The role of epidermal growth factor-like domain-related abnormalities, protein S Tokushima, and anti-protein S autoantibodies in pregnancy loss.

BACKGROUND: Protein S (PS) deficiency and autoantibodies that bind to PS (anti-PS) have been described in patients with adverse pregnancy outcomes, including pregnancy loss. PS Tokushima is a congenital abnormality of the second epidermal growth factor (EGF)-like domain, and anti-PS has been reported to recognize EGF-like domains. OBJECTIVES: We evaluated the role of PS Tokushima and anti-PS in patients with pregnancy loss. METHODS: Patients with recurrent early pregnancy loss (n = 324; group A), those with one or more mid-to-late pregnancy loss (n = 196; group B), and infertile women having no pregnancy loss (n = 650; group C) were screened for PS type II deficiency and anti-PS. Patients who were diagnosed with PS type II deficiency underwent genetic analysis for the detection of PS Tokushima. RESULTS: The incidence of patients with PS Tokushima was 1.85 %, 5.10 %, and 1.23 % in groups A, B, and C, respectively. The incidence of patients with PS Tokushima was significantly higher in group B (p = 0.0027) than in group C. The incidence of patients with anti-PS was 20.1 %, 23.0 %, and 19.2 % in groups A, B, and C, respectively. The incidence of patients with anti-PS was significantly higher in groups A (p = 0.0229), B (p = 0.0071), and C (p = 0.0288) than in previously reported healthy nonpregnant women (7.1 %, 4/56). CONCLUSIONS: Our data suggest that PS Tokushima is associated with mid-to-late pregnancy loss, while anti-PS are associated with recurrent early pregnancy loss, mid-to-late pregnancy loss, and infertility.

Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor-Like Domain.

Background Factor XII (FXII) deficiency and autoantibodies that bind to FXII (anti-FXII) have been described in patients with adverse pregnancy outcomes, including recurrent pregnancy loss. It has been reported that FXII functions not only as a coagulation protein but also as a growth factor. Objectives We studied the association between anti-FXII and the epidermal growth factor (EGF) system in patients with recurrent pregnancy loss. Patients/Methods We used synthetic peptides that span the second EGF-like domain in the heavy chain of FXII (EGF2) in inhibition and direct binding studies to determine if anti-FXII antibodies recognize EGF2. Furthermore, we examined whether anti-FXII antibodies, which recognize EGF2, also recognize recombinant EGF and heparin-binding EGF-like growth factor (HB-EGF). Results Among 100 patients with recurrent pregnancy loss, the plasma of 23 patients (23.0%) recognized the synthetic peptide ASQ41, which covers EGF2. Among the 23 anti-ASQ41-positive patients, plasma samples from 13 patients (56.5%) recognized the 22-residue segment C-terminal half of ASQ41. Among the 23 anti-ASQ41-positive patients, the plasma of 17 patients (73.9%) recognized recombinant human EGF. Affinity-purified anti-FXII antibodies, which recognize ASQ41, also recognized recombinant EGF family proteins such as EGF and HB-EGF. Conclusions The autoantibodies in patients with recurrent pregnancy loss recognized the EGF2 domain in FXII and other proteins of the EGF family. Since proteins in the EGF family play an important role in normal pregnancy, autoantibody-associated disruption of the EGF system may cause pregnancy loss.

Risk Factors and Outcomes of Recurrent Pregnancy Loss in Japan.

AIM: To clarify the risk factors and pregnancy outcomes for each risk factor of recurrent pregnancy loss (RPL) in Japan. METHODS: Using a prospective RPL database collected from 16 facilities in Japan, the prevalence of risk factors for RPL, their treatments and pregnancy outcomes were examined. RESULTS: Of 6663 patients registered in our database, 5708 patients had RPL. All examinations for risk factors were performed for 1340 patients (23.5%). The prevalences of positive antiphospholipid antibodies (aPL), malformation of the uterus, thyroid dysfunction, parental karyotype abnormality, factor XII deficiency, protein S deficiency and unknown risk factors were 8.7%, 7.9%, 9.5%, 3.7%, 7.6%, 4.3% and 65.1%, respectively. Although factor XII deficiency and protein S deficiency are not recognized as risk factors for RPL in general, low-dose aspirin (LDA) or unfractionated heparin + LDA therapy improved live birth rates. In transiently aPL-positive patients, the live birth rate with LDA therapy was similar to that with heparin + LDA. For unknown risk factors of RPL, the live birth rate in normal fetal karyotype in the none treatment group was similar to that in all other treatments group (81.3% vs 86.0%). Of 5708 RPL patients, pregnancy outcomes were known for 2261 patients and 1697 patients (75.1%) had at least one live birth. CONCLUSION: The risk factors and pregnancy outcomes for each risk factor of RPL are useful for clinicians and patients. Factor XII deficiency and protein S deficiency may be risk factors of RPL.

Antigenic binding sites of anti-protein S autoantibodies in patients with recurrent pregnancy loss.

BACKGROUND: Protein S (PS) deficiency is a risk factor for adverse pregnancy outcomes including recurrent pregnancy loss. Several studies have shown that the presence of anti-PS autoantibodies (anti-PS) leads to an acquired PS deficiency. Hence, an epitope mapping study was conducted to know the pathogenesis of anti-PS in patients with recurrent pregnancy loss. METHODS: PS was treated with thrombin to divide the protein into γ-carboxyglutamic acid (Gla) domain and Gla-domain free PS. For the preparation of fragments of epidermal growth factor (EGF)-like domains (EGF1-4), PS was subjected to proteolysis using lysyl endopeptidase. The epitopes were identified in immunoblot. Whether anti-PS recognized EGF family proteins in anti-PS-positive patients was also examined. RESULTS: Anti-PS recognized Gla-domain free PS, especially the three fragments of EGF-like domains, EGF1-2, EGF3-4, and EGF1-4. Anti-PS recognized recombinant human EGF. Anti-PS and polyclonal antibodies to recombinant human EGF recognized PS in the absence of Ca2+ but not in the presence of Ca2+. In competitive inhibition studies, polyclonal antibodies to recombinant mouse EGF blocked anti-PS binding to PS in a concentration-dependent manner. CONCLUSIONS: These results suggest that anti-PS in patients with recurrent pregnancy loss recognize EGF-like domains in PS. Interestingly, anti-PS also recognized EGF family proteins. Anti-PS in patients with recurrent pregnancy loss may be associated with not only thrombophilia but also the disruption of the EGF system.

Autoantibodies to Factor XII and Kininogen-Dependent Antiphosphatidylethanolamine Antibodies in Patients with Recurrent Pregnancy Loss Augment Platelet Aggregation.

PROBLEM: Numerous studies have suggested that factor XII (FXII) deficiency, autoantibodies to FXII (anti-FXII), and antiphosphatidylethanolamine antibodies (aPE) are associated with recurrent pregnancy loss (RPL). aPE in RPL patients recognize the LDC27 peptide of kininogen domain 3. Anti-FXII in RPL patients recognizes the heavy chain of FXII, especially the amino-terminal sequences IPP30 peptide. Previous studies suggested that LDC27 and IPP30 are the responsible sites competing for the same binding site on platelets and inhibiting augmentation of thrombin-induced platelet aggregation. Our aim was to study the influence of antibodies to LDC27 and IPP30 on platelet aggregation. METHODS OF STUDY: In fifteen healthy volunteers, platelet aggregation induced by γ-thrombin in the presence or absence of antibodies to LDC27 and IPP30 was measured. Sixteen RPL patients who were positive for anti-FXII were measured for spontaneous small platelet aggregate (SSPA) formation. RESULTS AND CONCLUSIONS: Antibodies to LDC27 and IPP30 markedly increased aggregation of normal platelets stimulated by γ-thrombin. Augmentation of SSPA formation was more frequent in the patients with RPL who were positive for anti-FXII than in the control group (P = 0.003). This study strongly supports the hypothesis that aPE and anti-FXII may cause RPL due to disruption of the normal antithrombotic effects of kininogens and FXII.

Kininogen-dependent antiphosphatidylethanolamine antibodies and autoantibodies to factor XII in patients with recurrent pregnancy losses.

Factor XII, plasma prekallikrein and high-molecular-weight kininogen were first identified as coagulation proteins in the intrinsic pathway because patients deficient in these proteins had marked prolongation of in vitro surface-activated coagulation time. However, deficiencies of these proteins are not associated with clinical bleeding. Paradoxically, studies suggest that these proteins have anticoagulant and profibrinolytic activities. In fact, association between deficiencies of these proteins and thrombosis has been reported. Recently, autoantibodies to these proteins and antiphospholipid antibodies are frequent coagulation-related abnormalities found in unexplained recurrent aborters. Evidence has accumulated for the presence of the kallikrein-kininogen-kinin system in the fetoplacental unit. The contact system, or kallikrein-kininogen-kinin system, in the reproductive tract plays an essential roll in the regulation of thrombosis, hemostasis, angiogenesis and in the defense against invasive bacterial infection. Autoantibodies to these proteins may be associated with pregnancy losses due to disruption of this system. These possibilities will be reviewed, the functions of the individual components will be summarized, and their role in blood coagulation and pregnancy discussed.

Expression of apoptosis in placenta of experimental antiphospholipid syndrome mouse.

PROBLEM: To study the histopathology and expression of apoptosis in placenta of pregnancy-complicated antiphospholipid syndrome (APS)-positive mouse models. METHOD OF STUDY: ICR mice were immunized with IgG isotype of human anticardiolipin (aCL) and/or lupus anticoagulant (LA). The pathological and apoptotic expression was studied in the placenta of positive APS mice and compared with respective control samples. RESULTS: Mice with passive immunization of human aCL and/or LA produced an increase in fetal resorption along with markedly induced thrombosis in the placenta associated with increased placental apoptosis. In addition, fewer mitotic cells, less trophoblast giant cell invasion, and more shrunken cells in the deciduas were seen. CONCLUSION: Our study showed the pathogenic role of aCL and LA in pregnancy complications.

A unique preliminary study on placental apoptosis in mice with passive immunization of anti-phosphatidylethanolamine antibodies and anti-factor XII antibodies.

PROBLEM: Antiphospholipid antibodies have been investigated both in humans and in animal models. In contrast, there are fewer reports describing anti-phosphatidylethanolamine (aPE) antibodies in humans, and there are no reports of animal studies with aPE till date. Clinically, FXII deficiency or anti-FXII antibodies are sometimes associated with aPE in patients with recurrent pregnancy loss. Therefore, we asked whether aPE and/or anti-FXII in mice could cause fetal resorption, placental thrombosis and apoptosis. Moreover, antibodies to respective target antigens (LDC27 or IPP30) could cause pregnancy failure as well. METHODS OF STUDY: Animal models were used to carry out these objectives. All the animals were immunized with different antibodies by passive immunization. Placental samples were used for various observations. RESULTS AND CONCLUSIONS: Mice with passive immunization of aPE (or anti-LDC27) and aFXII (or anti-IPP30) produced a slight increase in fetal resorption, but markedly induced thrombosis and hemorrhage in the placenta associated with lower platelet counts and increased placental apoptosis. In addition, fewer mitotic cells, less trophoblast giant cell invasion, and more shrunken cells in the deciduas were seen. Our study supports the pathogenic role of aPE and aFXII in pregnancy complications and also suggests a beneficial role of LDC27 and IPP30 antigens on pregnancy failures.

Antiphosphatidylethanolamine antibodies might not be an independent risk factor for further miscarriage in patients suffering recurrent pregnancy loss.

The prevalence of antiphosphatidylethanolamine antibodies (aPEs) is higher in recurrent pregnancy loss patients than that in women with normal pregnancy. We conducted a cohort study to examine the predictive value of aPE for recurrent pregnancy loss and to determine its clinical significance. We examined plasma protein dependent (P+) and independent (P-) aPE IgG and IgM antibodies in 367 women with two or more unexplained consecutive pregnancy losses. We also examined conventional antiphospholipid antibodies (aPL) such as beta2-glycoprotein I-dependent anticardiolipin antibodies (beta2GPI-dependent aCL), lupus anticoagulant with reference to the dilute activated partial thromboplastin time (aPTT) and the diluted Russell's viper venom time (RVVT). Subsequent pregnancy outcome without medication was examined, and patients with and without aPE were compared. Totals of 37 (10.1%), 14 (3.8%), 23 (6.3%), 6 (1.6%), 9 (2.5%), 10 (2.7%) and 50 (13.6%) of the 367 patients were, respectively, positive for P+aPE IgG, P-aPE IgG, P+aPE IgM, P-aPE IgM, beta2GPI-dependent aCL, lupus anticoagulant by RVVT and LA by aPTT. The patients with aPE differed from patients with beta2GPI-dependent aCL or lupus anticoagulant by RVVT. No difference in live birth rate was apparent between positive and negative aPE patients with no medication. The areas under the curves for each ROC curve for the four aPEs were 0.535, 0.612, 0.546 and 0.533, respectively, so there was no significant variation in diagnostic capacity. We did not obtain any evidence that aPE elevation is an independent risk factor to predict further miscarriage in recurrent pregnancy loss patients.

Subsequent pregnancy outcomes in recurrent miscarriage patients with a paternal or maternal carrier of a structural chromosome rearrangement.

Information concerning the prognosis of subsequent pregnancies in patients with reciprocal translocations is limited. This study was performed to determine the percentage success rate with first pregnancies after ascertainment of a carrier status. A total of 2,382 couples with a history of two or more consecutive miscarriages were studied in multicenters. The prevalence of an abnormal chromosome in either partner was examined, and subsequent success rates were compared between cases with and without an abnormal karyotype in either partner. A total of 129 couples (5.4%) had an abnormal karyotype in one partner excluding inversion 9 in 44 men and in 85 women. Thus, 2,253 couples had a normal karyotype in both partner. Eighty-five (3.6%) had translocations, 13 being Robertsonian translocations. Twenty-nine of the 46 cases (63.0%) who became pregnant with reciprocal translocations in either partner experienced a live birth with natural conception. In contrast, 950 of 1,207 cases (78.7%) with normal chromosomes had successful live births, the difference being significant (P = 0.019). No infant with an unbalanced translocation was found in 29 cases of successful pregnancy following recurrent miscarriage. Pregnancy prognosis was worsened with either maternal or paternal reciprocal translocations. Explanation of the success rate with natural conception should be provided before the subsequent pregnancy after ascertainment of carrier status.

Decreased factor XII activity is associated with recurrent IVF-ET failure.

PROBLEM: A decrease in factor XII (FXII) activity has been observed in women with unexplained recurrent miscarriages. Because of the many similarities between recurrent in vitro fertilization-embryo transfer (IVF-ET) failure and early pregnancy loss patients, we investigated whether women with recurrent IVF-ET failure had low FXII activity. METHOD OF STUDY: Blood from 110 patients with three or more IVF-ET failures was tested for FXII activity, autoantibodies and other coagulation parameters. These patients were compared with 191 recurrent miscarriage patients, 87 controls. FXII activity was measured by an activated partial thromboplastin time (APTT) assay. RESULTS: Factor XII activity of the IVF-ET failure group (median 79.5, range 37-150) was significantly lower than that of control group (median 109, range 35-225, P < 0.001 by Mann-Whitney test) or that of recurrent pregnancy loss (RPL) group (median 92, range 20-205). The FXII activity of IVF-ET failure patients was positively correlated with age, but not associated with the number of IVF-ET failures, infertile duration, autoantibodies, or other coagulation parameters. CONCLUSION: Decreased FXII activity may causally relate to reproductive failure.

The antigenic binding sites of autoantibodies to factor XII in patients with recurrent pregnancy losses.

Recently, numerous studies have suggested an association between factor XII (FXII) deficiency and recurrent pregnancy losses, and between autoantibodies to FXII and recurrent pregnancy losses. Autoantibodies to FXII rather than FXII deficiency may be a risk factor for recurrent pregnancy losses. To know the pathogenesis of autoantibodies to FXII, epitope mapping study was done. Seventeen anti-FXII antibody positive recurrent pregnancy loss patients were chosen for this study. We used synthetic peptides in inhibition and direct binding studies to identify the antigenic binding site of autoantibodies to FXII. Among plasmas from 17 recurrent pregnancy loss patients who were positive for autoantibodies to FXII, 13 patients (76.5%) recognized amino acids 1-30, the amino-terminal heavy chain region that is known as factor XII binding site to platelet glycoprotein Ibalpha.

IgG-antiphospholipid antibodies in follicular fluid of IVF-ET patients are related to low fertilization rate of their oocytes.

PROBLEM: Patients undergoing in vitro fertilization and embryo transfer (IVF-ET) failures show an increased incidence of antiphospholipid antibodies (aPL) in their blood. The physiological manifestations of aPL in this patient group are nonetheless controversial. Pathological effects of aPL on embryos in vitro have been documented. We questioned whether aPL if found in follicular fluids (FFs) could result in embryonic damage. METHOD OF STUDY: Blood from 44 patients with three or more IVF-ET failures were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of immunoglobulin (Ig)G, IgM and IgA aPL. Both the 29 aPL-positive and 15 aPL-negative patients gave permission for FF collection during their next IVF-ET attempt for additional aPL determinations. RESULTS: Patients with no aPL in their blood, had no aPL in their FFs. Patients with IgG and/or IgM aPL in their blood had IgG but not IgM in their respective FFs. CONCLUSIONS: The presence of IgG aPL in FFs and increased infertility length were significantly related to lower fertilization rates, independently. Follicular fluid IgG aPL appears as a risk factor in association with successful IVF-ET outcomes.

Abnormal placental cord insertion may induce intrauterine growth restriction in IVF-twin pregnancies.

BACKGROUND: This study determined whether twins conceived through assisted reproduction technology (ART) have an increased risk of perinatal complications compared with natural twin pregnancies and investigated potential associated major risk factors. METHODS: This retrospective study consisted of 199 twins born between 1994 and 2003. There were four groups according to conception modalities: 97 twins after spontaneous pregnancy, 24 after induced ovulation, 28 after intrauterine insemination (IUI) and 50 after IVF with embryo transfer. Analysis included preterm birth, Caesarean delivery, weight discordance, intrauterine growth restriction (IUGR), low-birth-weight, Apgar score, chorionicity, gross placental pathology and placental umbilical cord insertion (UCI) site. RESULTS: A significant difference was found in IUGR between the IVF group (7.0%) and spontaneous pregnancy group (14.9%). When maternal age was >30 years there was a 2.86-fold increase in the risk of IUGR. There was a 3.69-fold increased risk of IUGR in the presence of abnormal UCI (odds ratio 3.69, 95% CI 1.62-8.42) and a 2.18-fold increased risk of abnormal UCI in monochorionic twins when compared with dichorionic twins (odds ratio 2.18, 95% CI 1.30-3.66). CONCLUSION: Twins conceived through ART are not at an increased risk of perinatal complications. A relationship has been found between abnormal UCI and IUGR.

Preconception peripheral natural killer cell activity as a predictor of pregnancy outcome in patients with unexplained infertility.

PROBLEM: Preconception high peripheral natural killer (NK) cell activity in women with recurrent spontaneous abortion can predict subsequent miscarriages. We have examined prospectively, for the first time, the pregnancy rate in patients with unexplained infertility by measuring the peripheral NK activity. METHOD OF STUDY: We tested the peripheral NK activity of 94 infertile women who despite treatment were unable to conceive for 6 or more months (mean; 2.4 years). Peripheral NK activity was measured by a chromium-51 release cytotoxicity assay. Women were followed for 2 years and assessed. RESULTS: In 77 patients who were followed for 2 years, 28 had conceived but 49 did not. The peripheral NK activity of the group that became pregnant (mean +/- S.D.; 34.5 +/- 13.8%) was significantly lower than that of non-conception group (42.3 +/- 13.3%, P = 0.017). CONCLUSIONS: Our finding suggests that elevated peripheral NK activity in patients with unexplained infertility is a risk factor for attaining pregnancy success.

Platelet inhibition by single low-dose aspirin, using the newly developed aggregometry with the laser light scattering method.

Low-dose aspirin (acetylsalicylic acid 81 mg/day, LDA) is often used as an antiplatelet drug in the treatment of cardiovascular and cerebrovascular diseases as well as for patients with anti-phospholipid antibody syndrome. In this study, we explored the duration of the inhibitory effect of a single LDA on platelet aggregation, using the newly developed aggregometry with the laser light scattering method. Five healthy volunteers (females between 23 and 30 years old) ingested 81 mg of buffered aspirin. Platelet aggregation was measured with adenosine 5'-diphosphate before the ingestion and at the 1st, 2nd, 4th, 6th, and 8th day thereafter. The results showed that the effect of 81 mg of aspirin continues for at least 8 days, which suggested that the intermittent administration of 81 mg of aspirin (a few times a week) might be an alternative way to induce the anti-platelet effect.

Antiphosphatidylethanolamine antibodies in recurrent early pregnancy loss and mid-to-late pregnancy loss.

AIM: Associations have been reported between antiphospholipid antibodies (aPL), mainly anticardiolipin antibodies (aCL) and/or the lupus anticoagulant, and recurrent pregnancy losses (RPL). However, relatively few studies describing antiphosphatidylethanolamine antibodies (aPE) have been reported. We describe the prevalence of aPL to both cardiolipin and phosphatidylethanolamine in patients with RPL. METHODS: Patients with recurrent early pregnancy losses (n = 145) and mid-to-late pregnancy loss(es) (n = 26) were screened for aPE and aCL. RESULTS: In patients with recurrent early pregnancy losses, prevalence of immunoglobulin G (IgG) aPE (17.9%, P = 0.001) and immunoglobulin M (IgM) aPE (12.4%, P = 0.01) was significantly higher than in the control group. In patients with mid-to-late pregnancy loss(es), prevalence of IgM aPE (19.2%, P = 0.008) and IgG aCL (23.1%, P = 0.02) was significantly higher than in the control group. CONCLUSION: Our data suggest that aPE may be a risk factor in patients with mid-to-late pregnancy loss(es) as well as recurrent early pregnancy losses.

Antiphospholipid antibodies and kininogens in pathologic pregnancies: a review.

PROBLEM: Recently, evidence has accumulated for the presence of the kallikrein-kinin system or plasma contact system in the fetoplacental unit. The Kallikrein-kinin system or plasma contact system consists of three essential plasma proteins. These are coagulation factor XII, prekallikrein and high molecular weight kininogen. Deficiencies of these proteins and antiphospholipid antibodies are frequent hemostasis-related abnormalities found in unexplained recurrent aborters. METHOD OF STUDY: Review of existing data. RESULTS: Reports of antiphosphatidylethanolamine antibodies (aPE) with similar or identical pathogenic associations as those described for anticardiolipin antibodies (aCL) are found in the literature. We showed a strong association between recurrent pregnancy losses and aPE, the latter of which recognizes kininogens, and kininogen-binding proteins, factor XI and prekallikrein. The reports of aPE are reviewed, the function of the kininogens are summarized, and their role in pregnancy is discussed. CONCLUSIONS: Because kallikrein-kinin system may play an important role in pregnancy especially in fetoplacental unit, disruption of this system may be a risk factor for early gestational losses.