Low-molecular -weight heparin in pregnancies after ART -a retrospective study-.

INTRODUCTION: The utility of an antithrombotic prophylaxis in Assisted Reproductive Technologies (ART) is highly debated. It has been hypothesised that specific effects of heparin on the coagulation system during implantation can improve the number of clinical pregnancies and live births. MATERIALS AND METHODS: We studied a cohort of 327 women undergone at least 1 ART cycle before thrombophilia testing. Overall, a number of 751cycles was analysed. Low-Molecular-Weight Heparin (LMWH) and/or low-dose aspirin (ASA) were prescribed in 132 (17.6%) cycles. Furthermore, all the women underwent thrombophilia screening. RESULTS: The univariate analysis showed that LMWH with/without ASA was significantly associated with both the outcomes clinical pregnancy and live birth, while the use of ASA was not associated with live birth. The logistic regression showed that the use of LMWH was significantly associated with both the outcomes, clinical pregnancy (OR: 6.0, 95%CI: 2.8-15.6) and live birth (OR: 10.7, 95%CI: 3.2-36.1). The type of ART procedure significantly influenced the likelihood of achieving clinical pregnancy. CONCLUSIONS: Present findings suggest that LMWH alone or combined with ASA could have a role in fostering the implantation of embryos and improving the number of live births after ART.

Clinical Pregnancies and Live Births in women approaching ART: a follow-up analysis of 157 women after thrombophilia screening.

INTRODUCTION: The role of thrombophilia screening and antithrombotic therapy in unselected women undergone Assisted Reproductive Technologies (ART) is largely unknown. Nonetheless, in many Countries infertile women undergo thrombophilia screening and/or antithrombotic therapy. MATERIALS AND METHODS: We carried out a follow-up study. The original sample (n=1107) consisted of infertile women observed in 13 years. A cohort of 157 women with at least 1 cycle before thrombophilia test and 1 after test was investigated. All underwent thrombophilia screening; an antithrombotic treatment was prescribed in 216 out of 801 cycles. Clinical pregnancy and live birth rates were the main clinical objectives. RESULTS: Overall, 15 (9.6%) women carried thrombophilia. The Cox regression showed that LMWH alone or combined with ASA was significantly associated with the outcome "live birth" "live births" (p: 0.015, HR: 2.8, 95%CI: 1.2-6.6 for combined therapy), independently of the carriership of thrombophilia. Women with a lower number of attempts had a higher likelihood of delivering a live-born child using the combined therapy (p<0.001, HR: 0.7, 95%CI: 0.7-0.8), independently of the presence of thrombophilia. CONCLUSIONS: A potential benefit of LMWH in improving number of live births, independently of the presence of thrombophilia, is suggested. Universal thrombophilia screening before ART is not useful to discriminate women with a worse pregnancy prognosis.

The M2 haplotype in the ANXA5 gene is an independent risk factor for idiopathic small-for-gestational age newborns.

Hereditary thrombophilias can impair vascular placental functions and predispose to the birth of small-for-gestational age (SGA) babies. The placental anticoagulant protein annexin A5 (ANXA5) may contribute to this process. A functional haplotype (M2) within the ANXA5 gene is associated with fetal loss and venous thrombosis. This study investigated the prevalence of the M2 haplotype in a group of women with idiopathic SGA newborn babies. Seventy-eight women with at least one previous unexplained SGA birth and 195 controls all from Southern Italy were investigated. Hereditary thrombophilia was found in 13 (16.5%) cases and 21 (11%) controls (P < 0.05.). The M2 haplotype was found in 29% of cases (n = 23) and 15% of controls [n = 30; P = 0.001; OR = 2.3, 95% CI (1.17-4.48)]. Within the case group, 82.5% of the M2 haplotype carriers gave birth to babies with a birthweight below the 3rd percentile [P = 0.01; OR = 2.4, 95% CI (1.26-4.73)]. A logistic regression, corrected for age, parity and gravity showed that the M2 haplotype was independently associated with the delivery of an SGA new born [P = 0.029; OR = 2.6, 95% CI (1.1-6.0)]. In conclusion, the M2 haplotype of the ANXA5 gene confers a risk of delivering SGA babies.

Risk of obstetric and thromboembolic complications in family members of women with previous adverse obstetric outcomes carrying common inherited thombophilias.

BACKGROUND: Factor (F)V Leiden and the prothrombin 20210A mutation (PTm) are associated with the occurrence of obstetric complications, including pregnancy-related venous thromboembolism (VTE). It is not known whether family members of women with FV Leiden or PTm and previous obstetric complications have a higher risk of VTE or adverse obstetric outcomes. METHODS: A retrospective family study including 563 relatives of 177 women with previous adverse outcomes carrying FV Leiden or PTm, referred between April 1993 and June 2010. A history of obstetric complications and VTE was obtained. Prevalence of VTE and obstetric complications in relatives with and without inherited thrombophilias was compared. Adjusted odd ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression models that controlled for predictors (age, FV Leiden and PTm). RESULTS: Relatives carrying FV Leiden had a significant and independent risk for obstetric complications (OR: 1.98, 95% CI 1.03-3.83); this risk was not observed in the presence of PTm (OR: 1.03, 95% CI 0.46-2.32). The presence of FV Leiden or PTm in heterozygosis was significantly and independently associated with the occurrence of VTE (OR: 5.2, 95% CI: 1.70-15.91). Severe thrombophilias were strong risk factors for VTE (OR: 23.2, 95% CI: 6.0-89.85). Male gender was a significant and independent risk factor for VTE (OR: 3.49, 95% CI: 1.51-8.05). The risk did not change when relatives of women with a previous pregnancy-related VTE were excluded (OR: 3.49, 95% CI: 1.51-8.05). CONCLUSIONS: Knowledge of thrombophilia status may help to better define the obstetric and thromboembolic risks in asymptomatic family members of women who suffered from obstetric complications.

Subclinical atherosclerosis and genetic risk markers in healthy offspring of patients with premature myocardial infarction.

AIM: Healthy young subjects with parental history of premature myocardial infarction (PHPMI) might constitute a privileged population for the study of genetic risk markers (GRM) for atherosclerosis. Aim of this study was to evaluate which, if any, GRM atherosclerosis-associated in previous studies has increased prevalence in a selected population. METHODS: Twenty-four healthy young subjects (12 males and 12 females; mean age 18.0±8.0 years) with PHPMI and 24 age- (±1 year), sex-matched healthy subjects without PHPMI were enrolled in the study. They underwent: 1) fasting measurement of lipid profile, resting blood pressure and body mass index; 2) high resolution B-mode ultrasonographic evaluation of common carotid artery intima-media thickness (IMT); 3) evaluation of Single Nucleotide Polymorphisms (SNPs) for six candidate genes associated with preclinical atherosclerosis. RESULTS: Compared to controls, subjects with PHPMI had increased IMT of common carotid arteries (mean of combined sites: 0.535±0.171 mm versus 0.432± 0.133 mm in controls, P=0.017). Offspring of coronary patients showed an increased prevalence of the unfavourable chemochine (C-X-C motif) ligand 12 (CXCL12) SNP risk genotype (P=0.047). CONCLUSION: In healthy young subjects with PHPMI there is an increased prevalence of the unfavorable CXCL12 SNP risk genotype.

Impact of common thrombophilias and JAK2 V617F on pregnancy outcomes in unselected Italian women.

BACKGROUND: Although an association between thrombophilias and adverse pregnancy outcome has been shown, the influence of the most common inherited thrombophilias and the somatic mutation JAK2 V617F in determining an adverse outcome is questioned. OBJECTIVES: We examined the contribution of the factor V Leiden (FVL), the prothrombin G20210A (PTm) and the somatic JAK2 V617F mutations to adverse pregnancy outcome in an unselected cohort of pregnant women. PATIENTS/METHODS: During the study period, 5345 pregnant women were admitted to the 14 hospitals of the five provinces of the Campania region (Italy). Of these, 3097 samples were investigated and obstetric history collected. The presence of the FVL, PTm, and JAK2 V617F mutation was prospectively determined by polymerase chain reaction followed by TaqMan SNP genotyping assays. RESULTS AND CONCLUSIONS: We identified 119 (3.8%) women that carried FVL and 138 (4.4%) with the PTm. Only 4 (0.1%) women carried both mutations. Only one woman tested positive for the JAK2 V617F somatic mutation. The prevalence of a previous history of an adverse pregnancy outcome was similar in women with common thrombophilias as compared to those without. In the current pregnancy, there was no association of any of the genetic markers considered with any of the adverse outcomes investigated. Carriership of FVL or PTm showed a positive trend with delivery of a small for gestational age newborn (OR: 1.5, 95% CI: 0.9-2.5). Pregnancy outcomes in asymptomatic women with inherited thrombophilias are often uneventful. Therefore, in women at low-risk of an adverse pregnancy, neither screening for common thrombophilias nor administration of routine thromboprophylaxis are warranted.

[Anticoagulant prophylaxis in women affected by thrombophilia and previous obstetric complications]. / Profilassi antitrombotica in donne con diagnosi di trombofilia e precedenti complicanze ostetriche.

Pregnancy is a condition of excessive clotting due to a decrease of some coagulation factors and a reduction of anticoagulant proteins, such as protein S. It is known that the causes of congenital or acquired thrombophilia may be associated with an increased risk of venous thromboembolism during pregnancy and/or obstetric complications, such early or late fetal loss, intrauterine fetal deaths, pre-eclampsia, fetal growth restriction. During pregnancy the use of a prophylaxis with antithrombotic drugs is considered at present a promising opportunity to significantly reduce the prevalence of thromboembolic complications, improving maternal and fetal outcomes. This article is a review to most recent evidence of pregnant anticoagulant prophylaxis in women with previous thromboembolic events.

The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis.

BACKGROUND: Myeloproliferative disorders (MPDs) represent a risk factor for thrombosis in the portal, mesenteric, and hepatic districts. OBJECTIVE: We aimed to assess whether the Janus kinase 2 (JAK2) V617F mutation, an acquired mutation that occurs in MPD patients, is a risk factor for portal and mesenteric venous thrombosis (PMVT) independently of the presence of overt MPDs. PATIENTS AND METHODS: The medical histories of 99 patients presenting with PMVT were obtained. The presence of the JAK2 V617F and VHL 598C > T mutations was determined by polymerase chain reaction followed by restriction enzyme analysis and direct cycle sequence analysis. RESULTS: Over a 10-year period of observation, of the 99 patients presenting with PMVT, the JAK2 V617F mutation was detected in heterozygous state in 17 individuals [17.2%; 95% confidence interval (95% CI) 10.9-25.9]. None of the patients presenting with the JAK2 V617F mutation carried an inherited thrombophilic risk factor. Seven patients with (43.8%; 95% CI 19.8-70.1) and two without (2.4%; 95% CI 0.3-8.4) the JAK2 V617F mutation had a diagnosis of MPD at the occurrence of the venous thrombotic event. After a median follow-up of 41 months (range 3-114 months), three out of the 10 patients carrying the JAK2 V617F mutation were then diagnosed as having idiopathic myelofibrosis (n = 2) or polycythemia vera (n = 1), whereas in seven patients a MPD was not detected. Two of the 83 patients without the JAK2 V617F mutation went on to develop MPDs. CONCLUSIONS: Determination of the JAK2 V617F mutation may contribute to the search for genetic determinants of PMVT and may be useful to recognize patients who should be carefully observed for the subsequent development of overt MPDs.

Low protein Z levels and risk of occurrence of deep vein thrombosis.

BACKGROUND: Protein Z (PZ) serves as a cofactor for activated factor X inhibition by the PZ-dependent protease inhibitor. In vivo and in vitro studies aimed at investigating the role of PZ levels in venous thombosis have produced conflicting results. OBJECTIVES: We investigated whether reduced PZ levels and PZ gene common variants are associated deep vein thrombosis (DVT). PATIENTS AND METHODS: In 197 patients with DVT and in 197 age-matched and sex-matched controls, PZ plasma levels and gene polymorphisms were evaluated by means of an enzyme-linked immunosorbent assay and direct cycle sequence analysis. RESULTS: Similar PZ levels were found in controls (1.44; SD 0.63 microg mL-1) and in patients (1.44; SD 0.96 microg mL-1). The incidence of PZ levels below the 5.0 (0.52 microg mL-1) or the 2.5 percentile of controls (0.47 microg mL-1) was higher in patients (10.2% and 8.7%, respectively) than in controls {4.1% [odds ratio (OR) 2.7, 95% confidence interval (CI) 1.2-7.3], and 2.0% (OR 4.6, 95% CI 1.5-13.9), respectively}. This relationship was independent of the effect of age, sex, and factor V Leiden and FII A(20210) alleles [OR 2.8 (95% CI 1.1-7.3), and OR 4.9 (95% CI 1.4-17.3)]. PZ levels were associated with the intron C G-42A and the intron F G79A polymorphisms in cases (r2=0.129) and in controls (r2=0.140). However, frequencies of the PZ gene polymorphisms were similar in the two groups and were not associated with very low PZ levels. CONCLUSIONS: The present data suggest an association between very low PZ plasma levels and the occurrence of DVT, with PZ gene polymorphisms contributing little to this relationship.

Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia.

Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.

Age and homocysteine plasma levels are risk factors for thrombotic complications after ovarian stimulation.

BACKGROUND: The magnitude of thrombotic risk during ovarian stimulation cycles is not known. We calculated the magnitude of thrombotic risk in a cohort of women starting a new cycle of ovarian stimulation and investigated the role of inherited and acquired thrombophilia for these events. METHODS: This is an observational study involving outpatients of a clinical research centre. Consecutive women undergoing ovarian stimulation (n = 305) were enrolled. Blood samples for studying inherited and acquired thrombophilia were obtained > or = 2 months after the last cycle of treatment. Odds ratios (OR) and confidence intervals (CI) were determined for markers significantly associated with thrombotic events. Blood samples were analysed for inherited and acquired causes of thrombophilia (antithrombin, protein C, protein S, antiphospholipid antibodies, the Factor V Leiden and FIIA20210 mutations, the TT677 MTHFR genotype, and homocysteine plasma levels). RESULTS: Thrombotic events were observed in 4/747 cycles of ovarian stimulation, with a prevalence of 0.5%, corresponding to 1.6 per 100 000 cycles/woman. Age > or = 39 years and homocysteine plasma levels above the 97.5 percentile were significantly associated with thrombotic events during IVF [OR 15.2 (95% CI 2.0-115.0) and 14.4 (1.5-141.3) respectively]. CONCLUSIONS: Age > or = 39 years and mild hyperhomocysteinaemia are strongly associated with the occurrence of thrombotic events during IVF.

Is steam sterilization really making any difference in dialysis-induced cytokine release?

Ethylene oxide (ETO) is presently the most commonly used sterilization method for medical devices. Although alternative sterilization modes such as steam sterilization have been suggested, the effect of steam on dialysis-induced cytokine release is unknown. We enrolled 9 patients on chronic hemodialysis and evaluated at different intervals IL-1beta production while treated with ETO (NC 1785-Bellco) and steam sterilized NC 1785S-Bellco) Synthetically Modified Cellulose (SMC). A basal test during treatment with NC 1785 was performed (A); the same test was set up 4 weeks after treatment with NC 1785S (B) and, lastly, 4 weeks after returning to NC 1785 (C). Peripheral blood mononuclear cells (PBMC) were purified before and after the dialysis session, were isolated on a Ficoll/Hypaque gradient and incubated for 24 h. Spontaneous IL-1beta release was evaluated in the supernatant and in the lysate. In A, IL-1beta levels were (in pg/ml/10(6) cells, in supematant and lysate, respectively): 5.8 +/- 4.8 and 7.6+/-5.2 in pre-HD and 4.68 +/- 3.6 and 9.7 +/- 6.65 in post-HD. These levels showed a clear reduction in B: 2.5 +/- 2.2 and 4.4 +/- 3.1 in pre-HD, and 4.35+/- 6.6 and 7.52 +/- 7.22 in post-HD. In the C test, 4 weeks after the return to the ETO membrane, IL-1beta levels remained unchanged: 2.9 +/- 1.8 and 4.5 +/- 3.1 in pre-HD; and 2.6 +/- 3 and 5.7 +/- 6.6 in post-HD. Statistical analysis showed significant changes in the pre-HD levels both in supematant (p < 0.04) and in lysate (p < 0.04). Steam sterilization of SMC induced a lower spontaneous IL-1beta release, but this effect was not statistically significant due to the large inter-individual variation. Hence, contrary to claims of better biocompatibility, steam sterilization does not result in a reduced production of pro-inflammatory IL-1beta.

ACE gene polymorphism and insulin action in older subjects and healthy centenarians.

OBJECTIVES: To evaluate the possible relationship between angiotensin-converting enzyme (ACE) insertion-deletion (ID) genotype and insulin resistance in a population of healthy older Italian subjects. DESIGN: Prospective recruitment of a convenience sample. PARTICIPANTS: One hundred twenty-five subjects age 62 to 105 in good health and not taking any drug known to interfere with glucose metabolism. RESULTS: In the sample population, the relative frequencies of the ACE genotypes deletion-deletion (DD) (0.424), ID (0.400), and insertion-insertion (II) (0.176) were not significantly different from values predicted by Hardy-Weinberg equilibrium. The genotype distribution was similar in men and women. Subjects carrying the II genotype had a higher FPG (P <.001) and FPI (P <.001) than did subjects with DD or ID genotype. Subjects with II genotype also had a significantly higher HOMA index than did subjects with DD or ID genotype (P for trend <.002). In a multivariate stepwise regression analysis, the ACE ID polymorphism was significantly and independently associated with the HOMA index (P <.001). The same result was confirmed performing multivariate analysis in the younger group and centenarians separately. CONCLUSIONS: In an older population, the presence of II ACE genotype is associated with a high degree of insulin resistance independent of other anthropometric variables known to interfere with insulin action; this association is significant in both the younger subjects and the centenarians.

Familial thrombophilia and the occurrence of fetal growth restriction.

BACKGROUND AND OBJECTIVES: To evaluate the association between unexplained or gestational-hypertension-associated fetal growth restriction (FGR) and factor V Leiden, prothrombin A20210 mutations, and methylenetetrahydrofolate reductase (MTHFR) TT 677 genotype. DESIGN AND METHODS: Sixty-one women with a previous history of FGR and 93 parous women with uneventful pregnancies from the same ethnic background were investigated for the presence of factor V (FV) Leiden, prothrombin A20210 mutations, and MTHFR TT 677 genotype. Moreover, antiphospholipid antibodies, antithrombin, protein C, and total and free protein S antigen were determined in all patients. RESULTS: Among the controls, 2 (2.2%) carried the FV Leiden mutation, 19 (20.4%) were TT MTHFR homozygotes and 1 (1.6%) carried the prothrombin A20210 allele. The FV Leiden mutation was present in 8 women with FGR (13.1%, OR: 6.9, 95%CI 1.4-33.5), the TT MTHFR homozygosity in 17 (27.8%, OR: 1.5, 95%CI 0.7-3.2) and the A20210 prothrombin allele in 7 (11.5%, OR: 5.9, 95%CI 1.2-29.4). In six cases (9.8%) there was coexistence of more than one mutation (2 had the FV Leiden and the TT MTHFR genotype and 4 carried the A20210 prothrombin allele and TT MTHFR genotype). A logistic regression analysis showed that FV Leiden and A20210 prothrombin mutations were independently associated with the occurrence of FGR. INTERPRETATION AND CONCLUSIONS: Present data indicate an association between prothrombotic genetic factors and FGR.

[Hypersensitivity to oral anticoagulants: report of a case]. / Ipersensibilità agli anticoagulanti orali: descrizione di un caso.

We report a case regarding a 71 year-old Caucasian man with NYHA functional class III congestive heart failure, who was under warfarin treatment due to left ventricular thrombosis. After a few days, although the drug was not overdosed, the INR increased up to 11.68. Normal values were reestablished only after a 20-day pharmacological wash-out. Surprisingly, no episode of major or minor bleeding occurred. Gene typing of cytochrome P450 CYP2C9, a liver enzyme responsible for warfarin metabolism, showed that the patient was a carrier of both the mutant alleles (CYP2C9*2/*3) of this enzyme. This genetic defect caused a reduced catabolism of S-warfarin and excessive anticoagulation.