Recurrent pregnancy loss and frequency of eight antiphospholipid antibodies and genetic thrombophilic factors in Czech women

PROBLEM: The aim of this study was to investigate frequencies of eight antiphospholipid antibodies (aPLs) in serum, four genetic thrombophilic factors and their mutual relation in 206 patients with repeated pregnancy loss (RPL). METHOD OF STUDY: Enzyme-linked immunosorbent assay was used for detection of aPLs against ph-serine, ph-ethanolamine, ph-inositol, DL-glycerol, phosphatidic acid, anti-annexin V, cardiolipin, and beta2-GPI. FV 1691G>A (Leiden mutation), FII 20210G>A mutation, MTHFR 677C>T and MTHFR 1298A>C variant genotypes were determined using a melting curve analysis of the PCR amplification product detected by the fluorescence resonance energy transfer. Genotypic distribution and allelic frequencies were calculated. Correlation between aPLs and thrombophilic factors was tested by chi-square and Fisher exact test. RESULTS: Our results show significantly increased prevalence of aPLs against ph-inositol (17-19.6% dependent on number of spontaneous miscarriages) and against ph-serine (18-25%). aPLs in IgG prevail. In 96% of the studied group, at least one risk factor was found (either aPLs positivity or thrombophilic factor). Both aPLs and thrombophilic factors were present in 43%. In the group of women with three or more RPLs, strong positive correlation of aPLs positivity and thrombophilic risk factors was observed. CONCLUSION: Antiphospholipide antibodies and genetic thrombophilic factors are important risk factors in the pathogenesis of RPL. Both autoantibodies against various kinds of phospholipides and genetic thrombophilic factors must be studied together in diagnosis of RPL for appropriate treatment.     

Circulating cell-derived microparticles in women with pregnancy loss

Citation
Alijotas‐Reig J, Palacio‐Garcia C, Farran‐Codina I, Zarzoso C, Cabero‐Roura L, Vilardell‐Tarres M. Circulating cell‐derived microparticles in women with pregnancy loss. Am J Reprod Immunol 2011; 66: 199–208 Problem To analyze cell‐derived microparticles (cMP) in pregnancy loss (PL), both recurrent miscarriages (RM) and unexplained fetal loss (UFL). Method of study Non‐matched case–control study was performed at Vall d’Hebron Hospital. Cell‐derived microparticles of 53 PL cases, 30 with RM, 16 with UFL, and 7 (RM + UFL), were compared to 38 healthy pregnant women. Twenty healthy non‐pregnant women act as controls. Cell‐derived microparticles were analyzed through flow cytometry. Results are given as total annexin (A5+), endothelial‐(CD144+/CD31+ CD41?), platelet‐(CD41+), leukocyte‐(CD45+) and CD41? c‐MP/μL of plasma. Antiphospholipid antibodies (aPLA) were analyzed according to established methods. Results Comparing PL versus healthy pregnant, we observed a significant endothelial cMP decrease in PL. When comparing RM subgroup with controls, we observed significant decreases in endothelial cMP. When comparing the PL positive for aPLA versus PL‐aPLA‐negative, no cMP numbering differences were seen. Conclusion Pregnancy loss seems to be related to endothelial cell activation and/or consumption. A relationship between aPLA and cMP could not be demonstrated.     

Antiprothrombin antibodies are associated with pregnancy loss in patients with the antiphospholipid syndrome

OBJECTIVE: To document the clinical association between the history of pregnancy loss in patients with the diagnosis of primary or secondary antiphospholipid syndrome (APS) and the presence of different antiprothrombin antibody subtypes [immunoglobulin G (IgG), IgM and IgA] in a cohort of patients with APS. METHODS: Records of 170 female patients with primary APS, or APS secondary to systemic lupus erythematosus (SLE) or secondary to other autoimmune diseases were studied. RESULTS: In female APS patients with IgG antiprothrombin antibodies (n = 105) significant associations to pregnancy loss (p < 0.0001), early pregnancy loss (p < 0.0001) and a negative association to thrombocytopenia (p < 0.01) could be identified. In the group of patients with IgG antiprothrombin antibodies and at least one pregnancy (n = 84) a significant association with pregnancy loss (p < 0.005) and especially with early pregnancy loss (p < 0.0001) was demonstrated. No association with other immunoglobulin subtypes of antiprothrombin antibodies could be documented. In the subgroup of patients with primary APS and at least one pregnancy in the history, pregnancy loss (p < 0.005) and early pregnancy loss (p < 0.0001) were found to be highly associated with the presence of IgG antiprothrombin antibodies. IgG antiprothrombin antibodies represent the highest independent risk factor for pregnancy loss with an odds ratio of 4.5. There was no statistically significant association with venous or arterial thrombosis in all IgG antiprothrombin antibody positive patients. CONCLUSION: The results of this study document the association of IgG antiprothrombin antibodies with pregnancy loss and in particular early pregnancy loss in a large and well-characterized cohort of patients. We would recommend routine testing for antiprothrombin antibodies in young female patients with APS.     

The growth promoting effects of bFGF, PD-ECGF and VEGF on cultured postimplantation rat embryos deprived of serum fractions

Serum components in which embryos are cultured in vitro are very important for normal embryonic development. In this study, rat serum was fractionated using Macrosep filters to study the effect of a single growth factor. The fractionated serum, both that containing only material greater than 30 kDa molecular weight (> 30 kDa) and that from which material between 30 kDa and 50 kDa had been removed (< 30 kDa + > 50 kDa), caused significant embryonic growth retardation. Addition of different concentrations of basic fibroblast growth factor (bFGF, 18 kDa), vascular endothelial growth factor (VEGF, 45 kDa) and platelet-derived endothelial growth factor (PD-ECGF, 45 kDa), to fractionated serum (bFGF to > 30 kDa serum and VEGF or PD-ECGF to < 30 kDa + > 50 kDa serum) partially restored embryonic growth and development according to a morphological scoring system and protein assay. This restoration was clear by all criteria, as well as in yolk sac vascularisation and heart development. The growth promoting effects of all 3 factors were significant but did not reach the level seen in embryos grown in whole rat serum. The effect of these growth factors was also investigated on anembryonic yolk sac development using a concentration for which maximum whole embryonic growth was seen (128 ng/ml bFGF, 1.6 ng/ml VEGF and 4 ng/ml PD-ECGF), and significant anembryonic yolk sac development was found. These findings suggest that the angiogenic factors may have a growth promoting effect on total embryonic development and vascularisation.     

Autoantibodies in Argentine women with recurrent pregnancy loss

PROBLEM: To determine the presence or absence of subclinical autoimmunity in Caucasian Argentine healthy women with first trimester recurrent pregnancy loss (RPL), the sera of 118 healthy women with a history of three or more consecutive abortions and 125 fertile control women without abortions and two children were analyzed for the presence of autoantibodies: immunoglobulin (Ig)G and IgM anticardiolipin, antinuclear (ANA), antismooth muscle (ASMA), antimitocondrial (AMA), antiliver-kidney-microsomal fraction (LKM), antigastric parietal cells (GPC), antineutrophil cytoplasmatic (ANCA) and antibodies antigliadin type IgA and IgG and IgA antitransglutaminase related with celiac disease (CD). METHOD OF STUDY: ANA, ASMA, AMA, anti-LKM, antibodies to GPC and ANCA were determined by indirect immunofluorescence (IFI) and anticardiolipin, antigliadina and antitransglutaminase antibodies were measured by enzyme-linked immunosorbent assays (ELISA). RESULTS: There was no significant difference between controls and patients with ANA, ASMA, AMA, LKM, ANCA and GPC. The prevalence of anticardiolipin antibodies in RPL was significantly higher than controls (P < 0,01) and the prevalence of positive antibodies for antigliadina type IgA and IgG and IgA antitransglutaminase in RPL was significantly higher than controls (P < 0.04). CONCLUSION: We show that Caucasian Argentine women with RPL showed significantly higher incidence of anticardiolipin antibodies than normal controls and finally we recommended the screening of IgA and IgG antigliadina and IgA antitransglutaminase antibodies in pregnancy, because of the high prevalence of subclinical CD in RPL and the chance of reversibility through consumption of a gluten free diet.     

黄芪多糖对红斑狼疮小鼠6种抗磷脂抗体的影响

目的 :观察黄芪多糖 (PG2 )对红斑狼疮小鼠抗心磷脂 (anticardiolipin ,aCL)、抗磷脂酰胆碱 (antiphosphatidylcho line ,aPC)、抗磷脂酰丝氨酸 (antiphosphatidylserine,aPS)、抗磷脂酰肌醇 (antiphosphatidylinositol,aPI)、抗磷脂酸 (antiphosphatidicacid ,aPA)和抗磷脂酰乙醇胺 (antiphosphatidylethanolamine ,aPE) 6种自身抗体的影响。方法 :19只雌性NZB×NZWF1小鼠随机分为黄芪I组 (2 5mg kg) 7只 ,黄芪II组 (5 0mg kg) 6只及生理盐水组 (0 2ml d) 6只 ,日一次腹腔注射 ;雌性BXSB及C5 7BL 6小鼠作对照 ,用酶联免疫吸附 (ELISA)法测定各组小鼠的 6种抗磷脂抗体A值进行比较。结果 :NZB×NZWF1小鼠黄芪II组各种抗磷脂抗体A均值比生理盐水组明显降低 (P <0 0 5或P <0 0 1) ,与BXSB ,C5 7BL 6小鼠比无统计学差异 ;黄芪I组与生理盐水组比有所升高 ;黄芪I组及生理盐水组与BXSB、C5 7BL 6比明显升高 (P <0 0 5或P <0 0 1)。结论 :黄芪多糖低剂量有使抗磷脂抗体升高的趋势 ,高剂量可明显抑制抗磷脂抗体的产生     

Autoantibodies to plasminogen and tissue plasminogen activator in women with recurrent pregnancy loss

Reduced fibrinolytic activity has been described in primary anti-phospholipid syndrome (PAPS), and may be responsible for thrombotic events. Antibodies to tissue type plasminogen activator (t-PA) or plasminogen (PLG) might contribute to the hypofibrinolytic state in autoimmune diseases, but the clinical significance of these antibodies is still unclear in recurrent pregnancy loss (RPL). The aim of this study is to evaluate the prevalence and clinical significance of anti-PLG and anti-t-PA antibodies in 87 patients with a history of RPL: 54 women with well-defined PAPS (mean age 32.5 years; range 26-38) and 33 women with unexplained RPL (mean age 30 years; range 24-39). IgG anti-PLG antibodies were found in 20 and four patients from the group with RPL/PAPS and unexplained RPL, respectively; IgG anti-t-PA antibodies were found in 11 and two patients from the above two groups, respectively. IgG anti-PLG antibodies were associated with the high risk of RPL (OR 7.2, P = 0.004), especially with RPL/PAPS (OR 11.2, P < 0.001) evaluated by Fisher's exact test, while IgG anti-t-PA were associated with RPL/PAPS (OR 10.0, P = 0.01) but not with RPL (OR 6.8, P = 0.06). A significant inhibition of exogenous fibrinolysis was observed by IgG fractions from patients with anti-PLG or anti-t-PA antibodies on microplates and on the human umbilical vein endothelial cells, compared with those from healthy controls. The prevalence of IgG anti-PLG antibodies was high in RPL patients, especially in RPL/PAPS, while the prevalence of IgG anti-t-PA antibodies was high in RPL/PAPS but not in RPL, and some of them might inhibit fibrinolysis in patients.     

Immunoglobulin class and IgG subclass distribution of anticardiolipin antibodies in patients with systemic lupus erythematosus and associated disorders.

The class and subclass distribution of an antibody response may give insight into the stimulating mechanism and likely effector functions. IgA, IgG and IgM anticardiolipin antibodies (aCL) were quantified in a consecutive series of 200 samples sent to an autoimmune serology laboratory to determine the relationships between aCL responses of each of these antibody classes and, in particular, whether there was any utility in the measurement of IgA aCL. Positive results for one of the three aCL isotypes were found in 105 samples (53%), and in 41 samples IgA aCL was detected (21%). However, amongst these unselected samples, little additional information was obtained by measurement of IgA aCL, which was found in conjunction with IgM or IgG aCL in all but five samples, and in these the isolated elevation of IgA aCL was only slight, and showed no disease specificity. The levels of each of the four IgG subclasses of aCL were measured in a subgroup of serum samples from 28 patients with autoimmune disease and from 29 patients with syphilis. Amongst the SLE patients IgG1 and IgG3 aCL were the predominant IgG subclasses, consistent with an antigen-driven, T cell-dependent antibody response. However, a subgroup of eight of the autoimmune subjects had predominant elevation of IgG2 aCL, possibly implying a role for T cell-independent antibody production to cardiolipin. Amongst the syphilis patients IgG1 and IgG3 aCL were also the predominant subclasses of aCL but IgG4 aCL were also detected in the majority of subjects, consistent with prolonged antigenic stimulation.     

Pregnancy: Low-dose aspirin for prevention of pregnancy losses in women with primary antiphospholipid syndrome

Pregnancy loss, often recurrent, is one of the most importantclinical manifestations associated with the primary antiphospholipidsyndrome. In these cases, pregnancy wastage is related to thepresence of antiphospholipid antibodies, namely lupus anticoagulantand anticardiolipin antibodies, but patients do not have featuresof systemic lupus erythematosus or any other well-defined autoimmunedisease. We report here on the outcome of 21 consecutive pregnanciesin 18 patients with the syndrome who were treated with low-doseaspirin (100 mg/day) from 1 month before attempting conceptionand throughout the pregnancy. Low-dose prednisone (15–30mg/day) was added for potentially non-obstetric (autoimmune-related)reasons in six pregnancies. Patients were monitored as havinghigh-risk pregnancies. Prior to therapy, the rate of live-bornbabies was 6.1% (46 previous fetal losses and three live-bornbabies), and after therapy, it was 90.5% (21 pregnancies and19 live-born babies). Pre-term delivery due to maternal or fetalindications was required in 15% (3/20) of the viable pregnancies.Except for prematurity (20% of viable pregnancies) and its potentialassociated complications, there were no significant adverseeffects to either mothers or babies. Our treatment modalityis advocated for prevention of pregnancy losses in patientswith the ‘obstetric’ primary antiphospholipid syndrome.     

Elevated blood flow resistance in uterine arteries of women with unexplained recurrent pregnancy loss.

BACKGROUND: Uterine perfusion appears to regulate uterine receptivity. However, vascular changes in recurrent pregnancy loss (RPL) remain poorly studied. METHODS: One hundred and twenty one women were enrolled into this study: normal women with sterility caused by male factor (control group: n = 72) and women with RPL (n = 49). Women with uterine anomaly, impaired glucose tolerance, abnormal thyroid function, or anti-phospholipid antibodies were excluded from the study. In the mid-luteal phase of a non-pregnant cycle, transvaginal pulsed Doppler ultrasonography of the uterine artery was performed. Uterine arterial pulsatility index (PI), endometrial thickness, serum estradiol, progesterone, and nitrite/nitrate concentrations were determined. RESULTS: In the RPL group, the PI in the uterine artery of women with antinuclear antibodies was significantly higher than that of women without antinuclear antibodies (P < 0.05). Among women without antinuclear antibodies, the mean (+/-SD) uterine artery PI in the RPL group (2.44 +/- 0.41) was also significantly higher than in the control group (2.19 +/- 0.40; P < 0.01). The PI was inversely correlated with serum progesterone levels (r = -0.47, P < 0.01). CONCLUSIONS: Elevated uterine arterial impedance is associated with RPL. Pulsed Doppler ultrasonography is useful in identifying women with unexplained RPL who have impaired uterine circulation.     

The carriage of pro-inflammatory cytokine gene polymorphisms in recurrent pregnancy loss

PROBLEM: Recurrent pregnancy loss (RPL) affects 2-4% of couples, and remains largely unexplained. Recent studies have examined the role of cytokines in the maintenance of normal pregnancy, which is linked with an increased expression of Th2 cytokines. Overexpression of Th1 cytokines is associated with RPL. Knowing that functional polymorphisms exist for certain cytokines, it has therefore been suggested that women with RPL may have a genetic predisposition to overexpress Th1 cytokines. METHOD OF STUDY: The genes for interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) carry functional gene polymorphisms. In both cases these are biallelic polymorphisms that can be detected by polymerase chain reaction followed by restriction fragment length polymorphism. The aim of this pilot study was to assess whether carriage of the rarer alleles (TNF*2 and IL-1B*2) could act as independent risk factors in recurrent miscarriage. RESULTS: We found an increased incidence in the carriage of TNF*2, more pronounced in those women with two or more miscarriages. Carriage of the IL-1B*2 either alone or in association with TNF*2 was not associated with recurrent miscarriage. CONCLUSION: There may be a role for these cytokine gene polymorphisms in RPL.     

Association of recurrent pregnancy loss with chromosomal abnormalities and hereditary thrombophilias

Background

Recurrent pregnancy loss (RPL) which is generally known as >3 consecutive pregnancy losses before 20 weeks'' gestation is seen in 0.5–2% of women

Objective

To evaluate the association of parental and fetal chromosomal abnormalities with recurrent pregnancy loss in our area and to analyze the frequency of three types of hereditary thrombophilia''s; (MTHFR C677T polymorphisms, FV Leiden G1691A mutation and Prothrombin (factor II) G20210A mutation) in these female patients.

Methods

The present case-control retrospective study was performed between February 2007 and December 2011 on 495 couples, who had two or more consecutive pregnancy losses before 20 weeks'' gestation. We used conventional cytogenetic analysis and polymerase chain reaction-restriction fragment length polymorphism.

Results

Parental chromosomal abnormality was detected in 28 cases (2.8% of all cases, 5.7% of the couples) most of which (92.9%) were structural abnormalities. All of the structural abnormalities were balanced chromosomal translocations. Chromosomal analysis performed from the abortion materials detected a major chromosomal abnormality in 31.9% of the cases. The most frequently observed alteration in the hereditary thrombophilia genes was heterozygote mutation for the MTHFR C677T polymorphisms (n=55).

Conclusion

Balanced translocations are the most commonly detected chromosomal abnormalities in couples being evaluated for recurrent pregnancy loss and these patients are the best candidates for offering prenatal genetic diagnosis by the help of which there is a possibility of obtaining a better reproductive outcome.     

Pregnancy outcome in recurrent spontaneous abortion associated with antiphospholipid antibodies: a comparative study of intravenous immunoglobulin versus prednisone plus low-dose aspirin

PROBLEM: To compare the use of intravenous immunoglobulins (IVIG) with prednisone plus low-dose aspirin (LDA) in treating pregnant women with a history of recurrent fetal loss having the antiphospholipid antibody (aPL), in terms of live-birth rate and maternal and perinatal morbidity. METHOD: A prospective, two-centers trial study included 82 recurrent aborters with aPL syndrome. Twenty-nine were treated with prednisone and LDA in one center, 53 received IVIG in the other center. Maternal and fetal outcomes and pregnancy complications were compared between groups. RESULTS: Live-birth rates were equivalent between groups (78 vs 76%). Mean birth weight was higher in the IVIG group than in the prednisone plus LDA group. In the prednisone- plus LDA-treated patients, gestational hypertension and gestational diabetes were found significantly more often than in the IVIG-treated group (14 vs 5% and 14 vs 5%, respectively). CONCLUSION: In patients with aPL syndrome, IVIG treatment improved pregnancy outcome, with significantly lower pregnancy complication rates, when compared with prednisone plus LDA therapy.     

Apoptosis in the uterus of mice with pregnancy loss

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis-regulating gene products p53 and bcl-2. METHOD OF STUDY: Pregnancy loss was induced by LPS or CP on days 9 or 12 of pregnancy, respectively. LPS- or CP-associated apoptosis was assessed by the TdT mediated dUTP-biotin nick end labeling (TUNEL) method as well as by DNA fragmentation analysis, while p53 or bcl-2 expression was evaluated by immunohistochemistry. RESULTS: Lipopolysaccharide treatment initiated a resorption process that was accompanied by the appearance of apoptotic cells in the uterus, which increased in number by 24 hr after treatment. Induction of pregnancy loss with CP resulted in the appearance of some apoptotic cells in the uterus, reaching a peak at 72 hr after treatment. DNA fragmentation analysis revealed a DNA ladder at 24 hr after LPS as well as 72 hr after CP treatment. Immunohistochemical analysis demonstrated a continuous p53 expression in the uterus of LPS- or CP-treated mice, which was somewhat elevated at the peak of the apoptotic process. On the other hand, bcl-2 expression in LPS-treated mice could be reciprocally correlated with the apoptotic process, appearing only at its initiation or completion, while in CP-treated mice it was continuously expressed except for some elevation at the completion of the apoptotic process. CONCLUSIONS: Our results suggest a possible role for the apoptotic process in mechanisms mediating pregnancy loss and indicate an involvement of p53 and bcl-2 in its regulation.