142 THURSDAY, SEPTEMBER 7

increased in the PVN of the maternal brain, but was not changed significantly in the PVN of the fetal brain. In conclusion, CRF and CRF- RI mRNA were already expressed in the rat fetal brain by day 2- of gestation. In the rat fetal brain, LPS challenge increased CRF mRNA expression but didn’t change CRF-RI mRNA expression in the PVN. Conclusion: It is suggested that CRF upregulates its own receptor mRNA in the PVN of maternal brain, but the fetal brain doesn’t yet have this mechanism.

P4.16.09 FACTOR V LEIDEN IN PATIENTS WITH RECURRENT FETAL LOSS M. Drabkova’, M. Vojtiskovaz, A. Vasku3, T. Burnog’, P. Janku’, K.Kankova3 ‘Dept. of Obstet. Gynecol., ‘Dept. of Genetic, 31nstitute of Pathological Physiology, Medical Faculty, Masaryk University, Brno, Czech Republic, Europe.

Objectives: To determine the association of Factor V Leiden mutation (FVL) frequency and recurrent fetal loss. Study Methods: A total of 445 women 17 to 45 years old were included in this case-control study. We examined the prevalence of the point mutation in the factor V gene (R 506 Q or Leiden). 138 unselected women with a history of one or more (mean 2.5, range 1 to 5) spontaneous abortion or stillbirth of unexplained etiology. Mutation analysis was performed by polymerase chain reaction and restriction analysis with Mln I. The control group were 307 unselected women who gave birth at our department. The significance of the difference in both group was tested by the chi-square test. Results: The frequency of the FVL in the case group was 14.5 % (20/138), in the control group 7.0 % (221307) (p>O.O05). There were no differences in the group of the women with one abortion and control group (p= 0.121). The frequency of FVL in the group with two abortions was 14.0 % (10172) (p>O.O05), in the group with three and more abortions 18.0 % (8145) (p>O.O05). Significant increase of homozygous genotyp was found in the case group, especially in the group of women with repeated loss. Conclusions: population Czech women, a statistically significant association of the Leiden mutation with recurrent fetal losses was found. The risk for recurrence of fetal loss tended to be greater in homozygous carriers.

P4.16.10 FLUORESCENCE IN SITU HYBRIDIZATION ON UNCULTURED AMNIOCYTES O.Torok (a), I. Zsupan (a), Zs. Buezico (a), R. Adany (b), M.Balazs (b) (a) Dept. OB/GYN (b) Dept. of Hygiene (c) University of Debrecen, Hungary.

Objectives: To demonstrate the advantages of FISH in prenatal diagnosis of aneuploidies Study Methods: In an initial study the results of FISH on uncultured amniocytes were compared with standard karyotyping in 178 second trimester cases. Subsequently FISH was applied in cases undergoing amniocentesis for abnormal ultrasound finding or if the amniocentesis had to be repeated for the suspicion of low-level true mosaicism in culture of amniocytes. The results were controlled by standard karyotyping, Results: The hybridization was successful in 98% of cases and a good correlation was found in between FISH and the standard cytogenetic method. In 7 fetuses with major structural anomalies and in 4 cases with severe IUGR the correct diagnosis could be set up by FISH two days after the ammocentesis. In 5 samples with the suspicion of true mosaicism at least 200 interphase ammocytes could be scored using FISH. In 2 cases mosaicism of “trisomy 20” and “21” could be ruled out with high probability. In 2 cases FISH supported the presence of low level mosaic “trisomy 21” and “X” monosomy. Conventional cytogenetic analysis in the second amniotic fluid specimen confirmed the FISH findings. In one case contrary to FISH standard karyotyping from the second amniotic fluid sample did not reveal mosaicism. Conclusion: Our results suggest that in case of severe IUGR and structural anomalies detected by ultrasound the rapid result of FISH can be considered for further management decisions. In the diagnosis of chromosomal mosaicisms the role of FISH needs further examinations.

P4.16.11 MOLECULAR BIOLOGICAL CHARACTERIZATION OF SOLUBLE PLACENTAL TISSUE PROTEINS: PPlSIGALECTIN, PP17b/MANNOSE-6-PHOSPHATE RECEPTOR TRANSPORTER AND PP18/BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE N.G. Dept. Biochemistry, University of P&s, P&s, Hungary H. Bohn, Behringwerke AG, MarburgiLahn, Germany G.N. Than, Dept. OBIGYN, University of P&s, P&s, Hungary B. Siimegi, Dept. Biochemistry, University of P&s, P&s, Hungary

Objectives: Soluble placental tissue proteins fJ’Ps) are synthesized in increased amounts &ring pregnancy and play an important role in the development of the fetus and placenta or in the maintenance of pegnancy. From the 26 different PPs four have been recently cloned and characterized by our research team. Study Methods: cDNAs encoding for PPs were isolated from a placental cDNA library with monospecific antisera and sequence analyzed. RNA expression in different tissues were examined by Northern-blot analyses. PP contents in human sera and in different human healthy and tumorous tissues were detected by chemiluminescence Western-blot analysis. Structural and functional characteristics of PPs were also investigated. Results: We have cloned and sequenced cDNAs encoding for pP13, PP18 and two members of the pP17 potein Emily @Pl7a, PP17b). By our results pP13 $ a new member of the :galactoside binding Stype animal lectin (gale&in) Emily, pP17b turned out to be a mannose-6-phosphate receptor @fPR) cargo potein while PP18 $ a branched-chain amino acid transaminase @CAT). Besides 6mctional godies, secondary and tertiary structural characteristics of the poteins have been computed. Specific expression patterns of the PPs in different healthy and timorous tissues have been detected, as well as the possibility of seromonitorization of cervical carcinoma ptients with PP17-assay has turned op. Conclusions: Seemingly PPs have oncodevelopmental functions: PP13igalectin may be involved in cell-cell and cell-matrix interactions; PP17blMPR transporter may function in HSV-2 infection and cervical cancer genesis; PP181BCAT may act in the cell cycle regulated by the c- “zyc proto-oncogene.

P4.16.12 PATERNITY IN PRENATAL PERIOD .I. Jovanovic-Privrodski (11, S. Romac (2), Z. Belopavlovic (3), M. Bogavac (3), A. Krstic (l), V. Manasijevic (2) Institute of Child and Youth Health Care, Novi Sad, Yugoslavia. Institute for Biology, Belgrade, Yugoslavia. University Clinic of Obstetrics and Gynecology, Novi Sad, Yugoslavia.

The problem of paternity can exist in prenatal period like in other parts of life. This problem can be solved successfully using the most frequently used genetic marker - DNA probe. Most frequently the blood test is used for analyzing DNA, but sometimes the other genetic markers like chorion villi and amnion in prenatal period or (post-mortem) hair can be used as well. In this paper we describe prenatal exclusion of paternity using DNA. A pregnant women (20 weeks of pregnancy) came to visit genetic counseling. She wanted to solve the problem of paternity of her fetus (she had two partners but the blood could be gotten only from one of them). The fetal blood was gotten by cordocenthesis. Six gene locuses were determined using AmpliType PM+DQAI kit for human identification DNA. In this case paternity for the presumed father was excluded because the fetus had two gene locuses, but the presumed father did not.

P4.16.13 PHOSPHORYLATED INSULIN-LIKE GROWTH FACTOR (IGF) -BINDING PROTEIN-l (IGFBP-1) INHIBITS WHILE NONPHOSPHORYLATED IGFBP-1 STIMULATES IGF-I-INDUCED AMINO ACID UmAKE BY CULTURED TROPHOBLAST CELLS M. K. Tsuchiya, M. Iwashita, Y. Nakamura, Dept. OBIGYN, Kyorin University, School of Medicine, Tokyo, Japan

Objectives: IGFBP-1 has been found to be phosphorylated and four to five phosphorylated forms (pIGFBP-1) and one nonphosphorylated form

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(npIGFBP-1) have been identified in various biological fluids. To elucidate the biological effects of these phosphoisoforms, we studied effects of pIGFBP-1 and npIGFBP-1 on amino acid uptake induced by IG-I using cultured trophoblast cells. Study Methods: IGFBP-1 was purified from mid term amniotic fluid using ammonium sulfate precipitation followed by phenyl-Sepharose column. Purified IGFBP-1 was further purified by DEAE-cellulose column in which pIGFBP-1 and npIGFBP-1 were separated. Trophoblast cells obtained from term pregnancy were incubated with indicated concentration of pIGFBP-1 or npIGFBP-1 for 24 hr and further incubated with 10 nM IGF-I for 3 hr. Cells were then incubated with 3H-a-amino isobutyric acid (SH-AIB) for 30 min. Cells were solubilized and the radioactivity in cells was counted by a scintillation counter. Results: Both pIGFBP-1 and npIGFBP-1 alone had no effect on SH-AIB uptake, however, pIGFBP-1 inhibited IGF-I stimulated SH-AIB uptake with ED 50 of 0.26 nM while npIGFBP-1 potentiated SH-AIB uptake with ED50 of 0.27 nM. Maternal IGF-I promotes fetal growth by stimulating nutrients transport in the placenta. Conclusions: As shown in this study, pIGFBP-1 inhibits while npIGFBP-1 stimulates this IGF-I action in the placenta. Thus, it is suggested that IGFBP-1 phosphoisoforms are also involved in fetal growth by modulating IGF-I action in the placenta.

P4.16.14 PRENATAL DETERMINATION OF FETAL SEX AND RH D STATUS BY DNA ANALYSIS OF MATERNAL PLASMA K. Hiraki, H. Masuzaki, D. Nakayama, T. Ishimaru, Dept. OBIGYN, Nagasaki Univ Sch Med, Nagasaki, Japan

Objectives: The recent demonstration of fetal DNA in maternal plasma raises the possibility that fetal DNA analysis may be possible with the use of maternal plasma, without cell separating techniques. We report use of PCR to detect fetal Y chromosome and Rh D gene from maternal plasma. Study Methods: We used QIAamp DNA Blood Mini Kit to extract DNA from maternal plasma. We used conventional PCR to amplify a Y- specific DNA sequence in plasma DNA samples from 26 pregnant women who had a gestational age of 14 to 41 weeks, and a Rh D sequence from three Rh D-negative pregnant women of 14 to 34 weeks. Results: Y sequences were detected in all of the 11 maternal plasma samples from women bearing male fetuses. None of the 15 women bearing female fetuses had positive results. The plasma samples obtained from three Rh D-negative women were positive for Rh D PCR analysis, which were concordant with the results of serologic analysis of the neonates. Conclusions: Prenatal determination of fetal sex and Rh D status with the use of maternal plasma can be performed rapidly and reliably. Fetal DNA in maternal plasma may be a valuable source of material for nonivasive prenatal diagnosis.

P4.16.15 PRELIMINARY RESULTS ON THE REGULATION OF THE HUMAN FSH RECEmOR PROMOTER ACTIVITY BY SELECTED E2F FAMILY MEMBERS L. Jakowicki (l), L. Putowski (l), C. Lee (2), W. Schillings (3), P. Reddy (2) (1) Dept. Surgical Gynecology, University School of Medicine, Lublin, Poland. (2) Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA. (3) Women’s Health Care Associates Newton, NJ, USA.

Objective: Since E2F’s transcriptional factors regulate expression of several proteins involved in cell cycle regulation we underwent studies concerning the influence of this factor on he FSH receptor promoter activity. Study Methods: DNA fragment containing FSH receptor promoter was sublcloned into pGL3 vector. Construct alone or together with expression vectors for E2F factor was transfected into cultured CHO cells. Additionally pRL-CMV vector was co-transfected in order to normalize transfection efficiency. Promoter activity was estimated by the measurement of firefly luciferase activity in cell lyzate. Obtained

results were normalized to the renilla activity driven by co-transfected pRL-CMV vector. Results: E2Fl decreased promoter activity (0,43*0,001). E2F4 and E2F5 increased promoter activity (1.29*0.04 and 1.66*0.20). Conclusions: Surprisingly increase (1.66 fold) in the activity caused by E2F5 is not so dramatic as it was noted previously for rat FSH-R promoter under the same condition. Lack of the E2F binding site in the human FSH-R receptor promoter, whereas it is present in rat gene, is possible explanation of these differences. E2Fl decreases the FSH-R promoter activity. It is more likely, the influence of E2F family members on human FSH-R promoter seems to be an indirect effect of this proteins.

P4.16.16 RELATION BETWEEN BIRTH WEIGm AND GENETIC POLYMORPHISM OF THE INSULIN GENE REGION IN JAPANESE Y. H. Osada, H. Sekimoto, K. Masuda, K. Seki, S. Sekiya, Dept. OBIGYN, Chiba University School of Medicine, Chiba, Japan.

Objective: Fetal development depends not only on the intrauterine environment provided by the mother, but is closely associated with genetic factors from both parents. We examined the relation between birth weight and gene polymorphism in the IDDM2 region including the insulin gene in Japanese. Study Methods: We enrolled 211 neonates delivered to Japanese mothers after 36 weeks of normal pregnancy. DNA was extracted from the umbilical blood and also maternal peripheral blood cells. Polymorphic regions in the insulin-like growth factor II (IGF2) and tyrosine hydroxylase (TH) genes adjacent to the insulin gene were amplified by PCR. Birth weights were converted into standard deviation unit (SDU) based on gestational week, sex and para status. Results: For IGF2 polymorphism, SDU of birth weight were not significantly different among the 3 genotypes (-/-,+/-,+/+). For TH polymorphism the frequencies of neonates with alleles 6 to 11 were 25.9,24.9. 4.7,39.8,4.5 and 0.3%, respectively. Allele 10 has been reported to have strong linkage with class III allele that is a repeating sequence upstream of the insulin gene. The SDU was significantly higher in individuals with allele 10 than other alleles (p=O.O16). SDU were significantly different between neonates with and those without allele 10 in the genotype (p=O.O007). When classified further by parental origin of allele 10, a significant difference in SDU was observed only between paternal derived allele 10 (+) and allele 10 (-) groups (p=O.O027). Conclusion: This study showed that gene expression of the insulin gene region is one of the factors that regulate development in fetal stage.

P4.16.17 RELATIONSHIP BETWEEN GESTATIONAL AGE AND FREQUENCY OF FETAL TROPHOBLASTS AND NUCLEATED ERYTHROCYTES IN MATERNAL PERIPHERAL BLOOD A. Tan (l), T-H. Lim (l), V.H.H. Goh (2), (1) Singapore General Hospital, Outram Road, Singapore, 169608, (2) National University Hospital, Singapore.

The relationship between gestational age and frequency of fetal cells in the maternal blood was studied in order to determine the optimal time for cell recovery. Immunomagnetic colloid system was used to enrich nucleated erythrocytes (NRBCs) and trophoblasts from 20ml of maternal blood obtained between 9 and 35 weeks gestation (N=41). Nested polymerase chain reaction (PCR) for the Y chromosome of enriched NRBCs and trophoblasts showed decreasing negative predictive values with increasing gestational age. The sensitivity and th overall frequency for correct fetal gender diagnosis were the lowest in the third trimester. Fluorescence in situ hybridisation (FISH) using XY DNA-specific probes was used to determine the fetal gender of trophoblasts-enriched fraction. The fetal origin of enriched NRBCs was determined using simultaneous immunophenotyping for fetal hemoglobin and FISH with XY probes. The mean number and mean percentage purity for both fetal trophoblasts and NRBCs showed decreasing values with increasing gestational age. However, statistical analysis showed no relationship between gestational age and frequency of fetal cells even though more fetal cells tend to exist during the first trimester. Nevertheless, the first