Role of mu-calpain in human decidua for recurrent miscarriage.

PROBLEM: Earlier, we showed that calpain activated by increasing intracellular calcium ion concentration in endometrial cells, causes endometrial dysfunction for implantation and early pregnancy. In the present study, we investigated the existence and distribution of calpains, calpastatin, integrin beta3 and alpha-fodrin in decidua from patients with recurrent miscarriage. METHOD OF STUDY: Deciduae were surgically collected from 29 patients with recurrent miscarriage and 20 healthy women with informed consent. Immunohistochemistry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and western blot analysis were performed. RESULTS: Staining of mu-calpain, m-calpain, calpastatin, integrin beta3 and alpha-fodrin were observed in the cytoplasm of stromal and epithelial cells in decidua using immunohistochemistry. No significant differences were observed in staining patterns. Western blot analysis showed no significant differences in expression of m-calpain, calpastatin and alpha-fodrin, whereas mu-calpain was significantly higher and integrin beta3 was lower in subject. CONCLUSION: The results suggest that cleavage of integrin beta3 by mu-calpain may have an adverse effect on the mechanism of early pregnancy.

Occasional antiphospholipid antibody positive patients with recurrent pregnancy loss also merit aspirin therapy: a retrospective cohort-control study.

PROBLEM: It is well known that treatment with aspirin plus heparin is effective for patients with antiphospholipid syndrome (APS) to prevent pregnancy loss. However, it is unclear if occasional antiphospholipid antibodies (aPL) are a risk factor and whether patients with aPL at one time point but not diagnosed as APS should be treated. METHOD OF STUDY: We therefore studied whether aspirin alone is effective in patients with occasional aPL who did not meet the criteria for APS. We compared live birth rates between 52 patients with occasional aPL treated with aspirin and 672 unexplained patients with no medication. Patients in both group had a history of two or three pregnancy losses. RESULTS: In all, 44 of 52 patients (84.6%) with occasional aPL could experience live birth when treated with aspirin alone. 509 of 672 patients (75.7%) with unexplained pregnancy losses could have babies. When miscarriage cases caused by an abnormal embryonic karyotype were excluded, the success rates were 95.7% (44/46) and 81.2% (509/621), respectively. The live birth rate in patients with occasional aPL treated with aspirin was significantly higher than that in unexplained patients with no medication (P=0.008). CONCLUSION: We therefore conclude that aspirin is also useful in patients with occasional aPL but not APS.

Uterine cervical inflammatory cytokines, interleukin-6 and -8, as predictors of miscarriage in recurrent cases.

PROBLEM: Inflammatory changes frequently occur in cases of second trimester miscarriage or pre-term delivery, but little attention has been paid to this association with recurrent miscarriage. As interleukin-6 and interleukin-8 are inflammatory cytokines reported to be associated with bacterial vaginosis, intrauterine infections, and pre-term delivery, we here investigated whether they might have predictive value for spontaneous abortion in recurrent cases. METHOD OF STUDY: Cervical mucus and sera were collected at 4-5 weeks' gestation from a total of 59 patients with a history of two or more unexplained consecutive first trimester miscarriages, and examined by enzyme-linked immunosorbent assay. Patients then were followed up without medication and their pregnancy outcomes were compared with the test results. RESULTS: Of a total of 59 patients, 13 (22%) miscarried subsequently. Both IL-6 and IL-8 in cervical mucus were significantly higher in patients who miscarried subsequently than in those who had a live birth. In addition, there was no correlation between cervical mucus and serum concentrations of IL-6 and IL-8 take at the same time, and there was no relation with serum IL-6 and IL-8 levels between the two groups. CONCLUSION: Cervical IL-6 and IL-8 might have predictive value for cases of recurrent miscarriage.

Complement as a predictor of further miscarriage in couples with recurrent miscarriages.

BACKGROUND: The clinical significance of complement is unclear in patients with unexplained recurrent miscarriage, though low levels of complement 3 (C3) and/or complement 4 (C4) are reported to be associated with the antiphospholipid syndrome (aPS). We therefore investigated whether C3 and C4 have a predictive value for subsequent miscarriages. METHODS: In total, 215 patients with a history of two consecutive first-trimester miscarriages and no abnormal chromosomes in either partner, no uterine anomalies and no antiphospholipid (aPL) antibodies were examined. Blood tests for C3, C4, total haemolytic complement (CH50), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) were performed before subsequent pregnancy. Patients were then followed up without treatment, and their pregnancy outcomes were compared with their previous blood test results. RESULTS: From 215 pregnant patients, 45 subsequently miscarried, whereas the remainder had a live birth. There was no relation with serum CH50, IgG, IgA and IgM levels, but C3 and C4 levels in patients with subsequent miscarriage were significantly higher than in those whose pregnancy was successful. CONCLUSION: In patients with two previous miscarriages, C3 and C4 levels were higher in those women who had a third miscarriage, than in women that went on to have a live birth.

Elevated aldosterone in amniotic fluid and maternal blood has diagnostic potential in pregnancies complicated with a fetus of Bartter syndrome.

Pregnancies with fetuses affected with the Bartter syndrome, an autosomal recessive disorder of hyperreninism and hyperaldosteronism, are complicated by early onset of polyhydramnios which results in preterm deliveries. We have assessed biochemical changes in amniotic fluid and the mother's blood with a view to early diagnosis. Aldosterone levels of both amniotic fluid and the mother's blood were found to be increased at 27 weeks of gestation, while electrolyte levels did not differ significantly from those reported earlier for controls. After birth the baby suffered from polyuria with hyponatremia, hypomagnesemia and hypercalciuria which could be controlled by treatment with sodium chloride and magnesium. Elevated aldosterone thus might be a useful marker for early diagnostic purposes.

Role of cathepsins and cystatins in patients with recurrent miscarriage.

In the implantation, trophoblasts penetrate maternal decidua by secreting proteases. It has been reported that cathepsins are highly expressed in the mouse villi, and play an important role in normal embryonal growth and decidualization. In this study, we evaluated cathepsins and their endogenous inhibitors, cystatins, in tissue and serum of patients with recurrent miscarriage. Decidua and villi were surgically collected from 22 patients and 12 healthy women. Immunohistochemistry was performed with antibodies against cathepsins, stefin A (cystatin A), stefin B (cystatin B) and cystatin C. The concentrations of cathepsins, stefins and cystatin C were measured by Enzyme-linked immunosorbent assay. In addition, we measured the serum level of cystatin C in 85 Japanese women with recurrent miscarriage. Staining of cathepsin B, D, H, L, stefin B and cystatin C was observed in the cytoplasm of epithelial cells in decidua. Stefin A was expressed on the surface of the trophoblast. The concentration of cathepsin B and H in patients' decidua was significantly higher than in control individuals. The serum level of cystatin C was significantly lower in patients than in control individuals. Our findings suggest that the regulation of the cathepsin-cystatin system may play an important role in patients with recurrent miscarriage.

Pregnancy outcome in recurrent aborters is not influenced by Chlamydia IgA and/or G.

PROBLEM: It is unclear whether chlamydia infection influences the miscarriage rate and immunological factors in patients with recurrent miscarriage. METHOD OF STUDY: Chlamydia DNA, IgA and IgG to Chlamydia trachomatis, natural killer cell activity, complement 3 (C3), C4, hemolytic complement, antinuclear antibodies, antiphospholipid antibodies, prolactin, activated partial thromboplastin time, prothrombin time and fibrinogen were examined in 504 patients with a history of two or more consecutive first-trimester miscarriages. Subsequent pregnancy outcomes were compared between cases with and without antibodies to C. trachomatis. RESULTS: Totals of 10 of 30 and 48 of 201 patients receiving no medication miscarried subsequently with and without chlamydia infection. Chlamydia IgA and/or IgG were associated with a high level of C3 but not other immunological and coagulatory parameters. CONCLUSION: Antibodies to C. trachomatis do not influence subsequent pregnancy outcome in patients with a history of recurrent miscarriage.

No association of C677T methylenetetrahydrofolate reductase and an endothelial nitric oxide synthase polymorphism with recurrent pregnancy loss.

PROBLEM: It is controversial whether polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and the endothelial nitric oxide synthase (eNOS) are associated with recurrent pregnancy loss. METHOD OF STUDY: We studied the frequency of the C677T polymorphism of MTHFR and a eNOS gene polymorphism, as well as the plasma levels of homocysteine and NO, in 85 cases with a history of two or more unexplained embryonal losses, 40 patients suffering fetal loss and 76 controls. RESULTS: The frequency of the MTHFR gene T allele, which has been reported to be associated with miscarriages, in patients suffering fetal loss was rather significantly lower than in controls whereas there was no difference in the frequency of the eNOS gene A allele. There were no differences in the plasma homocysteine levels among the three groups. However, the NO concentrations in the embryonal loss and fetal loss groups were significantly higher than that in controls. CONCLUSION: We conclude that the NO concentration but not the polymorphism of MTHFR and eNOS gene and hyperhomocysteinemia are associated with recurrent pregnancy loss in Japanese.

The role of cathepsin B and cystatin C in the mechanisms of invasion by ovarian cancer.

OBJECTIVE: The aim of this study was to investigate the contribution of cathepsin B and cystatin C to the mechanisms of invasion by ovarian cancer. MATERIALS AND METHODS: Using surgical materials from patients with ovarian cancer, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis were performed using antibodies against cathepsin B or cystatin C. Serum levels of cathepsin B and cystatin C in patients with benign and malignant ovarian lesions were determined by enzyme-linked immunosorbent assay (ELISA). An invasion assay using an ovarian cancer cell line was performed by addition of cystatin C or specific inhibitors of cathepsin B. RESULTS: While immunohistochemical staining of cathepsin B and cystatin C was evident in cancer cells and associated stromal tissue, this was not the case in benign tumors. The malignancies were also found to be positive for cathepsin B and cystatin C by SDS-PAGE and Western blotting analysis. No significant difference in serum cathepsin B levels was observed between patients with benign and malignant disease. However, the concentration of cystatin C in cases with ovarian cancer was significantly higher in benign cases (P<0.0001) and in healthy controls (P<0.0001). Invasion by cancer cells was dose-dependently suppressed by cystatin C and cathepsin B inhibitors. CONCLUSION: The results provided convincing evidence that cathepsin B and cystatin C may contribute to the mechanisms of invasion of ovarian cancer.

Cleavage of integrin by mu-calpain during hypoxia in human endometrial cells.

PROBLEM: The distribution and activation of mu-calpain and possible cleavage of integrin in human endometrial cells under hypoxic condition were investigated. METHOD OF STUDY: Human endometrial epithelial and stromal cells were subjected to hypoxia, and subsequently used for immunostaining and western blot analysis. RESULTS: The proform of mu-calpain was detected in the cytoplasm of normal cells, and displayed a substantial decrease after hypoxia. Conversely, the active form of mu-calpain was not detected in normal cells, but was abundant after hypoxia. The cytoplasmic domain of integrin beta3 was also detected in the cytoplasm of endometrial cells. Western blot analysis confirmed that both the proform of mu-calpain and the integrin beta3 cytoplasmic domain decreased during hypoxia. CONCLUSIONS: Mu-calpain is activated in human endometrial cells during hypoxia and that subsequent cleavage of the integrin beta3 cytoplasmic domain may give some adverse effects to the function of human endometrium.

Conservative treatment by angiographic artery embolization of an 11-week cervical pregnancy after a period of heavy bleeding.

OBJECTIVE: To describe a rare case of conservative treatment of an 11-week cervical pregnancy after a period of heavy bleeding. DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A 33-year-old woman was admitted to our hospital for treatment of a cervical pregnancy. Two-and-a-half years thereafter, she gave birth to a healthy baby by vaginal delivery at 38 weeks of gestation. INTERVENTION(S): Systemic methotrexate treatment, ligation of descending branches of uterine arteries, cervical cerclage, and unilateral internal iliac artery embolization. MAIN OUTCOME MEASURE(S): Transvaginal ultrasound, magnetic resonance imaging, and arteriography findings. RESULT(S): The patient was successfully treated with unilateral internal iliac artery embolization on the same side as the pregnancy in the 11th gestational week. CONCLUSION(S): After failed methotrexate and vessel ligation in cervical pregnancy, unilateral internal iliac artery embolization is an effective and conservative treatment that allows preservation of reproduction potential.