'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Antibodies to phosphatidylserine/prothrombin (aPS/PT) enhanced the diagnostic performance in Chinese patients with antiphospholipid syndrome.

BACKGROUND: Increasing evidence has highlighted the role of non-criteria antiphospholipid antibodies (aPLs) as important supplements to the current criteria aPLs for the diagnosis of antiphospholipid syndrome (APS). In this retrospective study, we evaluated the clinical relevance of antibodies to phosphatidylserine/prothrombin (aPS/PT) in Chinese patients with APS. METHODS: A total of 441 subjects were tested, including 101 patients with primary APS (PAPS), 140 patients with secondary APS (SAPS), 161 disease controls (DCs) and 39 healthy controls (HCs). Serum IgG/IgM aPS/PT was determined by ELISA. RESULTS: The levels of IgG/IgM aPS/PT were significantly increased in patients with APS compared with DCs and HCs. IgG and IgM aPS/PT were present in 29.7% and 54.5% of PAPS, and 42.1% and 53.6% of SAPS, respectively. For diagnosis of APS, IgG aCL exhibited the highest positive likelihood ratio (LR+) of 21.60, followed by LA (13.84), IgG aß2GP1 (9.19) and IgG aPS/PT (8.49). aPS/PT was detected in 13.3% of seronegative PAPS patients and 31.3% of seronegative SAPS patients. LA exhibited the highest OR of 3.64 in identifying patients with thrombosis, followed by IgG aCL (OR, 2.63), IgG aPS/PT (OR, 2.55) and IgG aß2GP1 (OR, 2.33). LA and IgG aCL were correlated with both arterial and venous thrombosis, whereas IgG aPS/PT and IgG aß2GP1 correlated with venous or arterial thrombosis, respectively. CONCLUSIONS: Our findings suggest that the inclusion of IgG/IgM aPS/PT may enhance the diagnostic performance for APS, especially in those in whom APS is highly suspected, but conventional aPLs are repeatedly negative. In addition, IgG aPS/PT may contribute to identify patients at risk of thrombosis.

Interactions between rivaroxaban and antiphospholipid antibodies in thrombotic antiphospholipid syndrome.

INTRODUCTION: Rivaroxaban can affect lupus anticoagulant (LA) testing and antiphospholipid antibodies (aPL) may interfere with the anticoagulant action of rivaroxaban. AIMS: To establish the influence of rivaroxaban on LA detection and of aPL on the anticoagulant action of rivaroxaban. METHODS: Rivaroxaban and 52 IgG preparations (20 LA+ve, 12 LA-ve thrombotic antiphospholipid syndrome [APS] patients, and 20 normal controls [NC]) were spiked into pooled normal plasma (PNP) for relevant studies. LA detection was also studied in APS patients receiving rivaroxaban 20 mg once daily. RESULTS: In vitro spiking of samples with rivaroxaban showed no false positive LA with Textarin time, Taipan venom time/Ecarin clotting time (TVT/ECT), dilute prothrombin time (dPT) and in-house dilute Russell's viper venom time (DRVVT), but false positives in the majority of NC and LA negative IgG with two commercial DRVVT reagents at 250 ng/mL but not 50 ng/mL rivaroxaban. Ex vivo studies: six LA+ve patients on rivaroxaban remained LA positive with TVT/ECT and DRVVT at peak (162-278 ng/mL) and trough (30-85 ng/mL) rivaroxaban levels. Six LA-ve patients became (apparently) LA+ve with two DRVVT reagents (test/confirm ratio median [confidence interval], 1.6 [1.3-1.8], 1.6 [1.4-1.9]) but not with TVT/ECT at peak rivaroxaban levels, and remained LA-ve with both DRVVT reagents and TVT/ECT at trough levels. aPL positive IgG spiking of PNP had no effect on rivaroxaban's anticoagulant action on thrombin generation or rivaroxaban anti-Xa levels. CONCLUSIONS: The TVT/ECT ratio and Textarin time were not affected even at peak rivaroxaban levels, enabling detection of LA ex vivo. aPL had no effects on rivaroxaban's anticoagulant action in vitro.

False-positive lupus anticoagulant in patients receiving rivaroxaban: 24 h since the last dose are needed to exclude antiphospholipid syndrome.

Rivaroxaban, a direct factor Xa inhibitor, affects laboratory clotting tests. We report here 10 venous thromboembolism patients with false-positive lupus anticoagulant during rivaroxaban therapy. Two dilute Russell Viper Venom time (dRVVT)-based integrated assays, HemosIL dRVVT Screen/HemosIL dRVVT Confirm (Instrumentation Laboratory, LA1/LA2 (Siemens, Germany) and LA1/LA2 (Siemens, Marburg, Germany), showed that the patients were lupus anticoagulant-positive. Antiphospholipid antibodies were negative except for one patient. Screening activated partial thromboplastin time-based assay PTT lupus anticoagulant was lupus anticoagulant-positive, whereas the confirmatory Staclot lupus anticoagulant (both Diagnostica Stago, France) was lupus anticoagulant-negative. Re-examination after discontinuation of rivaroxaban (>24 h) ruled out the presence of lupus anticoagulant. Our data indicate that to reliably evaluate lupus anticoagulant in patients on rivaroxaban, blood should be drawn 24 h after the last dose.

Elevated serum anti-phosphatidylcholine IgG antibodies in patients with influenza vaccination-associated optic neuritis.

INTRODUCTION: Because the optic nerve is mainly comprised from phospholipids such as phosphatidylcholine, the association between optic neuritis, anti-phospholipids antibodies and vaccination was examined. SUBJECTS: Two female pediatric patients suddenly presented bilateral optic neuritis after administration of trivalent inactivated influenza vaccine. METHODS: These two patients and another 11 patients with central nervous system demyelinating diseases were examined these anti-phospholipids antibodies. And immune histopathology was examined using serum derived from a patient with optic neuritis. RESULTS: High serum titer of anti-phosphatidylcholine antibody levels were detected during acute phase in patients with optic neuritis. The patient's serum IgG antibodies were found to have stained the capillary endotheliums in the preserved autopsied optic nerve. Patients with optic neuritis had significantly elevated serum levels of anti-phosphatidylcholine antibody in comparison to the other patients without optic neuritis. CONCLUSION: Anti-phosphatidylcholine antibodies may be one of the causes of optic neuritis.

Pandemic influenza immunization in primary antiphospholipid syndrome (PAPS): a trigger to thrombosis and autoantibody production?

OBJECTIVE: The objective of this report is to conduct short- and long-term evaluation of a large panel of antiphospholipid (aPL) autoantibodies following pandemic influenza A/H1N1 non-adjuvant vaccine in primary antiphospholipid syndrome (PAPS) patients and healthy controls. METHODS: Forty-five PAPS and 33 healthy controls were immunized with H1N1 vaccine. They were prospectively assessed at pre-vaccination, and three weeks and six months after vaccination. aPL autoantibodies were determined by an enzyme-linked immunosorbent assay (ELISA) and included IgG/IgM: anticardiolipin (aCL), anti-beta2glycoprotein I (anti-ß2GPI); anti-annexin V, anti-phosphatidyl serine and anti-prothrombin antibodies. Anti-Sm was determined by ELISA and anti-double-stranded DNA (anti-dsDNA) by indirect immunofluorescence. Arterial and venous thrombosis were also clinically assessed. RESULTS: Pre-vaccination frequency of at least one aPL antibody was significantly higher in PAPS patients versus controls (58% vs. 24%, p = 0.0052). The overall frequencies of aPL antibody at pre-vaccination, and three weeks and six months after immunization remained unchanged in patients (p = 0.89) and controls (p = 0.83). The frequency of each antibody specificity for patients and controls remained stable in the three evaluated periods (p > 0.05). At three weeks, two PAPS patients developed a new but transient aPL antibody (aCL IgG and IgM), whereas at six months new aPL antibodies were observed in six PAPS patients and none had high titer. Anti-Sm and anti-dsDNA autoantibodies were uniformly negative and no new arterial or venous thrombosis were observed throughout the study. CONCLUSIONS: This is the first study to demonstrate that pandemic influenza vaccine in PAPS patients does not trigger short- and long-term thrombosis or a significant production of aPL-related antibodies (ClinicalTrials.gov, #NCT01151644).

Livedoid vasculopathy: clinical features and treatment in 24 Chinese patients.

Livedo vasculopathy (LV) is a chronic cutaneous disorder characterised by recurrent, painful ulceration ending in stellate scars. We have conducted a retrospective study of clinical features and treatment response of LV in 24 Chinese patients. LV occurred more frequently in women (male:female ratio 1:3). The peak age at onset of disease ranged from 14 to 20 years, younger than previously published data. 87.5% of the patients (21/24) showed significant summer exacerbation with ulcer formation. Out of 24 patients tested, 14 (58.3%) had positive antiphospholipid antibodies. Ten out of 14 patients (71.4%) were tested to be hypersensitive to multivalent insect antigens. Combinative anti-inflammatory therapy with steroids, tetracycline and Tripterygium glycosides plus antiplatelet/profibrinolytic drugs promoted quick healing of ulcer and reduce recurrence. The younger age of disease presentation and significant summer exacerbation are 2 novel clinical features observed in this study. These findings suggest that apart from procoagulation other risk factors may contribute significantly to the pathogenesis of LV. Although antiplatelet/profibrinolytic drugs are deemed as a first line therapy for LV, anti-inflammatory medications such as steroids, tetracycline and Tripterygium glycosides, from our experiences, are indispensable, especially for acute, ulcerative stage of disease.

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity.

STUDY QUESTION: Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? SUMMARY ANSWER: A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. WHAT IS KNOWN ALREADY: Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1ß, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. WIDER IMPLICATIONS OF THE FINDINGS: Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

European Forum on Antiphospholipid Antibodies: research in progress.

The research projects of the European Forum on Antiphospholipid Antibodies are representative of how dynamic is this area of investigation. The present review is focused on the most recent projects of the Forum on the aetiopathogenic aspects of the antiphospholipid syndrome (APS). Studies on the genetic background of the APS are ongoing in order to better define the proximity between APS and full-blown systemic lupus erythematosus. However, the analysis of the polymorphisms of genes coding for inflammatory mediators may offer new information on the role of inflammatory processes in triggering thrombotic events as well as the whole susceptibility for developing the vascular manifestations. A systematic and wide detection of serological markers of infectious processes will give new insight on the role of infectious agents in favouring autoimmunity in APS. Owing to the well-known role of vitamin D(3) defect in autoimmune disease, the detection of vitamin plasma levels in APS patients will offer the rationale for a possible therapeutic supplementation. Additional projects are aimed to better characterize the diagnostic/prognostic value of antiphospholipid antibodies (aPL) by defining their epitope specificity and binding avidity. Pregnancy complications represent the obstetric side of APS. Research projects are focussed on the role of complement activation in placenta damage and on the potential ability of aPL to affect the fertility. Finally, a study has been planned in order to draw definitive conclusions on the associations between aPL and atherosclerosis.

[Premature cardiovascular disease: no indication for the determination of antiphospholipid antibodies or lupus anticoagulant as a possible risk factor]. / Vroegtijdige hart-vaatziekte: geen indicatie voor het bepalen van antifosfolipideantilichamen of lupusanticoagulans als mogelijke risicofactor.

In patients with premature atherothrombotic disease, the antiphospholipid syndrome (APS, defined as any thrombosis plus the repeated presence ofantiphospholipid antibodies) is sometimes looked for, as observational studies have suggested a link between APS and premature atherothrombosis. However, recent reviews that evaluated the prognostic value of antiphospholipid antibodies have concluded that the prognostic significance ofAPS for recurrent thrombotic disease is, at best, unclear and that its presence has no therapeutic consequences. A study that compared high- versus standard-dosage warfarin in patients with APS found no additional benefit from high-dosage warfarin. A study in patients with a recent ischaemic stroke found no additional benefit from warfarin (standard dosage) versus aspirin in patients with or without antiphospholipid antibodies. Therefore, on the basis of the evidence at hand, screening for APS in patients with premature atherosclerosis is not considered to be useful.

Pregnancy outcome in patients with a history of recurrent spontaneous miscarriages and documented thrombophilias.

The aim of this study was to evaluate the effect of treatment in patients analyzed for recurrent spontaneous miscarriage with a diagnosis of a hereditary thrombophilia, the presence of antiphospholipid and/or autoimmune antibodies, and/or hyperhomocystinemia (HHC) with or without methylenetetrahydrofolate reductase (MTHFR) polymorphisms. In total, 76 women with 2 or more embryonic or fetal losses were analyzed. Of these, 49 (64.4%) women were found to have one or more thrombophilias and/or autoimmune antibodies, and 33 (43.4%) women were found to have a MTHFR polymorphism and/or HHC. Since completion of the recurrent miscarriage analysis, 39 women conceived again. All women with a thrombophilia were treated with low-dose aspirin plus low molecular weight heparin. All women with previously diagnosed HHC and/or MTHFR polymorphisms were treated with folate and vitamin B(6) and B(12) supplementation. In the thrombophilia group, 27 women conceived resulting in 20 successful pregnancies (74.1%) and 7 pregnancy losses (2 trisomy 16, 1 ectopic pregnancy and 4 unexplained miscarriages), i.e. an unexplained pregnancy loss rate of 14.8%. In the HHC/MTHFR group 22 women conceived, resulting in 17 successful pregnancies (77.3%) and 5 pregnancy losses (1 trisomy 16, 1 Turner syndrome and 3 unexplained miscarriages), i.e. an unexplained pregnancy loss rate of 13.6%.

Effect of sairei-to combined with aspirin and prednisolone on four recurrent reproductive failure women who are positive for anti-phospholipid antibodies.

We treated four pregnancy cases positive for antiphospholipid antibodies (APLs) who had experienced recurrent second trimester fetal or neonatal losses using a Japanese modified Chinese herbal medicine, Sairei-to, low dose aspirin and adrenocorticosteroid hormone. The clinical courses of their new pregnancies in conjunction with the dynamic changes in their APL titers are described in this paper, and the possible efficacy of this treatment is discussed. The concept that autoimmune abnormalities, especially positive APLs, are generative factors for a range of reproductive failures, such as recurrent abortions, intrauterine growth retardation, intrauterine fetal death and preeclampsia, is now attracting a great deal of attention in the fields of reproductive immunology and perinatal medicine (Yasuda et al., 1995). The main mechanisms in the generation of reproductive failures are considered to be direct damage to chorionic villi by APLs during the period of placentation (Rote et al., 1998), as well as the formation of thrombi intervillous spaces (Arakawa et al., 1999). Considering these mechanisms of the reproductive failure generation by APLs, the application of immune suppressive therapy in combination with anti-coagulation therapy should be reconsidered as a treatment option. In this context, we treated four pregnancy cases positive for APLs who had experienced recurrent second trimester fetal or neonatal losses using a Japanese modified Chinese herbal medicine, Sairei-to, low dose aspirin and an adrenocorticosteroid hormone.

Frequency and specificities of antiphospholipid antibodies (aPL) in volunteer blood donors.

In the State of Indiana, blood donors are screened by a written questionnaire prior to donation to identify potential exposures to infectious diseases and to assess medications. The potential donor is not asked about aPL-related events. Animal experiments have shown, however, that passive transfer of aPL can produce aPL-associated pathology in the recipient. We determined the incidence of antiphospholipid antibodies (aPL) in 775 volunteer blood donors in Indiana. Tubing segments containing 2 ml of anticoagulated blood were obtained from donor units. The average donor age was 43 years (range 17-82); 45% were female. Our in-house aPL ELISA tested for IgG, IgA and IgM to cardiolipin (aCL), phosphatidylserine (aPS), phosphatidylethanolamine (aPE) and phosphatidylcholine (aPC). The plasmas were tested with and without supplemental phospholipid (PL)-binding plasma proteins from adult bovine plasma (ABP). A total of 24 tests were performed for each donor. Normal ranges were determined for 98% of the donors by calculating multiples of the means (MoM) of OD values for each antigen-isotype combination. No decimals were used, all MoMs were rounded up to the next whole number. An additional two MoMs were added to the calculated cutoff values to eliminate borderline positive values. Results revealed that 63 (8.1 %) donors had positive findings for one or more aPL. The relatively high number of aPL-positive individuals reflects the observation that different donors were often in the highest 2% of each antigen-isotype combination tested. The aPL specificities, isotypes, and PL-binding protein dependence are summarized in this report.

Characterization of anti-phosphatidylcholine polyreactive natural autoantibodies from normal human subjects.

In 1990 our group reported a patient with autoimmune hemolytic anemia and high titers of IgM anticardiolipin antibodies that cross-reacted with phosphatidylcholine (PTC). These autoantibodies also recognized bromelain-treated erythrocytes (BrE) and in vitro aged erythrocytes. The epitope exposed with this treatment is PTC. To detect and characterize antiphosphatidylcholine antibodies (anti-PTC) in a normal human population, we studied by ELISA the presence of serum anti-PTC (IgG and IgM) in clinically healthy human subjects. The most representative samples were also studied for IgG or IgM activity against BrE by flow cytometry, rheumatoid factor activity, anti-dsDNA, anti-ssDNA by ELISA and by indirect immunofluorecence (IIF) using HEp-2 line and a healthy human fibroblast strain as substratum. Eighty five percent of sera had IgM anti-PTC and none had IgG. IgM antibodies against BrE were inhibited by PTC micelles (mPTC). Anti-PTC were also inhibited by phosphorylcholine and phosphatidic acid. Aggregated gammaglobulin (AGG) reactivity was inhibited by dsDNA and mPTC. The IgM anti-dsDNA activity was inhibited by soluble dsDNA, AGG and mPTC. All sera gave intermediate filaments pattern by IIF and reacted against purified vimentin by dot blot and Western blot.Our study shows hemolytic IgM anti-PTC present in normal human serum. The main epitope recognized by these autoantibodies is phosphorylcholine. The physicochemical characteristics, crossreactivity with self-antigens and functional properties are typical features of natural autoantibodies.

Antiphospholipid antibodies, ischemic stroke in young adults, and calcium supplementation: a hypothesis.

The proper treatment of younger patients who have suffered ischemic stroke and who have no stroke risk factors other than antiphospholipid antibodies is unsettled. We propose a rationale to support adding dietary supplementation with calcium and vitamin D to the present standard regimen of anticoagulant therapy for these patients. We expect that the benefits from this additional therapy will prove additive. Proving this hypothesis will require large numbers of patients unlikely to present to any one center. The military health care system is well suited to such a study.

Case-control study of the frequency of thrombophilic disorders in couples with late foetal loss and no thrombotic antecedent--the Nîmes Obstetricians and Haematologists Study5 (NOHA5).

BACKGROUND: Women with familial thrombophilia have an increased risk of still birth. We postulated that the presence of asymptomatic risk factors for venous thrombosis might be a risk factor for late foetal loss. METHODS: We performed a case-control study on the prevalence of heritable thrombophilic defects, of antiphospholipid-related markers and of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with at least one episode of late unexplained foetal loss and in control women with successful pregnancies. Partners of cases and controls were also studied. Written conclusions of the pathological examination of the placentas, when available, were also reviewed. RESULTS: We found at least one positive biological risk factor for venous thrombosis in 21.1% of the patients and in 3.9% of the controls (p < 10(-4)). In women, the crude odds ratio for still birth associated with any positive biological risk factor for venous thrombosis was 5.5, 95% confidence interval (95%CI) [3.4-9.0]. No difference was found between partners of cases and controls (5.2% and 4.7%). Using conditional logistic regression analysis, 4 adjusted risk factors for still birth remained: protein S deficiency, positive anti beta2 glycoprotein I IgG antibodies, positive anticardiolipin IgG antibodies and the factor V Leiden mutation. The C677T mutation in the MTHFR gene was not an individual risk factor but an homozygous genotype was strongly associated with the former 4 risk factors (16.8% of patients vs. 0.9% of controls). In women with such associations, still births always occurred in absence of folic acid supplementation during pregnancy. Available conclusions of pathological analysis of placentas were found to have a very high proportion of "maternal vascular disease of the placenta" in patients with at least one positive risk marker for thromboembolism, specially in case of association with the C677T MTHFR homozygous genotype, compared to patients with negative markers (p <10(-4)). CONCLUSIONS: Late foetal loss, through placenta thrombosis, may sometimes be the consequence of a maternal multifactorial prothrombotic state associating traditional heritable or acquired thrombosis risk factors to conditions predisposing to an acute mild hyperhomocysteinaemia (coexistence of a genetic predisposition with late pregnancy-related increased folate needs).

Ongoing prothrombotic state in patients with antiphospholipid antibodies: a role for increased lipid peroxidation.

We measured the urinary excretion of Isoprostane F2alpha-III and Isoprostane-F2alpha-VI, two markers of in vivo lipid peroxidation, and the circulating levels of the prothrombin fragment F1+2, a marker of thrombin generation, in 18 antiphospholipid antibodies-positive patients, in 18 antiphospholipid antibodies-negative patients with systemic lupus erythematosus, and in 20 healthy subjects. Furthermore, 12 patients positive for antiphospholipid antibodies were treated with (n = 7) or without (n = 5) antioxidant vitamins (vitamin E at 900 IU/d and vitamin C at 2, 000 mg/d) for 4 weeks. Compared with antiphospholipid antibodies-negative patients, antiphospholipid antibodies-positive patients had higher urinary values of Isoprostane-F2alpha-III (P =. 0001), Isoprostane-F2alpha-VI (P =.006), and plasma levels of the prothrombin fragment F1+2 (P =.0001). In antiphospholipid-positive patients, F1+2 significantly correlated with Isoprostane-F2alpha-III (Rho =.56, P =.017) and Isoprostane-F2alpha-VI (Rho =.61, P =.008). After 4 weeks of supplementation with antioxidant vitamins, we found a significant decrease in F1+2 levels (P <.005) concomitantly with a significant reduction of both Isoprostane-F2alpha-III (P =.007) and Isoprostane-F2alpha-VI (P <.005). No change of these variables was observed in patients not receiving antioxidant treatment. This study suggests that lipid peroxidation might contribute to the activation of clotting system in patients positive for antiphospholipid antibodies.