Association of Killer Cell Immunoglobulin-Like Receptors Gene Polymorphisms With Unexplained Recurrent Spontaneous Abortion

intracytoplasmatic sperm injection (ICSI) and to find out (i) the difference between the FF and serum concentration, (ii) the relationship between the etiology of sterility and these growth factors concentrations, (iii) the influence of these investigated growth factor on ICSI outcome. DESIGN: Prospective study. MATERIALS AND METHODS: 75 women undergoing ICSI therapy were included in this study. Tubar sterility (G.1⫽17), endometriosis (G.2 ⫽ 15) and male factor infertility (G.3. 43). After pituitary down regulation using Gn-RH a, the women underwent COH either with rFSH or HMG/FSH. Serum and FF were collected at the time of oocytes retrieval and were frozen at -80°C until the measurement of LIF; VEGF and NO concentration. LIF and VEGF concentration was determined by using enzyme-linked immunosorbent assay (ELISA). Whereas, the NO was measured using the Griess reaction as an indirect assessment of NO activity. RESULTS: LIF concentrations in FF of patients with tubal sterility, endometriosis, male factor sterility were (43.20⫾12.10, 43.6⫾18.9 and 50.0⫾23.70 pg/ml respectively) and the corresponding values in serum were (1.60⫾0.90; 1.50⫾0.96 and 2.30⫾2.4 pg/ml). VEGF concentrations in FF of the first, second and third groups were (3247.8⫾931.9; 3546.3⫾893.40 and 3358.1⫾894.3 pg/ml) and in serum as follows (375.3⫾180.5; 354.3⫾160.5 and 374.8⫾182.4). NO concentrations were at the same level in FF and serum in the first group (42.3⫾9.50 vs. 39.190⫾ 15.52, p⫽0, 26; 41.5⫾8.99vs.36.3⫾13.6 p⫽ 0.18) in the second group and 40.1⫾12.5 vs. 35.96⫾18.9 p⫽ 0.059 ␮mol/l in the third group. LIF concentration in FF was 43.2⫾12.1 and in 1.6⫾0.9 pg/ml in serum (p⬍0.001). VEGF in FF was 3247.8⫾931.9 and in serum 375.3⫾180.5 pg/m (p⬍0.001). NO concentrations in FF and serum were similar (42.3⫾9.5 vs. 39.19.52␮mol/l; p⫽0.26). The main number of retrieved, fertilized and transferred oocytes in the first group was (8.2⫾5.3; 4.7⫾3.7 and 2.0⫾0.8) the corresponding value in the second group was (5.0⫾3.6; 3.7⫾3.1 and 1.9⫾0.9) and in the third group (5.9⫾4.1; 3.4⫾2.7 and 1.7⫾0.9). No significance difference was shown between the groups in respect of the age of the patient (p⫽0.31), the main number of retrieved (p⫽0.132), fertilized (p⫽0.29) and transferred oocytes (p⫽0.73). Moreover, the fertilization rate, in all three investigated groups (64.0⫾2.9%, 81.0⫾3.1% and 60.0⫾4.5%, p⫽0.42), cleavage (60. 0⫾3.1%; 69.0⫾2.9% and 65.0⫾2.8%; p⫽0.73) and ongoing pregnancy rates (17.6%; 46.7%; 20.9% p⫽0.19) were similar. The concentration of NO and LIF in FF correlate negatively with fertilization (-0.012; -0.218) and cleavage rate (-0.10; -0.067) of the oocytes. CONCLUSION: The concentration of LIF, VEGF and NO in the investigated groups was similar. LIF and VEGF concentration was significantly higher in FF in comparison to serum, which indicated that these growth factors synthesised in the ovary and play an important role in ovarian physiology. Whereas, NO concentration was at the same levels in FF and serum. Besides, a negative correlation was shown between NO concentration in FF, serum and fertilization and cleavage rates of the fertilized oocytes. Supported by: None

Wednesday, October 19, 2005 4:45 p.m. O-272 Association of Killer Cell Immunoglobulin-Like Receptors Gene Polymorphisms With Unexplained Recurrent Spontaneous Abortion. S. Wang, Z. J. Chen, Y. R. Zhao, Y. L. Jiao, L. C. Wang, C. Y. Xu. The Center for Human Reproduction, Shandong University and Shandong Provincial Hospital, Jinan, China; Central Laboratory, Shandong Provincial Hospital, jinan, China. OBJECTIVE: To investigate the association of Killer cell immunoglobulin-like receptors (KIR) polymorphisms with the susceptibility to unexplained recurrent spontaneous abortion (RSA). DESIGN: Prospective study. MATERIALS AND METHODS: We chose 100 of childless women with two or more early abortions characterized as unexplained RSA after extended evaluation (exclusion of anatomical, chromosomal, hormonal, metabolic, infectious, autoimmune and thrombophilic etiologies) as patient

S112

Abstracts

group and 112 of healthy women as control. Genomic DNA from patients and controls were genotyped for presence or absence of the following KIR genes: 2DL1,2DL2,2DL3,2DL4,2DL5,2DS1,2DS2,2DS3, 2DS4, 2DS5, 3DL1,3DL2,3DL3,3DS1,and KIRZ. Genotyping was performed using polymerase chain reaction of sequence specific primers (PCR-SSP). The primers used for the detection of KIR loci were based on those have been previously described (Maureen P.et al. Susceptibility to Psoriatic Arthritis: Influence of Activating Killer Ig-Like Receptor Genes in the Absence of Specific HLA-C Alleles. J Immunol.2002; 169: 2818-2822).The genotypic frequencies of each KIRs was calculated as the percentage of positive numbers among all specimens. KIR genotypic frequency differences significance between patients and controls were tested by the two-tailed Fisher’s exact test (the SAS system).The different significance of average activating KIR numbers between RSA patients and control subjects were tested by t-test. RESULTS: The results showed increased numbers of activating KIR genes in patients compared with control subjects (p⫽0.002).Individuals who possessed three or less than three activating KIR gene were more frequently control subjects than patients. In contrast, individuals with more than three activating KIR genes were more frequently patients than control subjects (60.0% vs. 41.9%, respectively, p⫽0.009). Genotypic frequencies of KIR2DL5 (75.0% vs. 54.5%, p⫽0.003), KIR2DS2 (51.0% vs.32.1%, p⫽0.008) and KIR2DS1 (70.0% vs.50.0%, p⫽0.003) were much higher in patients with RSA compared to that in controls. Furthermore, individuals’ positive for activating KIR2DS3 and KIR2DS5 were more frequently patients than control subjects, but these differences did not reach statistical significance. No significant differences were detectable between patients and control subjects for activating KIR2DS4, KIR3DS1. CONCLUSION: The results suggest that the genetic variation at the KIR locus influences the susceptibility to unexplained RSA in Chinese population. The increased frequencies of KIR2DS2 and KIR2DS1 may be one of the reasons of unexplained RSA.The function of these three genes KIR2DS2, KIR2DS1 and KIR2DL5 need to be further investigated. It is conceivable that distinct combinations of KIR genes may have a combined effect on unexplained recurrent spontaneous abortions susceptibility. A genetic imbalance between activating and inhibitory KIR genes may influence the pathogenesis of RSA. Supported by: This work is supported by the National Natural Science Foundation of China (30371304).

Wednesday, October 19, 2005 5:00 p.m. O-273 Multiple Thrombophilic Gene Mutations Are Risk Factors for Implantation Failure. C. B. Coulam, R. S. Jeyendran, L. A. Fishel, R. G. Roussev. Center for Pregnancy Success, Chicago, IL; Andrology Laboratory Services, Chicago, IL; Millenova Immunology Laboratories, Chicago, IL. OBJECTIVE: While the role of inherited thrombophilia has been accepted as a cause of recurrent pregnancy complications, the contribution of mutated thrombophilic genes to implantation failure has not been studied. Proteins involved in fibrinolysis are necessary for trophoblast invasion into the endometrium. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent implantation failure after IVF/ET with fertile control women. DESIGN: Prospective observational study MATERIALS AND METHODS: 42 women with a history of implantation failure after IVF/ET and 20 fertile control women had buccal swabs taken for DNA analyses. The prevalence of 10 gene mutations (Factor V G1691A, Factor V H1299R (R2), Factor V Y1702C, Factor II Prothrombin G20210A, Factor XIII V34L, ␤-Fibrinogen -455G⬎A, PAI-1 4G/5G, HPA1 a/b (L33P), MTHFR C677T, MTHFR A1298C) was compared between women experiencing recurrent implantation failure after IVF/ET and controls. RESULTS: Women with a history of implantation failure after IVF/ET displayed a higher prevalence of PAI-1 4G/5G polymorphisms than controls (P⫽0.007). No differences in the frequency of the other specific gene mutations were detected. However, the prevalence of total gene mutations among patients with implantation failure was significantly higher than

Vol. 84, Suppl 1, September 2005