P-538

reaching oocyte pick-up again. Nevertheless, although not significant, the higher implantation rate and pregnancy rate per embryo transfer seen in these patients determine that pregnancy probability per cycle initiated is not significantly different between patients receiving low and high gonadotrophin dose regimens. Supported by: None. P-535 WITHDRAWN P-536 DOES ASSISTED HATCHING BENEFIT PATIENTS WITH AN ELEVATED BASELINE FOLLICLE STIMULATING HORMONE (FSH)? A. Assemi, K. S. Richter, M. J. Tucker, A. W. Sagoskin. Shady Grove Fertility Reproductive Science Center, Rockville, MD. OBJECTIVE: The indications for assisted hatching remain controversial, with the exception of a history of repeated failed implantation despite the transfer of good quality embryos. It has been suggested that patients with an elevated FSH level may benefit from assisted hatching. However, there is very little published data comparing outcomes of transfers with and without assisted hatching based only on elevated FSH, independent of other possible indications such as treatment history or advanced maternal age. The goal of the current study was to evaluate assisted hatching among patients whose only indication for its use was an elevated baseline serum FSH level (greater than or equal to 10 IU/L). DESIGN: Retrospective analysis. MATERIALS AND METHODS: Records of treatment cycles of patients under the age of 39 years, with a baseline serum FSH of 10 IU/L or higher, and undergoing their first cycle of in vitro fertilization (IVF) were reviewed. Assisted hatching is routinely offered to patients with an elevated FSH at the study center, but some patients choose to decline hatching. Patient characteristics and cycle outcomes were compared between cycles with and without assisted hatching. Clinical pregnancy was defined by identification of an intrauterine gestational sac on ultrasound examination four to six weeks after oocyte retrieval. RESULTS: A total of 154 first IVF cycles by patients under 39 years and with an FSH of 10 IU/L or greater were identified. Assisted hatching was performed in 110 of these cycles, while 44 cycles did not have assisted hatching. Patient age and baseline FSH were similar between cycles with and without assisted hatching. There was a slight but statistically significant trend toward the transfer of a greater number of embryos in the hatching group. Clinical pregnancy rates were not significantly different, although the pregnancy rate was lower for the hatched compared to the un-hatched group.

OBJECTIVE: Embryonic implantation - the process by which the embryo orientates, attaches, and invades the underlying maternal endometrial tissue - is a critical feature of mammalian reproduction. Progranulin, one of the several growth factors and cytokines implicated in the dramatic morphological and physiological changes that occur during endometrial preparation and implantation, is found in epithelial endometrial cells, glands, and stromal cells of the mouse uterus; its expression is modulated by ovarian steroids. Progranulin has an essential role in promoting the growth and differentiation of preimplantation mouse embryos; its expression increases dramatically during the pre- and peri-implantation periods and drops precipitously following implantation. Furthermore, DNA microarray studies of endometrial biopsies from patients with and without endometriosis have recently shown that levels of progranulin mRNA are lower in women with endometriosis, implicating this gene in the etiology of implantation failure in women with this disorder. Based on these studies, we hypothesize that progranulin is important for human embryo implantation. Our objective was to translate our findings in the mouse to the human to form the baseline for further studies of the role of this protein in human embryo implantation. DESIGN: This is a prospective experimental study using immunoblotting to detect the presence of progranulin in tissues and biological fluids, and immunohistochemistry to define the localization of the protein in the human uterus. MATERIALS AND METHODS: After Lankenau Hospital IRB approval, tissue sections from paraffin blocks (from the Pathology department archives) obtained after endometrial biopsies for different clinical indications. Specimens reported as normal by the pathology department were subjected to immunohistochemistry with anti-progranulin antibody. Fresh tissue from endometrial biopsies, and endometrial fluid obtained after diagnostic hysteroscopy or hysterosonography were subjected to protein extraction, electrophoresis and immunoblotting with the same antibody. RESULTS: Analysis of protein extracts from fresh tissues subjected to immunoblotting with anti-progranulin antibody demonstrated that the human endometrium expressed a 67,000 Mr band corresponding to progranulin. We also identified a 67,000 Mr band corresponding to progranulin in fluid samples of uterine flushings, confirming progranulin is a secreted endometrial protein. Human endometrial tissue sections analyzed by immunohistochemistry revealed that progranulin was localized mainly in endometrial epithelial cells and more diffusely in stromal cells; control sections (preimmune serum substituted for anti-progranulin antibody) did not show any immunoreactivity. CONCLUSION: The human progranulin protein, like its mouse homologue, is expressed by the endometrium, where it is localized mainly in epithelial cells and more diffusely in stromal cells. In addition, analyses of uterine flushings indicate that progranulin is secreted into the endometrial lumen. These results are consistent with previous data from animal studies and with DNA microarray analyses of human samples, and suggest that progranulin is a marker for human embryo implantation. Supported by: Supported in part by NIH HD-06274 (GLG) and Fogarty International Center grant 5-D43-TW 00671 (CMP).

P-538 SPP1 GENE EXPRESSION IN FEMALE INFERTILITY. H. Wu, C. Huang, N. M. Wang. Changhua Christian Hospital, Changhua, Taiwan; National Changhua Univ. of Education, Changhua, Taiwan.

CONCLUSION: Results of this retrospective study suggest that there is no benefit from assisted hatching performed on the basis of an elevated baseline FSH alone. Larger prospective randomized studies are needed to confirm whether or not there is any benefit to assisted reproduction for this patient population. Supported by: None. P-537 PROGRANULIN CHARACTERIZATION IN HUMAN ENDOMETRIUM: TRANSLATIONAL STUDIES FROM MOUSE TO HUMAN. C. M. Perez, C. Minimo, P. Caballero-Campo, P. Rinaudo, J. Orris, G. L. Gerton. Lankenau Hospital, Wynnewood, PA; Center for Research on Reproduction and Women’s Health, Dept. of Obstetrics and Gynecology, Univ. of Pennsylvania., Philadelphia, PA; Univ. of California. Dept. of Obstetrics and Gynecology., San Francisco, CA.

FERTILITY & STERILITY威

OBJECTIVE: To investigate possible factors that might contribute to implantation failure in IVF programs. Animal model suggested a role of secreted phosphoprotein 1 (SPP1), expressed in the pregnant porcine uterus, in uterine receptivity. In this study we evaluated both SPP1 and CYP19, a key enzyme in the synthesis of estrogens from androgens, gene expressions in infertile patients treated with either anastrozole or clomiphene citrate. DESIGN: This is a prospective randomized trial. A total of 33 female infertile patients (ages 25-41 years) were included in this study approved by the Institutional Review Board (IRB No: CCH-93-02-03). Group A consisted of 14 patients received anastrozole 1 mg daily; and group B consisted of 19 patients received clomiphene citrate 100 mg daily from cycle day 3 to day 7 for ovulation induction. HCG (5,000 IU) was given when one dominant follicle reached 20 mm or two follicles reached 18 mm, followed by IUI. Oral micronized progesterone 100 mg twice per day was given to all patients for 2 weeks after IUI. Blood samples were taken on cycle day 3, day 8, day 10, IUI (intrauterine insemination) day, post-IUI day 7, and post-IUI day 14.for serial hormonal profiles and gene expression analyses of CYP1 and SPP1 using real-time PCR.

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MATERIALS AND METHODS: Hormonal profiles of FSH, LH, E2, T, and P were determined using ELISA. Total RNA from each blood sample was isolated using Trizol reagent (Invitrogen). cDNA synthesis and PCR were performed using SuperScript II RNase H- reverse transcriptase and the cDNA cycle kit (Invitrogen) according to the manufacturer’s protocol. Real-time quantitative reverse transcription-PCR (RT-PCR) was performed using the Roche LightCycler (Roche, Mannheim, Germany) and the FastStart DNA Master SYBR-green 1 system (Roche). RESULTS: The hormone profiles were similar in two groups. The anastrozole group has a significantly higher LH level, but a significantly lower estradiol level in the stimulation cycle. The SPP1 expression pattern of normal female is similar to CYP19. Interestingly, there was no detectable SPP1 in all infertile patients. The CYP19 in patient treated with aromatase inhibitor was totally depleted at day 8 and gradually increasing post IUI. The CYP19 expression patterns after IUI are the same in both study groups. Figure Legend: Real time PCR reactions of SPP1 gene from a pregnant woman (lane 1), post parturition woman (lane 2), and an infertile patient (lane 3). Lane 4 is a PCR reaction amplified SPP1 coding region from the same infertile patient. The size of amplified products from real time PCR is 194 bp and the PCR product is 885 bp. M, 100 bp-marker.

which are secreted by human embryo and supposed to be supporting implantation. On the other hand Leukemia Inhibiting Factor (LIF) and Transforming Growth Factor (TGFb) are factors secreted from human endometrium in perimplantation phase. The aim of this study is to investigate the effect of IL-1, LIF, TGFb and IFNg on TIMP-2 levels in choriocarcinomal cell lines. DESIGN: Experimental study. MATERIALS AND METHODS: Choriocarcinoma cell lines JEG-3 and Jar are cultured in RPMI 1640. IL-1, LIF, TGFb and IFNg are added in different concentrations into culture consisting 10x105 number of cells. Cultures without added cytokines are used as controls. After 24 hours of culture period, the supernatants had been frozen to -80 C. TIMP-2 levels are measured by ELISA assay. The minimal detectable value was 3.0 ng/ml. In statistical analysis, one way ANOVA test is used. P⬍ 0,05 value is appreciated significant in statistical aspect. RESULTS: IL-1,LIF, TGFb and IFNg decreases TIMP-2 levels significantly (p⬍0.05) in both JEG-3 and Jar cell lines. Decrease by TGFb occurs dose dependet and IFNg decreases the TIMP-2 levels most strongly. CONCLUSION: Interleukin-1, Leukemia Inhibiting Factor, Transforming Growth Factor and Interferon gamma decreases TIMP-2 levels. Consequently these cytokines can be considered supporting trophoblastic invasion and taking part in a successful implantation. Supported by: None

P-540 FAILED FRESH EMBRYO TRANSFER IS ASSOCIATED WITH HIGHER PREGNANCY RATES THAN SUCCESSFUL FRESH PREGNANCY IN GOOD PROGNOSIS PATIENTS DURING FROZEN-THAWED EMBRYO TRANSFER CYCLE. Z. P. Nagy, T. Taylor, G. Wright, H. I. Kort, D. Mitchell-Leef, D. B. Shapiro. Reproductive Biology Associates, Atlanta, GA.

CONCLUSION: The results of this preliminary study showed that SPP1 may be an important factor contributing to uterine receptivity possibly by interaction with ␣v␤3 integrin and/or other extracellular matrix proteins. Future studies with increased sample size are required to confirm these preliminary findings. Supported by: This study was supported by a grant from Changhua Christian Hospital (CCH# 9210).

P-539 EFFECT OF SOME CYTOKINES ON TIMP-2 LEVELS IN CHORIOCARSINOMA CELL LINES. H. Ozornek, E. Bilgin, U. Koldovsky. EUROFERTIL, Istanbul, Turkey. OBJECTIVE: Matrixmetalloproteinases constitute invasion of trophoblastic cells to maternal endometrium by degrading extracelluler matrix. However this process has to be balanced by metalloproteinase inhibitors. This balance is necessary for a successful implantation. Choriocarcinoma cell lines JEG-3 and Jar are widely used as a model in trophoblast research. Human trophoblast cells present an invasive character by secreting some metalloproteinases. This invasiveness of trophoblasts are blocked by metalloproteinase inhibitors. Tissue inhibitor of metalloproteinase-2 (TIMP-2) is a type of these inhibitor and to be secreted by human endometrium and decidua. Interleukin-1 (IL-1) and interferon gamma (IFNg) are cytokines

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Abstracts

OBJECTIVE: Pregnancy ensues when viable embryos are transferred into a receptive uterine environment. Although a high proportion of young, good prognosis patients achieve pregnancy in a fresh embryo transfer cycle (ET), a number of them will not get pregnant. It may be hypothesized that lower receptivity and/or asynchrony between endometrial and embryonic development is responsible for lack of pregnancy (possibly as a result of fresh cycle characteristics). Frozen-thawed cycles are distinctly different and therefore may offer advantage to patients who failed in their fresh cycle. Therefore, the objective of the present study was to compare pregnancy outcomes of frozen-thawed cycles based on the pregnancy outcome of the fresh embryo transfer cycle. DESIGN: Longitudinal, observational study of frozen-thawed embryo transfer cycles. MATERIALS AND METHODS: A total of 73 patients were included in the study who underwent IVF-ET cycles from January to December 2004 and returned for a frozen-thawed embryo transfer cycle. Patients were included if the maternal age was below 35 years (at the time of the fresh ET) and had at least 5 good quality embryos frozen. Patients in the fresh cycle underwent controlled ovarian hyperstimulation using recombinant FSH after pituitary down-regulation with long leuprolide acetate protocol. Supernumerary embryos were cryopreserved on day 3. Embryos were thawed after 4 days of progesterone administration in a leuprolide acetate and oral micronized estradiol replacement cycle. All embryos were submitted to assisted hatching followed by the removal of degenerated blastomeres (if present). For statistical analysis Kruskal-Wallis and chi-square tests were applied whenever appropriate. RESULTS: The mean age of patients undergoing transfer was 31.0 (⫹/-2.8 ) and 31.3 (⫹/-2.3) years in the negative (36 cases) and positive (37 cases) cryo pregnancy groups respectively (at the time of the egg retrieval; NS). All important clinical and laboratory procedures were standardized and the parameters were similar for all patients in the fresh and in the frozenthawed embryo transfer cycles.The mean number of pre-embryos thawed was 4.7 (⫹/-1.3) and 4.8 (⫹/-1.2; NS), survival rate was 82% and 79%; embryos transferred was 2.4 (⫹/-0.7) and 2.7 (⫹/-0.6) in the negative and positive cryo pregnancy groups respectively. Initial pregnancy rates were 42% and 65% (P⬍0.05) and implantation rates were 15% and 28% (P⬍0.05) in the previously negative (fresh ET) and previously positive (fresh ET) pregnancy groups, respectively. CONCLUSION: The results demonstrate that initial pregnancy and implantation rates in patients with failed fresh embryo transfer is significantly

Vol. 86, Suppl 2, September 2006