- Email: [email protected]
Aberrant expression of homeobox genes adversely affects implantation in endometriosis
endometrial biopsies, and the ICSI outcomes for patients presenting or not recurrent implantation failure (RIF). DESIGN: Cohort study. MATERIALS AND METHODS: This study included 211 patients submitted to infertility screening at a private reproductive medicine center. All patients underwent office hysteroscopy followed by endometrial biopsy, and received prophylactic antibiotic therapy with vibramycin. 105 patients were submitted to ICSI cycle. The ovarian stimulation protocol used GnRH-agonist or antagonist, recombinant-FSH (Gonal-F, Serono), and recombinant-hCG (Ovidrel, Serono). ICSI outcomes were evaluated according to groups: patients without previous IVF (FIRST-IVF: n ¼ 36), RIF patients defined as at least 3 previous unsuccessful IVF (RIF: n ¼ 18), and patients who underwent previous low complexity or 1-2 IVF cycles (INTER: n ¼ 51). RESULTS: The mean age of patients were similar between groups (FIRST IVF: 34.8 4.6; RIF: 36.5 5.7; INTER: 36.4 4.7; P¼0.280). The number of previous ICSI cycles was 4.1 1.4 for RIF patients and 0.4 0.7 for INTER group. In spite of prevalence of endometrial alteration at hysteroscopy in the RIF group had been higher (66.7%) than FIRST IVF (27.3%; P¼0.006) and INTER (37.0%; P¼0.032) groups, the prevalence of alterations at pathologic analysis was not significant (P¼0.592). After prophylactic antibiotic therapy with vibramycin, patients underwent ICSI cycles (1.5 0.5 cycles per patient), and accumulated ongoing pregnancy rate was 30.6% in FIRST-IVF, 29.4% for RIF and 42.9% in INTER groups (P¼0.411). CONCLUSION: Endometrial alteration at hysteroscopy was higher in the RIF group. After hysteroscopy and prophylactic antibiotic therapy, the accumulated ongoing pregnancy rate was 30% in RIF, which was similar to other groups without RIF. Our results suggest that hysteroscopy and prophylactic antibiotic therapy should be favorable in patients with RIF.
P-571 Wednesday, October 19, 2011 LETROZOLE USE IN FROZEN EMBYRO TRANSFER (FET) CYCLES: CLINICAL PREGNANCY OUTCOMES IN PATIENTS WITH AND WITHOUT ENDOMETRIOSIS. B. G. Patel, G. Bushnell, H. L. Higdon, III, P B. Miller, D. A. Forstein, B. A. Lessey. Obstetrics and Gynecology Division Reproductive Endocrinology and Infertility, Greenville Hospital System, Greenville, SC; Department of Public Health Sciences, Clemson University, Clemson, SC. OBJECTIVE: Aromatase has been associated with poor reproductive outcome during In Vitro Fertilization (IVF) and aromatase inhibitors like letrozole appear to be helpful in poor responders. The purpose of this study was to examine the effectiveness of letrozole, administered during a frozen embryo transfer (FET) cycle in women with and without diagnosed endometriosis. DESIGN: Retrospective case control study on FET cycles between January 2003 and December 2010. MATERIALS AND METHODS: The study population comprised 173 first time FET cycles. Overall, 61 of 173 subjects (35.3%) were diagnosed with endometriosis. Of the 112 patients without a diagnosis of endometriosis 27/112 (24.1%) had a prior laparoscopy. Twenty-two of 173 (12.7%) received letrozole (2.5 to 5 mg on days 3 to 7 of their FET cycle) along with the standard hormone replacement. Of the patients on letrozole, 17/22 (77.3%) had endometriosis. The control group included 105 patients without a diagnosis of endometriosis who received no letrozole. Statistical analyses included the Pearson chi-square test for outcome and demographic variables and the student t-test for continuous demographic variables. RESULTS: Demographics were similar between groups. Of the patients with endometriosis that received letrozole, clinical pregnancy was achieved in 10/17 (58.8%) compared to 30 of 105 (28.6%) in the control group (P¼0.02). None of 5 patients without known endometriosis who received letrozole during their FET cycle conceived. The quality of transfer was examined between groups. When only excellent transfers were included, the clinical pregnancy rate difference (10/15; 66.7% vs. 23/75; 30.3%, respectively), was significantly in favor of letrozole (P¼0.008). CONCLUSION: Letrozole is an effective adjunct therapy for women undergoing FET with a history of endometriosis. These women were more likely to have a clinical pregnancy compared to patients without endometriosis not receiving letrozole during FET, possibly through improvement in endometrial receptivity.
S276
Abstracts
P-572 Wednesday, October 19, 2011 ABERRANT EXPRESSION OF HOMEOBOX GENES ADVERSELY AFFECTS IMPLANTATION IN ENDOMETRIOSIS. S. K. Jana, P. Banerjee, V. Pramanik, P. Pasricha, K. Chaudhury, B. Chakravarty. School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India; Department of Biotechnology, National Institute of Technology, Durgapur, West Bengal, India; Reproductive Health, Institute of Reproductive Medicine, Kolkata, West Bengal, India. OBJECTIVE: To investigate Homeobox genes HOXA-10 and HOXA-11 mediated endometrial molecular defects during implantation window in endometriosis-associated infertility cases. DESIGN: A prospective comparative study conducted on 31 women with endometriosis (age<35 years) during their mid-secretory phase of the menstrual cycle and 26 age-matched fertile women as controls reporting to the Institute of Reproductive Medicine, India. The effect of endometrial HOXA-10 and HOXA-11 genes on the mediators of matrix turnover, MMP-2 and -9 and on endometrial receptivity markers including avb3 integrin, leukemia inhibitory factor (LIF) and pinopodes were explored in women with endometriosis. MATERIALS AND METHODS: Endometrial tissue samples collected during implantation window from both endometriotic and normal women were divided into four parts: stromal and epithelial cells were isolated from the first part for flow cytometric analysis of avb3 integrin and LIF expression, the second part was fixed to examine pinopodes expression by scanning electron microscopy, and mRNA were isolated from the third part to study HOXA-10 and HOXA-11 gene expression by Real Time PCR. The fourth part was used to study the expression of MMP-2, -9, HOXA-10 and -11 by Western blot. Data were compared by independent two sample ‘t’ test, and chi-square test, as applicable. Statistical significance was defined as P%0.05. RESULTS: A significantly higher endometrial expression of HOXA-11, MMP-2 and -9 were observed in women with endometriosis when compared with controls. Interestingly, expression of HOXA-10 gene was significantly less in endometriosis which is known to regulate the expression of endometrial receptivity markers. CONCLUSION: The findings suggest that aberrant expression of HOXA10 and -11 genes adversely affects endometrial remodeling and expression of receptivity markers. Suitable HOX gene therapy to improve implantation appears to be a promising area of research in the subsequent years. Supported by: Saikat k Jana was supported by a fellowship from ICMR, India.
P-573 Wednesday, October 19, 2011 BALANCED LYCAT GENE EXPRESSION IS CRUCIAL FOR FEMALE REPRODUCTION. L. Ni, W. Wang, Y. Tang, H.-C. Liu. Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, New York, NY. OBJECTIVE: To test our hypothesis that lysocardiolipin acyltransferase (Lycat) is a key regulator controlling the developmental potential of early embryos and uterine receptivity of the mothers. DESIGN: Experimental study. Transgenic (Tg) mouse lines over-expressing Lycat were generated. Our hypothesis was tested by analyzing the phenotype and the underlying molecular mechanism leading to infertility. MATERIALS AND METHODS: Full length cDNA and ires.lacZ were inserted into the 2nd exon of a Lycat BAC clone by ET-cloning. Tg lines were generated by pronuclear injection. Litter size from intercross breedings of Tg mice was documented. Embryos were collected at different stages to detect the onset of degeneration. Fully expanded WT and Tg blastocysts were placed on slides and fixed with 4% PFA. Cell number was obtained by counting DAPI-stained nuclei. WT, Tg blastocysts as well as endometrial stromal cells (ESCs) were boiled in water for 40 min. to release ATP. Equal volume of rL/L reagent from Promega ENLITEN ATP system was added. RLU was measured with GloMax20/20. RESULTS: Lycat is a cardiolipin remodeling enzyme. Elevate level of cardiolipin antibodies in humans results in impaired implantation. The female Tg mice exhibit reduced fertility as they either produce fewer pups per litter (Wt, 8.3 1.2; n ¼ 16. vs Tg, 3.6 1.6, n ¼ 32; P<0.01), or are infertile. Degeneration of Tg embryos started at implantation stage (E4.5) and completed by E11.5. Tg blastocysts contain fewer cells than the WTs (Tg, 69.5 6.4,n ¼ 6. vs.WT, 81.5 4.2, n ¼ 6; P<0.05). ATP assay shows that
Vol. 96., No. 3, Supplement, September 2011
P-571 Wednesday, October 19, 2011 LETROZOLE USE IN FROZEN EMBYRO TRANSFER (FET) CYCLES: CLINICAL PREGNANCY OUTCOMES IN PATIENTS WITH AND WITHOUT ENDOMETRIOSIS. B. G. Patel, G. Bushnell, H. L. Higdon, III, P B. Miller, D. A. Forstein, B. A. Lessey. Obstetrics and Gynecology Division Reproductive Endocrinology and Infertility, Greenville Hospital System, Greenville, SC; Department of Public Health Sciences, Clemson University, Clemson, SC. OBJECTIVE: Aromatase has been associated with poor reproductive outcome during In Vitro Fertilization (IVF) and aromatase inhibitors like letrozole appear to be helpful in poor responders. The purpose of this study was to examine the effectiveness of letrozole, administered during a frozen embryo transfer (FET) cycle in women with and without diagnosed endometriosis. DESIGN: Retrospective case control study on FET cycles between January 2003 and December 2010. MATERIALS AND METHODS: The study population comprised 173 first time FET cycles. Overall, 61 of 173 subjects (35.3%) were diagnosed with endometriosis. Of the 112 patients without a diagnosis of endometriosis 27/112 (24.1%) had a prior laparoscopy. Twenty-two of 173 (12.7%) received letrozole (2.5 to 5 mg on days 3 to 7 of their FET cycle) along with the standard hormone replacement. Of the patients on letrozole, 17/22 (77.3%) had endometriosis. The control group included 105 patients without a diagnosis of endometriosis who received no letrozole. Statistical analyses included the Pearson chi-square test for outcome and demographic variables and the student t-test for continuous demographic variables. RESULTS: Demographics were similar between groups. Of the patients with endometriosis that received letrozole, clinical pregnancy was achieved in 10/17 (58.8%) compared to 30 of 105 (28.6%) in the control group (P¼0.02). None of 5 patients without known endometriosis who received letrozole during their FET cycle conceived. The quality of transfer was examined between groups. When only excellent transfers were included, the clinical pregnancy rate difference (10/15; 66.7% vs. 23/75; 30.3%, respectively), was significantly in favor of letrozole (P¼0.008). CONCLUSION: Letrozole is an effective adjunct therapy for women undergoing FET with a history of endometriosis. These women were more likely to have a clinical pregnancy compared to patients without endometriosis not receiving letrozole during FET, possibly through improvement in endometrial receptivity.
S276
Abstracts
P-572 Wednesday, October 19, 2011 ABERRANT EXPRESSION OF HOMEOBOX GENES ADVERSELY AFFECTS IMPLANTATION IN ENDOMETRIOSIS. S. K. Jana, P. Banerjee, V. Pramanik, P. Pasricha, K. Chaudhury, B. Chakravarty. School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India; Department of Biotechnology, National Institute of Technology, Durgapur, West Bengal, India; Reproductive Health, Institute of Reproductive Medicine, Kolkata, West Bengal, India. OBJECTIVE: To investigate Homeobox genes HOXA-10 and HOXA-11 mediated endometrial molecular defects during implantation window in endometriosis-associated infertility cases. DESIGN: A prospective comparative study conducted on 31 women with endometriosis (age<35 years) during their mid-secretory phase of the menstrual cycle and 26 age-matched fertile women as controls reporting to the Institute of Reproductive Medicine, India. The effect of endometrial HOXA-10 and HOXA-11 genes on the mediators of matrix turnover, MMP-2 and -9 and on endometrial receptivity markers including avb3 integrin, leukemia inhibitory factor (LIF) and pinopodes were explored in women with endometriosis. MATERIALS AND METHODS: Endometrial tissue samples collected during implantation window from both endometriotic and normal women were divided into four parts: stromal and epithelial cells were isolated from the first part for flow cytometric analysis of avb3 integrin and LIF expression, the second part was fixed to examine pinopodes expression by scanning electron microscopy, and mRNA were isolated from the third part to study HOXA-10 and HOXA-11 gene expression by Real Time PCR. The fourth part was used to study the expression of MMP-2, -9, HOXA-10 and -11 by Western blot. Data were compared by independent two sample ‘t’ test, and chi-square test, as applicable. Statistical significance was defined as P%0.05. RESULTS: A significantly higher endometrial expression of HOXA-11, MMP-2 and -9 were observed in women with endometriosis when compared with controls. Interestingly, expression of HOXA-10 gene was significantly less in endometriosis which is known to regulate the expression of endometrial receptivity markers. CONCLUSION: The findings suggest that aberrant expression of HOXA10 and -11 genes adversely affects endometrial remodeling and expression of receptivity markers. Suitable HOX gene therapy to improve implantation appears to be a promising area of research in the subsequent years. Supported by: Saikat k Jana was supported by a fellowship from ICMR, India.
P-573 Wednesday, October 19, 2011 BALANCED LYCAT GENE EXPRESSION IS CRUCIAL FOR FEMALE REPRODUCTION. L. Ni, W. Wang, Y. Tang, H.-C. Liu. Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, New York, NY. OBJECTIVE: To test our hypothesis that lysocardiolipin acyltransferase (Lycat) is a key regulator controlling the developmental potential of early embryos and uterine receptivity of the mothers. DESIGN: Experimental study. Transgenic (Tg) mouse lines over-expressing Lycat were generated. Our hypothesis was tested by analyzing the phenotype and the underlying molecular mechanism leading to infertility. MATERIALS AND METHODS: Full length cDNA and ires.lacZ were inserted into the 2nd exon of a Lycat BAC clone by ET-cloning. Tg lines were generated by pronuclear injection. Litter size from intercross breedings of Tg mice was documented. Embryos were collected at different stages to detect the onset of degeneration. Fully expanded WT and Tg blastocysts were placed on slides and fixed with 4% PFA. Cell number was obtained by counting DAPI-stained nuclei. WT, Tg blastocysts as well as endometrial stromal cells (ESCs) were boiled in water for 40 min. to release ATP. Equal volume of rL/L reagent from Promega ENLITEN ATP system was added. RLU was measured with GloMax20/20. RESULTS: Lycat is a cardiolipin remodeling enzyme. Elevate level of cardiolipin antibodies in humans results in impaired implantation. The female Tg mice exhibit reduced fertility as they either produce fewer pups per litter (Wt, 8.3 1.2; n ¼ 16. vs Tg, 3.6 1.6, n ¼ 32; P<0.01), or are infertile. Degeneration of Tg embryos started at implantation stage (E4.5) and completed by E11.5. Tg blastocysts contain fewer cells than the WTs (Tg, 69.5 6.4,n ¼ 6. vs.WT, 81.5 4.2, n ¼ 6; P<0.05). ATP assay shows that
Vol. 96., No. 3, Supplement, September 2011