1. RECURRENT PREGNANCY LOSS (RPL) AND ITS RELATION TO COMBINED PARENTAL THROMBOPHILIC GENE MUTATIONS DOÇ. DR. A. GONCA İ M İ R YEN İ CESU CUMHUR İ YET ÜN İ VERS İ TES İ KADIN HASTALIKLARI VE DO Ğ UM AD. 18.04.2015G. Yenicesu UTD 20111

2. RPL RPL is classically defined as the occurrence of three or more consecutive losses of clinically recognized pregnancies prior to the 20th week of gestation (ectopic and molar pregnancies are not included). The ASRM defines RPL as two or more failed pregnancies (by ultrasound or histopathological examination) and suggests some assessment after each loss with a thorough evaluation after three or more losses. Practice Committee of the American Society for Reproductive Medicine. Definitions of infertility and recurrent pregnancy loss. Fertil Steril 2008; 89:1603. 18.04.2015G. Yenicesu UTD 20112

3. Approximately 15 % of pregnant women experience sporadic loss of a clinically recognized pregnancy. Just 2 % of pregnant women experience two consecutive pregnancy losses and only 0.4 to 1% have three consecutive pregnancy losses. 18.04.2015G. Yenicesu UTD 20113

4. Recurrent pregnancy loss (RPL) is a significant clinical problem with many etiologies. In recent years there are many studies that examined the incidence of specific thrombophilic gene mutations in couples with unexplained RPL. 18.04.2015G. Yenicesu UTD 20114

5. Thrombophilic mutations such as; FV L, prothrombin G20210A, MTHFR lead to enhanced blood coagulation and are involved in folic acid metabolism which influences DNA methylation. An association with RPL has also been reported for the Val34Leu polymorphism of the FXIII gene by leading to fibrin degradation. Wang XP, 2004; 39:238–241, Wolf CE 2003, Coulam CB 2006, Muszbek L 2000. 18.04.2015G. Yenicesu UTD 20115

6. Apo E gene encodes three alternative alleles –E2, E3 and E4 that play a crucial role in cholesterol - triglycerides metabolism(27). Homozygous individuals harbouring the E4 allele have a higher total cholesterol level, hence cardiovascular risk (1,28,29). 18.04.2015G. Yenicesu UTD 20116

7. We have previously reported the high frequency of ApoE4 gene polymorphisms as a risk factor for RPL. Goodman et al also reported the high frequency of Apo E4 genotype in women suffering from RPL. Yenicesu GI, et al; A prospective case-control study analyzes 12 thrombophilic gene mutations in Turkish couples with recurrent pregnancy loss. A J Reprod Immu 2010; 63(2):126-136. Goodman C. Am J Reprod Immunol 2009; 61:34–38. 18.04.2015G. Yenicesu UTD 20117

8. In this study, we compare the prevalence of 12 thrombophilic gene mutations in a series of patients with two or more consecutive, first-trimester losses and fertile couples with no history of miscarriages. 18.04.2015G. Yenicesu UTD 20118

9. The study was performed in gynecology clinics and genetic departments at Cumhuriyet University Hospital and Canakkale Onsekiz Mart University Hospital. The 543 women with RPL ( two or more consecutive RPL at 5–12 weeks of gestation) 327 RPL male partners 106 couples with no history of RPL and at least one normal pregnancy 18.04.2015G. Yenicesu UTD 20119

10. Chromosome analyses were performed in all RPL and control couples. Women with RPL were examined by US or HSG, for immunologic risk factors including APA, ANA, antithyroid antibodies, and LA. 18.04.2015G. Yenicesu UTD 201110

11. Exclusion criteria: anatomic abnormalities endocrinologic dysfunction autoimmune disease urogenital infections 18.04.2015G. Yenicesu UTD 201111

12. Trombophilic genes 1. factor V G1691A (FV Leiden) 2. factor V H1299R 3. factor II prothrombin G20210A 4. factor XIII V34L 5. β -fibrinogen − 455G>A 6. plasminogen activator inhibitor-1 (PAI-1) 7. GPIIIa L33P (HPA-1 a/b L33P) 8. MTHRF C677T 9. MTHFR A1298C 10. ACE I/D 11. Apo B R3500Q 12. Apo E (E2, E3, E4) 18.04.2015G. Yenicesu UTD 201112

13. Total genomic DNA was extracted by the Magna Pure Compact (Roche) (Invitek). Twelve thrombophilic genes were simultaneously amplified in a biotin- labelled single multiplex amplification reaction which is based on the reverse- hybridization principle and by Real Time PCR, LightCycler 2.0 methods (Roche). 18.04.2015G. Yenicesu UTD 201113

14. The frequencies of homozygous and heterozygous thrombophilic gene mutations, the frequencies of allelic mutations in female and male participants a heterozygous mutation was considered as one gene mutation a homozygous mutation was considered as two gene mutations. 18.04.2015G. Yenicesu UTD 201114

15. We have previously reported that 3/12 thrombophilic mutations (PAI-1 4G⁄ 5G, FVL, and homozygous MTHFR C667T, correlated significantly with RPL when compared to fertile controls. Yenicesu GI, et al; A prospective case-control study analyzes 12 thrombophilic gene mutations in Turkish couples with recurrent pregnancy loss. A J Reprod Immu 2010; 63(2):126-136. 18.04.2015G. Yenicesu UTD 201115

16. Tablo 1. Demographic data 18.04.2015G. Yenicesu UTD 201116

17. SONUÇLAR The frequencies of allelic mutations in women for FVL, FVR2, MTHFR C677T, MTHFR A1298C, PAI 1 5G/4G, ACE I/D and APOE in the RPL women (20.02%, 13.3%, 57.4%, 63.0%, 83.2%, 86.9% and 35.0%) respectively were significantly higher than in the fertile control women respectively. The rest of thrombophilic gene mutations were the same as control women. 18.04.2015G. Yenicesu UTD 201117

18. FVL-FVR2 18.04.2015G. Yenicesu UTD 201118

19. FVL-FVR2 18.04.2015G. Yenicesu UTD 201119

20. MTHFR 18.04.2015G. Yenicesu UTD 201120

21. MTHFR 18.04.2015G. Yenicesu UTD 201121

22. The frequencies of allelic mutations in men for FVL, FVR2, MTHFR C677T, MTHFR A1298C, PAI 1 5G/4G, ACE I/D and APOE in the RPL men (19.2%, 17.8%, 55.6%, 56.0%, 89.3%, 81.3% and 36.7%, respectively) were statistically higher than in the fertile control males. 18.04.2015G. Yenicesu UTD 201122

23. PAI 18.04.2015G. Yenicesu UTD 201123

24. PAI 18.04.2015G. Yenicesu UTD 201124

25. APOE E3/E2/E4 18.04.2015G. Yenicesu UTD 201125

26. APOE E3/E2/E4 18.04.2015G. Yenicesu UTD 201126

27. The total frequencies of allelic mutations in RPL subjects were due to FVL, 19.7% (176/870), FVR2, 15.0% (131/870), MTHFR C677T, 56.7% (489/870), MTHFR A1298C, 60.3% (525/870), PAI 1 4G/5G, 85.5% (744/870), ACE I/D 84.8 (738/870), and APOE 35.6% (310/870) respectively. The total frequencies of allelic mutations in fertile couple subjects were due to FVL, 2.0%(5/212), FVR2, 1.4%(3/212), MTHFR C677T, 26.9%(57/212), MTHFR A1298C, 34.0%(72/212), PAI 1 4G/5G, 66.0%(140/212), ACE I/D 68.3(145/212), and APOE 21.3% (45/212) respectively. 18.04.2015G. Yenicesu UTD 201127

28. To further define the relationship between some specific target genes with RPL, we also evaluated the allelic frequencies of 4G for PAI-1, D for ACE, C677T and A1298C for MTHFR and E4 for Apo E in two different populations of women experiencing RPL. 18.04.2015G. Yenicesu UTD 201128

29. The frequency of E3/E2, E3/E4 and E2/E4 profiles for Apo E were 20.9%,12.8% and 1.9% respectively in the RPL couples, and 10.9 %, 10.4 % and 0.0 % in fertile couples. while ApoE3 allele frequency was 0.81 in RPL and 0.89 in fertile couples the ApoE2 frequency was 0.11 for RPL and 0.06 for fertile couples. ApoE4 allele frequency was detected as 0.08 for RPL and 0.05 for fertile couples. 18.04.2015G. Yenicesu UTD 201129

30. The frequencies of heterozygous mutations for PAI-1, MTHFR C677T, MTHFR A1298C, ACE I/D and Apo E were significantly higher in RPL couples when compared to fertile control couples. 18.04.2015G. Yenicesu UTD 201130

31. The frequency of homozygous mutation for PAI-1 was significantly higher in RPL men compared with control men. The frequency of homozygous mutation for MTHFR C677T was 15.9% in the RPL men, but no homozygous mutation was detected in the control men. 18.04.2015G. Yenicesu UTD 201131

32. The presented preliminary results showed that ApoE2 allele profile may contribute to the thrombophilic risk factors for RPL. 18.04.2015G. Yenicesu UTD 201132

33. Increased T allele frequency of C677T polymorphism in MTHFR gene was detected in RPL patients (0.36) when compared to fertile couples (0.13). Also present results showed increased C allele frequency of A1298C mutation in RPL patients (0.37) when compared to the fertile couples (0.17). Difference in both common mutated alleles in the MTHFR gene were statistically significant. 18.04.2015G. Yenicesu UTD 201133

34. For the ACE I/D gene, the frequency of heterozygous and homozygous mutations in RPL couples were significantly higher than the fertile couples. D allele frequency for ACE was detected as 0.62 for RPL and 0.44 for fertile couples(P<0.0001). Current results also showed increased 4G allele frequency for PAI in RPL (0.55) when compared to healthy controls (0.38). 18.04.2015G. Yenicesu UTD 201134

35. CONCLUSION Briefly, the homozygosity of 4G in PAI-1 and MTHFR C677T genes in women with recurrent pregnancy loss, and heterozygosity of FV Leiden, FVR2, ACE and Apo E2 and ApoE4 genes in both parents play crucial roles in RPL and should be considered as risk factors in RPL. 18.04.2015G. Yenicesu UTD 201135

36. CONCLUSION Current results showed the maternal mutated thrombophilic genes’ profiles mainly have a potential risk for RPL but paternal mutation profiles in specific genes may also have a combined effect on RPL phenomenon. Study of mutations in a wide range of thrombophilic genes associated with the disease might be clinically useful as a marker to assess couple’s risk for RPL. 18.04.2015G. Yenicesu UTD 201136