOMETRIAL STROMAL CELLS AND ALTERED BY SUPEROVULATION IN A MURINE MODEL. S. White P. T. Jimenez. Obstetrics & Gynecology, University of Texas Southwestern Medical Center, Dallas, TX. OBJECTIVE: The purpose of this study was to determine the expression and hormonal regulation of the microRNA (miRNA, miR) -16 family in endometrial stromal cells during embryo implantation. DESIGN: Experimental laboratory study in CD1/ICR mice. MATERIALS AND METHODS: To determine the differential expression of miRNAs before and at the time of implantation, eight week old CD1/ICR female mice were mated with males of known fertility and sacrificed at 0.5 dpc (morning of vaginal plug) or 4.5 dpc. RNA from endometrial stromal cells isolated from the whole uterus at 0.5 dpc and from implantation sites at 4.5 dpc of three mice at each time point underwent microRNA microarray analysis in triplicate. A separate cohort of mice was sacrificed at 0.5, 2.5, 4.5, and 8.5 dpc to confirm the microarray results. To analyze hormonal regulation of the miRNAs, ovariectomized mice were subcutaneously injected with vehicle (sesame oil) or 1 mg estradiol-17b for 48 hr. Another cohort of mice was either injected with 10 IU PMSG followed by 10 IU hCG 48 hrs later to induce superovulation or allowed to ovulate spontaneously. Endometrial stromal cells were isolated. After RNA isolation, RT-qPCR was performed for the three members of the miR-16 family, miR-15a, -15b, and -16, as well as for their predicted targets, Indian Hedgehog (IHH), HOXA10, and PTCH1. GraphPad Prism 5 was used for statistical analysis. P value < 0.05 was considered significant. RESULTS: In the miRNA array, miR-16 was significantly downregulated at 4.5 dpc versus 0.5 dpc; this was confirmed by RT-qPCR. The miR-16 family members, miR-15a and -15b were also decreased at 4.5 dpc. The putative targets of the miR-16 family, IHH, HOXA10 and PTCH1, were significantly, but transiently upregulated at 2.5 dpc. miR-16 family members were increased with estradiol treatment in the ovariectomized mice. On the other hand, IHH and PTCH1 were significantly downregulated following 48 hr of estradiol treatment. Superovulation also increased miR-16 family expression and downregulated the predicted target IHH. CONCLUSION: The miR-16 family is gradually downregulated following ovulation with a maximal decrease at implantation (4.5 dpc). The coordinate increase in the miR-16 targets may serve an important role in embryo implantation. By contrast, superovulation or estradiol treatment caused rapid and profound upregulation of miR-16 family expression. Our findings suggest that aberrant induction of the miR-16 family by estradiol treatment or superovulation may alter the expression of its targets necessary for successful implantation. Supported by: K12 HD000849-25 (PTJ). P-471 Wednesday, October 22, 2014 HOMEOBOX TRANSCRIPTION FACTOR MSX1 IS REDUCED IN HUMAN ENDOMETRIAL BIOPSIES OF WOMEN FROM INFERTILE COUPLES. A. D. Bolnick,a J. M. Bolnick,a B. A. Kilburn,a J. Oakes,a J. Dai,a M. P. Diamond,b S. K. Dey,c D. R. Armant.a aObstetrics and Gynecology, Wayne State University, Detroit, MI; bObstetrics and Gynecology, Georgia Regents University, Augusta, GA; cDivision of Reproductive Sciences, Cincinnati Children’s Hospital, Cincinnati, OH. OBJECTIVE: To evaluate MSX1 protein expression in human endometrial biopsies from fertile and infertile patients across the secretory phase of the menstrual cycle.
e293
DESIGN: Control Case Study. MATERIALS AND METHODS: MSX1 protein expression was evaluated in endometrial biopsies obtained throughout the secretory phase from women of fertile (N¼89) and infertile (N¼89) couples using high-throughput immunohistochemistry. Samples were grouped into early (ESP; cycle days 14-19), mid (MSP; cycle days 20-24), and late (LSP; cycle days 25-28) secretory phases of the menstrual cycle. Stain intensity was evaluated separately in the luminal epithelium, glands and stroma by quantifying MSX1 protein expression using image analysis. Comparisons were made by ANOVA with Tukey post-hoc analysis. A Student’s t-test was used to determine the significance between fertile and infertile groups for each cell type throughout the secretory phase. RESULTS: Significant cyclic and tissue-specific regulation of MSX1 protein was found, as well as its dysregulation in women of infertile couples. MSX1 was expressed in all cell types across the secretory phase and was highest during the MSP for fertile patients. There was a significant decrease in stain intensity from 32.8 2.33 to 24.7 2.07 in the glands between the MSP and LSP of fertile patients (p<0.05). Infertile patients demonstrated a significant reduction in MSX1 expression throughout the endometrium during the secretory phase compared to fertile couples (p<0.05), with the greatest reductions (3-fold) in glands and stroma. CONCLUSION: The MSX genes encode homeobox transcription factors that are crucial during embryonic development. In fertile patients, MSX1 protein accumulated during the window of receptivity at MSP. MSX1 protein expression was significantly reduced in infertile patients, suggesting that its target genes might be critical for blastocyst implantation and decidualization similar to that found in mice. Supported by: NICHD National Cooperative Reproductive Medicine Network grant HD039005 (MPD) and NIH grant HD068524 (SKD). P-472 Wednesday, October 22, 2014 MICRORNAS AND THEIR TARGET GENES RELATED TO ENDOMETRIAL DECIDUALIZATION. H. Tochigi,a,b T. Kajihara,a Y. Mizuno,b S. Tamaru,a,b Y. Kamei,a Y. Okazaki,b,c O. Ishihara.a aObstetrics and Gynecology, Saitama Medical University, Moroyama-machi Irumagun, Saitama, Japan; bDivision of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Hidaka City, Saitama, Japan; cDivision of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka City, Saitama, Japan. OBJECTIVE: Endometrial decidualization is the essential step for successful implantation of embryo, however, molecular mechanism of the change is still under debate. The aim of this study is to investigate the role of microRNAs and their target genes at decidualization in human endmetrial stromal cells (HESCs). DESIGN: In vitro experiment on primary culture of HESCs. MATERIALS AND METHODS: Human endometrial tissues were obtained at the time of hysterectomy for uterine fibroids from normally cycling premenopausal women. The patients were not on hormonal treatment at the time of surgery. All the samples were collected during the proliferative phase of the cycle. Written informed consent for the study was obtained before the operation and the study protocol was approved by Institutional Review Board of Saitama Medical University Hospital. Human endometrial stromal cells (HESCs) were isolated and cultured as previously described. The HESCs was treated with or without 0.5 mM 8-bromo-cAMP (cAMP) and 10-6 M medroxyprogesterone acetate (MPA) for 6 days. The significantly altered expression of miRNAs target genes were identified with using miRNA microarray, mRNA microarray and RT-qPCR assay. We then investigated the roles of the miRNAs and the predicted target gene IGFBP1 known as a decidualization marker using gene overexpression experiments. Luciferase reporter assay was conducted to confirm whether the miRNA directly regulates the expression of IGFBP1. RESULTS: Nine miRNAs significantly altered at decidualization were identified by microarray experiments. Among them, one miRNA was up-regulated and eight were down-regulated including 0.27 times expression of miR-542-3p that predicted target gene as IGFBP1. The level of IGFBP1 mRNA expression was significantly reduced in decidualized HESCs by overexpression of the miR-542-3p. In addition, overexpression of miR-542-3p in HESCs inhibited morphorogical decidualization by the stimulation with cAMP and MPA. Luciferase reporter assay confirmed that the 3’-UTR of IGFBP-1 mRNA is directly targeted by the miR542-3p.
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ASRM Abstracts
CONCLUSION: These results suggest that miR-542-3p play an important role in endometrial decidualization through regulating IGFBP1 expression. P-473 Wednesday, October 22, 2014 EFFECTS OF LEVONORGESTREL-RELEASING INTRAUTERINE SYSTEM ON THE EXPRESSION OF STEROID RECEPTOR COREGULATORS TIF-2, AIB-1 AND NCOR IN ADENOMYOSIS. M. K. Kim, B. H. Yun, Y. S. Choi, S. K. Seo. Obstetrics and Gynecology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. OBJECTIVE: In the present study, the expressions of steroid receptor coregulators, transcriptional intermediary factor-2 (TIF-2), amplified in breast cancer-1 (AIB-1) and nuclear receptor corepressor (NCoR) in endometrial tissues from control group, untreated adenomyosis group and levonorgestrel-releasing intrauterine system (LNG-IUS)-treated adenomyosis group are immunohistochemically evaluated. DESIGN: Clinical study. MATERIALS AND METHODS: Tissue samples of endometrium were obtained from 38 premenopausal women with symptomatic adenomyosis, and 23 normal ovulatory women who had carcinoma in situ of uterine cervix without any other significant uterine abnormalities after hysterectomy. Seventeen women with adenomyosis were treated with LNG-IUS. Immunostaining for TIF-2, AIB-1 and NCoR was performed and assessed semiquantitatively. RESULTS: TIF-2 was distributed diffusely in endometrial glandular cytoplasm and stromal cell irrespective of menstrual phase. The expression of TIF-2 in glandular and stromal cell showed strong intensity in the control group and untreated adenomyosis group. The endometrium from LNGIUS-treated adenomyosis group demonstrates significantly decreased expression of TIF-2 compared with those from control group and untreated adenomyosis group. The expression of AIB-1 was observed diffusely in endometrial stromal cells and negligible in glandular cells. AIB-1 expression in stromal cell was greatest in untreated adenomyosis group. The endometrium from untreated adenomyosis group demonstrated significantly higher expression of AIB-1 compared with those from control group and LNG-IUS treated adenomyosis. The immunoreactivity of NCoR was observed in both endometrial glandular and stromal cell throughout the menstrual cycle. NCoR expressions in endometrial glandular nucleus were significantly decreased in untreated adenomyosis group compared with normal control group, and significantly increased in LNG-IUS-treated group compared with untreated adenomyosis group during the proliferative phase. CONCLUSION: The alteration of expression of TIF-2, AIB-1 and NCoR in adenomyosis may be a possible underlying pathogenetic mechanism of adenomyosis and these coregulators may also be associated with the treatment mechanism of LNG-IUS in adenomyosis. P-474 Wednesday, October 22, 2014 LIPID PROFILE AS A NON-INVASIVE TOOL TO PREDICT ENDOMETRIAL RECEPTIVITY – A PILOT STUDY. D. P. A. F. Braga,a,b A. S. Setti,a,b M. N. Eberlin,c E. Cabral,c E. Lo Turco,d A. Iaconelli, Jr.,a,b E. Borges, Jr.,a,b aFertility - Centro de Fertilizac¸~ao Assistida, S~ao Paulo, SP, Brazil; bInstituto Sapientiae, S~ao Paulo, SP, Brazil; cThoMSon Mass Spectrometry Laboratory, Bar~ao Geraldo, SP, Brazil; dUniversidade Federal de S~ao Paulo - UNIFESP, S~ao Paulo, SP, Brazil. OBJECTIVE: To make use of the analytical power of mass spectrometry (MS) to characterize the lipid profile of the receptive endometrial fluid of patients undergoing frozen–thawed embryo transfer cycles. DESIGN: Prospective study. MATERIALS AND METHODS: For this study 22 samples of endometrial fluid were collected from patients undergoing hormonal replacement therapy for frozen-thawed embryo transfers. Samples were split into groups according to the pregnancy result: Positive, the Receptive-EndometriumGroup (RE-Group, n¼13) and negative, the Non-Receptive-EndometriumGroup (NRE-Group, n-9). To avoid the bias of the embryo quality to the implantation success, the study included exclusively frozen-thawed cycles with blastocyst embryo transfer in which one or two top quality embryos were transferred. The lipid profiles of samples from the RE-Group were compared with the lipid profiles of samples from the NRE-Group, aiming to identify a possible biomarker of the receptive endometrium. Mass spectra fingerprinting were acquired using a 7.2T LTQ FT Ultra-MS, equipped with
Vol. 102, No. 3, Supplement, September 2014