Assessment of Red Blood Cell Aggregation in Preeclampsia by Microfluidic Image Flow Analysis-Impact of Oxidative Stress on Disease Severity.

Preeclampsia (PE) is a hypertensive disease characterized by proteinuria, endothelial dysfunction, and placental hypoxia. Reduced placental blood flow causes changes in red blood cell (RBC) rheological characteristics. Herein, we used microfluidics techniques and new image flow analysis to evaluate RBC aggregation in preeclamptic and normotensive pregnant women. The results demonstrate that RBC aggregation depends on the disease severity and was higher in patients with preterm birth and low birth weight. The RBC aggregation indices (EAI) at low shear rates were higher for non-severe (0.107 ± 0.01) and severe PE (0.149 ± 0.05) versus controls (0.085 ± 0.01; p < 0.05). The significantly more undispersed RBC aggregates were found at high shear rates for non-severe (18.1 ± 5.5) and severe PE (25.7 ± 5.8) versus controls (14.4 ± 4.1; p < 0.05). The model experiment with in-vitro-induced oxidative stress in RBCs demonstrated that the elevated aggregation in PE RBCs can be partially due to the effect of oxidation. The results revealed that RBCs from PE patients become significantly more adhesive, forming large, branched aggregates at a low shear rate. Significantly more undispersed RBC aggregates at high shear rates indicate the formation of stable RBC clusters, drastically more pronounced in patients with severe PE. Our findings demonstrate that altered RBC aggregation contributes to preeclampsia severity.

Blood Plasma Calorimetric Profiles of Women with Preeclampsia: Effect of Oxidative Stress.

Preeclampsia is a pregnancy-related disease with poor placentation and presents itself through hypertension and proteinuria. The disease is also associated with the oxidative modification of proteins in maternal blood plasma. In this work, we combine differential scanning calorimetry (DSC), capillary electrophoresis, and atomic force microscopy (AFM) to evaluate the changes in the plasma denaturation profiles of patients with preeclampsia (PE) as compared with those of pregnant controls. Our results demonstrate that the last trimester of pregnancy substantially affects the main calorimetric characteristics of blood plasma from pregnant controls relative to nonpregnant women. These variations correlate well with the changes in protein levels determined by electrophoresis. DSC analysis revealed significant deviations in the plasma heat capacity profiles of preeclamptic patients from those of pregnant controls. These alterations are expressed mainly in a substantial reduction in albumin-assigned transitions and an upward shift in its denaturation temperature, lower calorimetric enthalpy changes, and a reduced ratio of heat capacity in the albumin/globulin-assigned thermal transitions, which are more pronounced in severe PE cases. The in vitro oxidation model shows that the alteration of PE thermograms is partly related to protein oxidation. AFM data detected numerous aggregate formations in the plasma of PE samples and fewer small ones in the pregnant controls, which are not found in healthy nonpregnant samples. These findings could serve as a basis for further investigations to reveal the possible relationship between albumin thermal stabilization, the increased inflammatory state and oxidative stress, and protein misfolding in preeclampsia.

Membrane Lesions and Reduced Life Span of Red Blood Cells in Preeclampsia as Evidenced by Atomic Force Microscopy.

Preeclampsia (PE) presents with maternal de novo hypertension and significant proteinuria and is one of the leading causes of maternal and perinatal morbidity and mortality with unknown etiology. The disease is associated with inflammatory vascular response and severe red blood cell (RBC) morphology changes. This study examined the nanoscopic morphological changes of RBCs from PE women versus normotensive healthy pregnant controls (PCs) and non-pregnant controls (NPCs) applying atomic force microscopy (AFM) imaging. The results revealed that the membrane of fresh PE RBCs differed significantly from healthy ones by the presence of invaginations and protrusions and an increased roughness value (Rrms) (4.7 ± 0.8 nm for PE vs. 3.8 ± 0.5 nm and 2.9 ± 0.4 nm for PCs and NPCs, respectively). PE-cells aging resulted in more pronounced protrusions and concavities, with exponentially increasing Rrms values, in contrast to the controls, where the Rrms parameter decreased linearly with time. The Rrms, evaluated on a 2 × 2 µm2 scanned area, for senescent PE cells (13 ± 2.0 nm) was significantly higher (p < 0.01) than that of PCs (1.5 ± 0.2 nm) and NPCs (1.9 ± 0.2 nm). Furthermore, the RBCs from PE patients appeared fragile, and often only ghosts were observed instead of intact cells at 20-30 days of aging. Oxidative-stress simulation on healthy cells led to RBC membrane features similar to those observed for PE cells. The results demonstrate that the most pronounced effects on RBCs in PE patients are related to impaired membrane homogeneity and strongly altered roughness values, as well as to vesiculation and ghost formation in the course of cell aging.

Altered Mesenchymal Stem Cells Mechanotransduction from Oxidized Collagen: Morphological and Biophysical Observations.

Extracellular matrix (ECM) provides various mechanical cues that are able to affect the self-renewal and differentiation of mesenchymal stem cells (MSC). Little is known, however, how these cues work in a pathological environment, such as acute oxidative stress. To better understand the behavior of human adipose tissue-derived MSC (ADMSC) in such conditions, we provide morphological and quantitative evidence for significantly altered early steps of mechanotransduction when adhering to oxidized collagen (Col-Oxi). These affect both focal adhesion (FA) formation and YAP/TAZ signaling events. Representative morphological images show that ADMSCs spread better within 2 h of adhesion on native collagen (Col), while they tended to round up on Col-Oxi. It also correlates with the lesser development of the actin cytoskeleton and FA formation, confirmed quantitatively by morphometric analysis using ImageJ. As shown by immunofluorescence analysis, oxidation also affected the ratio of cytosolic-to-nuclear YAP/TAZ activity, concentrating in the nucleus for Col while remaining in the cytosol for Col-Oxi, suggesting abrogated signal transduction. Comparative Atomic Force Microscopy (AFM) studies show that native collagen forms relatively coarse aggregates, much thinner with Col-Oxi, possibly reflecting its altered ability to aggregate. On the other hand, the corresponding Young's moduli were only slightly changed, so viscoelastic properties cannot explain the observed biological differences. However, the roughness of the protein layer decreased dramatically, from RRMS equal to 27.95 ± 5.1 nm for Col to 5.51 ± 0.8 nm for Col-Oxi (p < 0.05), which dictates our conclusion that it is the most altered parameter in oxidation. Thus, it appears to be a predominantly topographic response that affects the mechanotransduction of ADMSCs by oxidized collagen.

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

Altered Thermal Behavior of Blood Plasma Proteome Related to Inflammatory Cytokines in Early Pregnancy Loss.

Early pregnancy loss (EPL) is a relatively common pathology of which almost 50% of cases remain idiopathic. In the search for novel biomarkers, differential scanning calorimetry (DSC) is intensively used to characterize the thermodynamic behavior of blood plasma/serum proteome in health and disease. Herein, for the first time, we investigate the DSC denaturation profiles of blood plasma derived from patients suffering EPL compared to healthy pregnant and non-pregnant women. Data analysis reveals that 58% of the EPL thermograms differ significantly from those of healthy pregnant women. Thermal stabilization of a fraction of albumin-assigned transition with concomitant suppression of the major and enhancement of the globulin-assigned transition are characteristic features of EPL calorimetric profiles that could be used as a new indicator of a risk pregnancy. The presented results suggest an altered composition or intermolecular interactions of the plasma proteome of women with EPL. In addition, the alterations of the EPL thermograms correlate with the increased blood levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and a higher prevalence of the polymorphism in the plasminogen activator inhibitor type-1 (PAI-1) gene, suggesting an expression of an overall enhanced immune response. The concomitant changes in plasma thermograms confirm the potential of the DSC approach for distinguishing changes in the pathological state of the blood plasma proteome.

Morphometric and Nanomechanical Features of Erythrocytes Characteristic of Early Pregnancy Loss.

Early pregnancy loss (EPL) is estimated to be between 15 and 20% of all adverse pregnancies. Approximately, half of EPL cases have no identifiable cause. Herein, we apply atomic force microscopy to evaluate the alteration of morphology and nanomechanics of erythrocytes from women with EPL with unknown etiology, as compared to healthy pregnant (PC) and nonpregnant women (NPC). Freshly isolated erythrocytes from women with EPL differ in both the roughness value (4.6 ± 0.3 nm, p < 0.05), and Young's modulus (2.54 ± 0.6 MPa, p < 0.01) compared to the values for NPC (3.8 ± 0.4 nm and 0.94 ± 0.2 MPa, respectively) and PC (3.3 ± 0.2 nm and 1.12 ± 0.3 MPa, respectively). Moreover, we find a time-dependent trend for the reduction of the cells' morphometric parameters (cells size and surface roughness) and the membrane elasticity­much faster for EPL than for the two control groups. The accelerated aging of EPL erythrocytes is expressed in faster morphological shape transformation and earlier occurrence of spiculated and spherical-shaped cells, reduced membrane roughness and elasticity with aging evolution. Oxidative stress in vitro contributed to the morphological cells' changes observed for EPL senescent erythrocytes. The ultrastructural characteristics of cells derived from women with miscarriages show potential as a supplementary mark for a pathological state.

Mesenchymal Stem-Cell Remodeling of Adsorbed Type-I Collagen-The Effect of Collagen Oxidation.

This study describes the effect of collagen type I (Col I) oxidation on its physiological remodeling by adipose tissue-derived mesenchymal stem cells (ADMSCs), both mechanical and proteolytic, as an in vitro model for the acute oxidative stress that may occur in vivo upon distinct environmental changes. Morphologically, remodeling was interpreted as the mechanical rearrangement of adsorbed FITC-labelled Col I into a fibril-like pattern. This process was strongly abrogated in cells cultured on oxidized Col I albeit without visible changes in cell morphology. Proteolytic activity was quantified utilizing fluorescence de-quenching (FRET effect). The presence of ADMSCs caused a significant increase in native FITC-Col I fluorescence, which was almost absent in the oxidized samples. Parallel studies in a cell-free system confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase with statistically significant inhibition occurring in the oxidized samples. Structural changes to the oxidized Col I were further studied by differential scanning calorimetry. In the oxidized samples, an additional endotherm with sustained enthalpy (∆H) was observed at 33.6 °C along with Col I's typical one at 40.5 °C. Collectively, these data support that the remodeling of Col I by ADMSCs is altered upon oxidation due to intrinsic changes to the protein's structure, which represents a novel mechanism for the control of stem cell behavior.

Morphometric and Nanomechanical Features of Platelets from Women with Early Pregnancy Loss Provide New Evidence of the Impact of Inherited Thrombophilia.

Pregnancy is associated with hypercoagulation states and increased thrombotic risk, especially in women with thrombophilia. We combine atomic force microscopy (AFM) and flow cytometry to examine the morphology and nanomechanics of platelets derived from women with early pregnancy loss (EPL) and control pregnant (CP) and non-pregnant (CNP) women. Both control groups exhibit similar morphometric parameters (height and surface roughness) and membrane stiffness of platelets. EPL patients' platelets, on the other hand, are more activated than the control groups, with prominent cytoskeletal rearrangement. In particular, reduced membrane roughness (22.9 ± 6 nm vs. 39.1 ± 8 nm) (p < 0.05) and height (692 ± 128 nm vs. 1090 ± 131 nm) (p < 0.05), strong alteration in the membrane Young modulus, increased production of platelets' microparticles, and higher expression of procoagulant surface markers, as well as increased occurrence of thrombophilia (FVL, FII20210A, PLA1/A2, MTHFR C677T or 4G/5G PAI-1) polymorphisms were found. We suggest that the carriage of thrombophilic mutations triggers structural and nanomechanical abnormalities in platelets, resulting in their increased activation. The activation state of platelets can be well characterized by AFM, and the morphometric and nanomechanical characteristics might serve as a new criterion for evaluation of the cause of miscarriage and offer the prospect of an innovative approach serving for diagnostic purposes.

PlA2 Polymorphism in Glycoprotein IIb/IIIa Modulates the Morphology and Nanomechanics of Platelets.

Glycoprotein IIb/IIIa (GPIIb/IIIa) is the most abundant platelet surface receptor for fibrinogen and von Willebrand factor. Polymorphism PlA1/A2 in the gene of GPIIb/IIIa is among the risk factors for the development of arterial and venous thrombosis. The aim of this study is to evaluate the effect of the carriage of PlA1/A2 on the size, topographic features, and membrane stiffness of platelets from healthy controls and patients with deep venous thrombosis (DVT). Atomic force microscopy (AFM) imaging and nanoindentation (force-distance curves) were applied to investigate the morphological and nanomechanical properties (Young's modulus) of platelets immobilized on glass surface. The surface roughness ( Ra) and height ( h) of platelets from patients with DVT, carriers of mutant allele PlA2 ( Ra = 30.2 ± 6 nm; h = 766 ± 182 nm) and noncarriers ( Ra = 28.6 ± 6 nm; h = 865 ± 290 nm), were lower than those of healthy carriers of allele PlA2 ( Ra = 48.1 ± 12 nm; h = 1072 ± 338 nm) and healthy noncarriers ( Ra = 49.7 ± 14 nm; h = 1021 ± 433 nm), respectively. Platelets isolated from patients with DVT, both carriers and noncarriers, exhibit much higher degree of stiffness at the stage of spreading ( E = 327 ± 85 kPa and 341 ± 102 kPa, respectively) compared to healthy noncarriers ( E = 198 ± 50 kPa). In addition, more pronounced level of platelet activation was found in polymorphism carriers. In conclusion, the carriage of PlA2 allele modulates the activation state, morphology, and membrane elasticity of platelets.

Rs5918ITGB3 Polymorphism, Smoking, and BMI as Risk Factors for Early Onset and Recurrence of DVT in Young Women.

OBJECTIVE: To evaluate the contribution of rs5918ITGB3 on the incidence and recurrence of deep venous thrombosis (DVT) in women and the relationship with body mass index (BMI) and smoking and to compare with data in men. RESULTS: Rs5918(C) polymorphism in ITGB3 gene was assessed in 224 patients diagnosed with DVT and 216 controls. Thrombophilic genetic variant rs5918(C) was significantly pronounced in women (χ2 =7.565, P = .008) and total patients (χ2 = 9.266, P = .002) but not in men. Women patients (<45 years) who were carriers of rs5918ITGB3 polymorphism had an early onset of DVT (34.5 vs 39.4 years, χ2 = 7.027, P = .008) as analyzed by Kaplan-Meier and a higher risk of the recurrent event (χ2 = 3.405, odds ratio = 2.581, P = .044). The period before recurrent venous thromboembolism event was related to smoking status and BMI in young female who were carriers of rs5918 polymorphism but not in the males. CONCLUSIONS: Carriage of genetic variant rs5918(C) polymorphism in ITGB3 gene in women contributes to higher risk of single and recurrent DVT events at younger age.

Interleukin-17 producing T cells could be a marker for patients with allergic rhinitis.

BACKGROUND: Interleukin-17A (I-17A)-producing CD4+T helper cells have been implicated in allergic inflammation; however, the role of IL-17A in allergic rhinitis (AR) patients with different degrees of atopy and airway reactivity to methacholine (Mch) has not been examined. OBJECTIVES: To explore IL-17A-producing CD3+CD4+T cells in peripheral blood of patients with persistent AR and assess the degree of atopy, eosinophil count (Eo count), and bronchial hyper-responsiveness (BHR) to methacholine. METHODS: The study involved 61 patients and 30 controls. The percentage of CD3+CD4+IL-17A+T cells in peripheral blood was measured by flow cytometry, bronchial challenges with Mch were performed, as were skin prick tests with standard inhalant allergens, and Eo count was measured. Atopic status was determined by the number of positive SPT results and wheal mean diameter. RESULTS: A statistically significant difference in Th17 cell percentage was found in the AR and control groups (2.59 +/- 1.32% and 1.24 +/- 0.22% respectively, P = 0.001). Forty-one patients (67.2%) were polysensitized to indoor and outdoor allergens, while 20 (32.8%) had positive skin prick tests to indoor allergens. CD4+T cells were significantly higher in the patient group compared to the control group (2.91 +/- 1.5% versus 1.91 +/- 0.62%, P = 0.005), as was Eo count (4.48 +/- 2.13 vs. 2.32 +/- 1.83) (P = 0.0001). Forty-one in the AR group (67%) and 7 (23%) in the control group were Mch-positive (P = 0.001). The percentage of IL-17A-producing CD4+T cells was significantly higher in males compared to females (3.15 +/- 1.8% versus 2.31 +/- 0.9%, P = 0.02) CONCLUSIONS: Polysensitized AR patients exhibited higher IL-17A-producing CD4+T cell levels and eosinophil counts. Male patients displayed a higher frequency of IL-17A-producing T cells.

IL-17 producing T cells correlate with polysensitization but not with bronchial hyperresponsiveness in patients with allergic rhinitis.

BACKGROUND: Th2-type T cell response has a considerable role in atopic diseases. The involvement of Th17 and IL-17 in atopy process provided new understanding of allergic diseases. Bronchial hyperresponsiveness is quite common in allergic rhinitis. We aimed to explore the expression of IL-17 producing CD3+ CD4+ T cells in peripheral blood of rhinitic patients, with/without bronchial hyperresponsiveness and sensitized to common allergens, as this relationship has not been examined. METHODS: Sixty one patients with allergic rhinitis and thirty controls were examined. IL-17 producing T cells were detected by flow cytometry, IL-17, IL-4 and IL-13 levels in peripheral blood were evaluated by ELISA. Bronchial hyperresponsiveness was investigated with methacholine challenge test. Atopy was evaluated by skin prick tests with common allergens. RESULTS: IL-17 producing T cell percentage of AR group was significantly higher: 2.59 ± 1.32 than in controls 1.24 ± 0.22, (p = 0.001). Significant sex related difference in CD3+ CD4+ IL-17 T cells was observed: respectively in male patients versus female 3.15 ± 1.8% and 2.31 ± 0.9%, (p = 0.02). Rhinitics had greater bronchodilator responses compared to controls (p = 0.001), however the percentages of T cells in both groups appeared equal. Serum IL-17 levels in AR group were significantly higher (5.10 ± 4.40) pg/ml than in controls (3.46 ±1.28) pg/ml, (p = 0.04). IL-4 levels (0.88 ± 1.27) and IL-13 levels (3.14 ± 5.85) in patients were significantly higher than in control's (0.54 ± 0.10) pg/ml, (p = 0.001) and (1.19 ± 0.64) pg/ml; (p = 0.001) respectively.The percentages of T cells in patients sensitized to 5 allergens (group I) were significantly lower (1.91 ± 0.62) than those sensitized to more than 5 allergens (group II) (2.91 ± 1.5) (p = 0.004). CONCLUSIONS: The observed higher levels of IL-17 producing T cells in polysensitized males suggest a role of IL-17 in pathogenesis of AR. The higher airway responsiveness in AR may not be Th17 dependent. The higher serum values of IL-17, IL-4 and IL-13 demonstrate the presence of cytokine balance in atopic diseases.

Further insights into the role of the annexin A5 M2 haplotype as recurrent pregnancy loss factor, assessing timing of miscarriage and partner risk.

OBJECTIVE: To study the influence of M2/ANXA5 for recurrent pregnancy loss (RPL), according to the timing of miscarriages and assess the male partner risk. DESIGN: Genetic association study. SETTING: Academic research center. PATIENT(S): Female patients from two academic centers in Germany and Bulgaria with two or more unexplained miscarriages were selected for this study. Male partners were available for a part of the German sample. Population controls were recruited from healthy individuals of respective populations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Incidence of M2 carriage and odds ratios were calculated between patient and control groups, and RPL risk was evaluated. RESULT(S): The M2 haplotype in ANXA5 was associated with greater overall RPL risk in German and in Bulgarian women, and a trend of higher prevalence was seen for male partners of German RPL patients. The highest relative risk of M2 carriage was observed in women of both populations with "early" fetal losses between the 10th and 15th gestational weeks, which was significant in the meta-analysis. CONCLUSION(S): M2 carriage seems to have an RPL risk role mostly for early abortions, gestational weeks 10-15. In the first phase of pregnancy this correlates with vascular remodeling to accomplish the transition from high- to low-resistance blood vessels.

Inherited thrombophilia and IVF failure: the impact of coagulation disorders on implantation process.

In connection with the embryo acceptance process after IVF procedure, endometrial cells surface receptors, extracellular matrix (ECM) molecules, endothelium and blood circulation factors were involved in remodelling of endometrium. Plasminogen activator inhibitor type 1 plays a significant role during the early phases of placental vascular remodelling and regulates the trophoblast invasion through controlling plasmin activity. Endometrial cell surface protein integrin alphaV/beta3, responsible for the adhesion of the embryo, has had also the same subunit beta3, which is component of integrin alphaIIb/beta3 connected with platelet aggregability. Prothrombin, furthermore, has had a debatable effect upon endothelial and mesenchymal cells and possible contribution on embryo vascular development. Confoundable data have been present about the role of coagulation factor V and its role for implantation. These and other coagulation factors have relatively common gene polymorphisms that enhanced their activity. This review discusses the effect of increased coagulation activity on implantation process, which is not yet fully determined. The establishment of the positive or negative impact of mother hypercoagulability on the success of embryo implantation after assisted reproduction technology could determine the timing of preventing anticoagulant therapy in women with history of early embryo loss.

Polymorphism A1/A2 in the cell surface integrin subunit ß3 and disturbance of implantation and placentation in women with recurrent pregnancy loss.

Polymorphism A1/A2 in the ß3 subunit of integrins αIIb/ß3 and αV/ß3 is implicated in the risk of development of embryonic and fetal recurrent pregnancy loss (RPL). In 191 women with RPL, polymorphism A1/A2 was statistically significantly associated with RPL at <10 weeks of gestation (29.3% versus 16.4% in controls), but it was much more pronounced in 67 women with RPL between 10 and 20 weeks of gestation (41.8%), illustrating its role in recurrent fetal loss.

Association of inherited thrombophilia with embryonic and postembryonic recurrent pregnancy loss.

To investigate the impact of maternal-inherited thrombophilia: effects of factor V Leiden (FVL) and prothrombin gene mutation (FII 20210G>A) on the development of recurrent pregnancy loss in embryonic and postembryonic periods. A total of 153 patients were analysed for FVL and FII 20210G>A according to placenta gestation: 94 women with embryonic loss prior 10 weeks of gestation and 59 women with postembryonic (early fetal) loss occurring between 10 and 14 weeks of gestation. The control group consisted of 100 healthy women, with at least one uncomplicated full-term pregnancy. FVL prevalence was not significantly associated with pregnancy loss prior to 10 weeks of gestation (9.6%) compared with controls (7%) [odds ratio (OR) 1.41; 95% confidence interval (CI) 0.454-4.416, P > 0.05], but it was much more pronounced in women with postembryonic loss (10-14 weeks of gestation) - 18.6% (OR 3.05; 95% CI 1.010-9.387, P = 0.047). FII 20210G>A was significantly higher in both groups with embryonic (17%) and early fetal losses (16.9%) as compared to controls (3%) (OR 6.63; 95% CI 1.731-29.752, P = 0.003; OR 6.60; 95% CI 1.572-31.856, P = 0.006). FII 20210G>A is significantly associated with an increased risk of early recurrent pregnancy loss throughout the entire first trimester. FVL was significantly higher only in early fetal period after starting of the placentation process, but not associated with embryonic recurrent pregnancy loss. These results suggested that the first trimester should be viewed rather as a heterogeneous interval, with different relation to FVL in the embryonic and postembryonic fetal period. Genetic testing should be applied according to the diverse contribution of thrombophilic markers to embryonic and postembryonic period.

Impact of thrombophilic genetic factors on pulmonary embolism: early onset and recurrent incidences.

The importance of genetic thrombophilic factors in the development of venous thromboembolism has been increasingly recognized. Factor V Leiden (FVL), prothrombin gene mutation G20210A (FII G20210), genetic variant C677T of the methylentetrahydrofolate reductase (MTHFR), as well as the polymorphism A2 (PlA2) in platelet glycoprotein IIb/IIIa were recently discussed. We analyzed the contribution of genetic thrombophilic factors to the pathogenesis of pulmonary embolism (PE) and their association with the early onset and recurrence of PE using DNA analysis methods. In this case control trial we found thrombophilic genetic variants in 58.8% of 51 patients with PE. FVL was found in 23.5% of the patients versus 7.1% of the 98 controls (p=0.01), PlA2 IIb/IIIa was found in 35.3% vs. 14.3% (p=0.03), and FII G20210A was found in 5.9% vs. 2.0% (NS). Patients with recurrent PE had a very high prevalence of genetic factors, 70.4%. High prevalence of FVL was found in patients under 45 years of age: 39.3% (OR=14.23, 95% CI=1.58-330.03, p=0.01) as well as in patients with recurrent incidence (37%, OR=7.647, 95% CI=2.27-26.44, p=0.001). FVL was also significantly higher in the subgroup of patients with PE combined with deep venous thrombosis (OR=6.500, 95% CI=1.81-23.76, p=0.002) in comparison with patients with isolated PE (OR=2.261, 95% CI=0.50-9.69). The carriers of FVL are at higher risk for early and recurrent PE events. High prevalence of PlA2 in PE patients evidently shows the impact of this polymorphism in PE development. A different treatment should be considered in carriers of thrombophilic defects.