Prostacyclin Agonist (Iloprost) Enhances Human Embryo Development

during the transition of the pre-receptive (early luteal phase, day 16) to the receptive endometrium (mid-luteal phase, day 21) in natural cycles; and (2) during the putative window of implantation (day 21) of gonadotropinstimulated controlled ovarian hyperstimulation (COH) cycle of oocyte donors in GnRH agonist or antagonist cycles compared to temporally-matched natural cycles. DESIGN: Prospective, randomized and blinded clinical research study. MATERIALS AND METHODS: RNA was extracted from endometrial specimens; for global gene expression profiling cRNA was generated and hybridized to Affymetrix Hu95A microarrays. Statistical analysis of the genomic data was performed using pair wise multiple group comparison (SAM software). To validate microarray findings we quantified the expression of selected genes using semiquantitative RT-PCR. We retrieved a 3500 bp-long sequence encompassing 3000 bp upstream and 500 bp downstream of the transcriptional start site of each gene. The search for putative EREs was carried out with ERE finder software with a range of sensitivity threshold from 0.75 to 0.87. The presence of potential EREs was also investigated using the MATCH program using nucleotide distribution matrix for ER with core similarity set to the stringency levels optimal to reduce false positives. The search for putative PREs was conducted using a patternbased method implemented in the GeneQuest module of Lasergene software. RESULTS: (1) Candidate ERE sequences were found in the 5’-flanking regions of 10 genes among 12 genes analyzed that were differentially expressed when comparing the receptive versus the pre-receptive endometrium; 8 of such genes (CD55, SERPING1, ID4, GATA2,MAPK3K5, CIR, MSX1 and GATA 2) had multiple ERE-like sequences. Seven genes had putative PREs. (2) Linear discriminant analysis identified 4 genes (OPTN, SNX7, PCOLN3, and COX 17) significantly up regulated in COH versus natural cycles with maximal potential functional significance. For these genes, the presence of ERE-like sequences was significantly higher than an average for the human genome. Of major interest was the discovery of an identical ERE-like sequence (TGGCCA-NNN-TGGTCT) in the promoter regions of extended homology of these genes. PREs were identified in two genes. CONCLUSIONS: This study identified a number of genes that may have functional significance for the opening and maintenance of the window of receptivity. Although induction of expression by steroid hormones has been previously detected for some of these genes, the mechanism of their transcriptional activation remains unknown. It is reasonable, however, to assume that in the endometrium, transcription of the implantation-related genes is directly regulated by nuclear estrogen and/or progesterone receptors. Our results demonstrated that most of these genes depict putative EREs and PREs. The identification of a unique pattern of ERE-like sequence in a subset of genes upregulated during COH cycle provides strong support for their functional significance. Supported by: None

P-245 Prostacyclin Agonist (Iloprost) Enhances Human Embryo Development. G. M. Grunert, R. C. Dunn, C. T. Valdes, L. Schenk, R. K. Mangal, W. A. Wun. OB and GYN Associates, Houston, TX. OBJECTIVE: It has been reported that human and mouse fallopian tubes contained high concentration of prostacyclin after ovulation. The supplement of Iloprost significantly enhanced mouse embryo development to and beyond blastocyst stage (Huang et al, 2004a). Transfer of Iloprost-treated mouse blastocysts to gestational carriers, both the implantation rate and viable pulp rate were significantly increased and no significant difference on the body weight between the control and experimental group (Huang et al, 2004b) . This study examines whether the Iloprost can enhance the development of human embryo or not. DESIGN: A prospective pilot study. MATERIALS AND METHODS: With the patients’ written instructions, 101 frozen zygotes from 14 couples and 52 frozen blastocysts were used. Those embryos were frozen during the period of January, 1993 to November, 2003. After thawing zygotes and overnight culture, only those embryos developed to 2-cell stage were used. For each patient, those 2-cell stage embryos were split into control pool or experimental pool. All embryos were cultured in Gobal medium (a modified KSOM medium from IVF online) for 5 days with change medium at 2.5 day. The experimental group supplemented with 0.1 uM of Iloprost (Cayman Chemical, Ann Arbor, MI).

FERTILITY & STERILITY威

The control group supplement with vehicle (0.000001% DMSO). ChiSquare test were used to compare the number of blastocyst formed. The quality (size, inner cell mass, trophectoderm) of blastocysts was analyzed by Wilcoxon Rank Sum Test. The statistical significance was defined at P⬍0.05. Forty-five thawed blastocysts were recovered. Those blastocysts were immediately cultured with/without Iloprost. The re-expansion of blastocoel was checked 18 hours later. The analysis was the same as for the frozen zygotes study. RESULTS: As shown in Table, Iloprost marginally enhanced the blastocyst formation and significantly enhanced the blastocyst quality.

CONCLUSIONS: Due to the limitation of human embryos, the blastocyst formation rate was at border line(P⫽0.0506). We have reported that the size and quality of TRO correlates with pregnancy. Low quality of ICM correlates with spontaneous abortion. The significant enhancing of these 3 quality indicators suggest a possible increase in implantation and viable pregnancy rates by Iloprost treatment in human ART cases. Although it looked Iloprost might help the thawed blastocyst, the trend was not significant. There are lots of possible mechanism may responsible for the non significant difference. In summary, Iloprost enhances the quality of blastocysts. It waits more study to clarify the action mechanism of prostacyclin and its application to human ART. Supported by: None P-246 Uterine Leiomyomas and Their Effect on In Vitro Fertilization Outcome: A Controlled Retrospective Study. S. D. Henderson, B. Rizk, M. Mulekar. University of South Alabama, Mobile, AL. OBJECTIVE: To investigate the effect of uterine leiomyomas on pregnancy and implantation rates, and pregnancy outcome in an infertile patient population with age adjusted for undergoing in vitro fertilization-embryo transfer (IVF-ET). DESIGN: A case controlled retrospective study. Study group: patients with intramural and subserosal leiomyomas with normal endometrial cavity undergoing IVF-ET. Control group: patients without uterine leiomyoma. MATERIALS AND METHODS: A total number of 134 patients undergoing 188 IVF cycles were reviewed. Documented fibroids by pelvic ultrasound results were used to determine presence, size and location of fibroids (intramural, subserosal and submucosal). In addition, patients with prior myomectomy whose ultrasound showed no fibroids were considered unexposed (group without fibroids). Exclusion criteria included donor eggs, frozen embryos, and severe male factor. After exclusion, a total of 120 patients and 160 IVF-ET cycles remained. Of the 120 patients, 25 patients were identified with fibroids, and the remaining 95 patients were without fibroids. IVF-ET first cycles only (total of 120) were included in the analysis. Controlled ovarian hyperstimulation protocols were used The main outcome measures were pregnancy and implantation rates. Secondary outcome measures included pregnancy outcome (spontaneous abortion, preterm labor and delivery), and determination of whether fibroid location and size have any effect on IVF outcome. RESULTS: Pregnancy rates were 40.0% and 33.7% for the fibroid and non-fibroid groups, respectively. Implantation rates (per embryo) were 17.6% and 13.5% for the fibroid and non-fibroid groups, respectively. There was no significant difference after adjusting for age between the two groups in pregnancy rate, implantation rate, and pregnancy outcome, including spontaneous abortions, preterm labor and deliveries. The mean patient age in the fibroid group was 37.88 (SEM⫽0.94), and the mean age in the

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