【参考文献】American Journal of Reproductive
Antiphospholipid Antibodies in Recurrent Early Pregnancy Loss and Mid to Late Fetal Loss*
Toshitaka Sugi, M.D., Ph.D., Hidehiko Matsubayashi, M.D., Ph.D.,
Tsunehisa Makino, M.D., Ph.D..
From the Department of Obstetrics and Gynecology, Tokai University School of Medicine, Isehara, Japan.
*Report of the Japan Antiphospholipid Project Group
PROBLEM: 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.
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 IgG aPE (17.9%, p=0.001) and 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.
CONCLUSIONS: Our data suggest that aPE may be a risk factor in patients with mid to late fetal loss(es) as well as recurrent early pregnancy losses.
Antiphosphatidylethanolamine antibodies, anticardiolipin antibodies, kininogen, antiphospholipid syndrome
Antiphospholipid syndrome has been described as the association of thromboembolic events and/or pregnancy loss with presence of lupus anticoagulant and antiphospholipid antibodies (aPL) to anionic phospholipids (PL) such as cardiolipin.1,2 Besides cardiolipin, other PL have been used in ELISA plates, both anionic and neutral, or even zwitterionic as phosphatidylethanolamine (PE).
Recent evidence shows that many aPL to negatively charged PL do not target anionic PL per se, but are specific for anionic PL-binding plasma proteins. At present, the most common and best characterized plasma protein aPL antigenic targets are b2-glycoprotein I (b2GPI) and prothrombin.3 We also reported that certain antiphosphatidylethanolamine antibodies (aPE) are not specific for PE per se, but are directed to PE-binding plasma proteins; for example, high molecular weight kininogen (HK), low molecular weight kininogen (LK) and proteins in complex with HK, factor XI or prekallikrein.4,5
The female reproductive tract is the second richest site for kininogen and its metabolic products in the body.6-9 The kininogen concentration in reproductive tissues and plasma was reported to fluctuate during ovulation, pregnancy, and parturition.6,9 Why the female reproductive system is so rich in kininogen and what governs the fluctuation of kininogen concentrations at the local level remains to be elucidated.
It is noteworthy that aPE have been described as the sole aPL in patient with thrombotic diseases.10-16 Recently, we reported a stronger association between recurrent pregnancy losses (RPL) and aPE than between RPL and antibodies to anionic PL for early gestational losses.17 A positive test result for aPE was more frequent in the patients with recurrent early pregnancy losses than in the members of the control group (p=0.0002). In contrast, there was no statistically significant difference in the incidence of positive test results for antibodies to anionic PL between the recurrent early pregnancy loss group and the control group. In this study, 21 patients were positive for plasma protein-dependent IgG aPE. Nineteen (90.5%) of these 21 patients were kininogen-dependent. Gris et al. also reported that aPE were found to be independent risk factor for unexplained early fetal loss.18 Since PE is a major component of both the outer and inner leaflets of cell plasma membranes, autoantibodies to PE and/or PE binding proteins might exert an effect on both resting and activated cells or cell fragments.
Fetal loss, occurring mostly in the second and third trimesters of pregnancy in aCL and/or lupus anticoagulant positive mothers, is one of the hallmark clinical manifestations of the antiphospholipid syndrome.19-21 There is however no available data concerning the prevalence of aPE in patients with mid to late pregnancy loss. In this study, we showed data on patients with recurrent early pregnancy losses and mid to late fetal loss.
MATERIALS AND METHODS
Patients and Controls
From January 2001 to December 2001, serum samples were obtained from 171 nonpregnant patients with a history of pregnancy loss(es) who referred to the SRL laboratory (Tokyo, Japan) for detection of aPL. All patients were referred by obstetricians of the 9 hospitals, who were involved in our abnormal pregnancy program. Among the patients, 2 groups were defined according to clinical data. The main biological and clinical characteristics of these groups of patients are given in Table I.
Early pregnancy loss group consisted of 145 patients with at least 2 episodes of unexplained early pregnancy loss defined as pregnancy loss before the 10th week of pregnancy; exclusive of ectopic pregnancy and elective abortion. None of them had any episode of mid to late fetal loss defined as fetal loss at or beyond the 10th week of pregnancy.
Mid to late pregnancy loss group consisted of 26 patients with one or more unexplained death(s) of a morphologically normal fetus at or beyond the 10th week of pregnancy.
The exclusion criteria were: any presumptive cause found for pregnancy loss(es) after routine evaluation for detection of uterine factors (i.e., normal hysterosalpingography and ultrasound examinations); any chromosomal abnormalities for both partners; any endocrine factors (normal prolactin and progesterone levels and normal thyroid function); any infectious factors (no group B streptococcal or Chlamydia trachomatis infection); any diabetes mellitus.
One hundred twenty two age matched, healthy, nonpregnant female volunteers with no history of miscarriage were tested as controls.
Sera were collected by clear venipuncture from the antecubital vein and stored at -80°C until use.
The aPE ELISA followed a previously described procedure.17 Briefly, Immulon 1, microtiter plates (Dynatech Laboratories, Chantilly, VA, USA) were coated with 30 ml of 50 mg/ml PE (Avanti Polar Lipids, Birmingham, AL, USA) diluted in chloroform : methanol (1:3) per well and dried under nitrogen. Each well was blocked for 1h with 10% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) in Tris buffered saline (TBS). To detect aPE ELISA reactivity, 50 ml of the patient sera diluted 1/100 in TBS containing 10% adult bovine plasma (ABP; Sigma Chemical Co.) was incubated for 1h. aPE was assessed by using alkaline phosphatase conjugated monoclonal antibody to human IgG and IgM (Sigma Chemical Co.). The plates were washed 3 times with TBS after PE coating, blocking, serum and conjugate incubations. Color development, produced by paranitrophenyl phosphate substrate, was measured by optical density at 405nm. Color development was stopped with 75 ml of 3N NaOH when the positive controls reached an optimal density of 1.0 at 405nm. Nonspecific binding control wells (without phospholipid coating) were processed in parallel with the ELISA for aPE, and the background values were subtracted. Positive values were determined as previously described.17 Positive samples were retested to confirm the results.
Serum IgG and IgM aCL were measured by a commercially available ELISA (Mesacup, MBL, Nagoya, Japan). This kit contains 10% adult bovine serum in its dilution buffer. Calibration was performed using a dilution curve prepared with the provided commercial calibrator which had been previously evaluated against standards from the Antiphospholipid Standardisation Laboratory, University of Louisville, Kentucky, USA. A normal range was established using the control individual sera with the cut-off corresponding to the 95th percentile of the obtained control values. Thus, IgG-aCL<10 GPL units/ml and IgM-aCL <8 MPL units/ml were considered negative.
Serum IgG to b2GPI-cardiolipin complex (aCL-b2GPI, IgG) was measured by a commercially available ELISA (Yamasa Corporation, Tokyo, Japan) according to the manufacturer's recommendations. This kit contains purified human b 2GPI (1.51 mg/well) in the dilution buffer. Positive values were determined by previously described methods.17
Differences between the two groups were analyzed for statistical significance (p<0.05) with the c2test.
Table I. Characteristics of the 3 groups of women in the study
pregnancy loss group Mid to late pregnancy
loss group Control women
N 145 26 122
Age (mean+SD) [range] 31.9+4.4 [21-43] 32.4+3.9 [25-39] 32.5+4.4 [26-41]
Number of pregnancy losses (mean+SD) [range]
Before the 10th week of pregnancy 2.8+1.2 [2-10] 1.7+1.3 [0-7] 0
At or beyond the 10th week of pregnancy 0 1.5+1.6 [1-3] 0
Prevalence of aPL in the early pregnancy loss group
Patients with recurrent early pregnancy losses (n=145) were screened for aPE. The prevalence of aPL in the early pregnancy loss group is reported in Table II and compared to that of normal controls. Twenty six (17.9%) and 18 (12.4%) patients of these 145 patients were positive for IgG and IgM aPE, respectively. Positive test results for IgG aPE [odds ratio: 4.22, 95% confidence interval (1.68-10.64), p=0.001] and IgM aPE [odds ratio: 4.18, 95% confidence interval (1.38-12.71), p=0.01] were more frequent in the early pregnancy loss group than in the control group. Four patients were positive for both IgG and IgM, aPE. Therefore, 40 (27.6%) patients were positive for IgG and/or IgM, aPE. The higher retrospective risk for recurrent early pregnancy loss was associated with positive IgG aPE.
The same 145 patients with recurrent early pregnancy losses also were tested for aCL. IgG and IgM aCL were measured with Mesacup kit and aCL-b2GPI, IgG was measured with Yamasa kit. As shown in Table II, 9 patients (6.2%) and 4 patients (2.8%) were positive for IgG and IgM aCL, respectively. Five patients (3.5%) were positive for aCL-b2GPI, IgG. There was no statistically significant difference in the incidence of positive test results for aCL between the early pregnancy loss group and the control group. Only 4 patients out of 40 aPE positive patients were also positive for aCL.
Prevalence of aPL in the mid to late pregnancy loss group
Patients with mid to late pregnancy loss(es) (n=26) were screened for aPE. The prevalence of aPL in the mid to late pregnancy loss group is reported in Table III and compared to that of normal controls. Three (11.5%) and 5 (19.2%) patients of these 26 patients were positive for IgG and IgM aPE, respectively. Two patients were positive for both IgG and IgM aPE. A positive test result for IgM aPE was more frequent in the mid to late pregnancy loss group than in the control group [odds ratio: 7.02, 95% confidence interval (1.74-28.32), p=0.008]. A positive test result for IgG aPE was not statistically more frequent in the patients with mid to late pregnancy loss than in the members of the control group.
The same 26 patients with mid to late pregnancy loss(es) also were tested for aCL. IgG and IgM aCL were measured with Mesacup kit and aCL-b2GPI, IgG was measured with Yamasa kit. As shown in Table III, 6 patients (23.1%) and 3 patients (11.5%) were positive for IgG and IgM aCL, respectively. Three patients (11.5%) were positive for aCL-b2GPI, IgG. A positive test result for IgG aCL was more frequent in the mid to late pregnancy loss group than in the control group [odds ratio: 4.28, 95% confidence interval (1.34-13.64), p=0.02]. There was no statistically significant difference in the incidence of positive test results for IgM aCL or aCL-b2GPI, IgG between the mid to late pregnancy loss group and the control group. The higher retrospective risk for mid to late pregnancy loss was associated with positive IgM aPE. Only one patient out of 6 patients, who were positive for IgG and/or IgM aPE, was positive for aCL.
Comparison of positive rates for various antiphospholipid antibodies in the early pregnancy loss gourp (n=145)
Early pregnancy loss
group (n=145) Control women
n (%) Odds ratio 95%CI p n (%)
aPE, IgG 26 (17.9) 4.22 1.68-10.64 0.001 6 (4.9)
aPE, IgM 18 (12.4) 4.18 1.38-12.71 0.01 4 (3.3)
aCL, IgG 9 (6.2) 0.94 0.35-2.52 NS 8 (6.6)
aCL, IgM 4 (2.8) 0.83 0.2-3.42 NS 4 (3.3)
aCL-b2GPI, IgG 5 (3.5) 1.42 0.33-6.05 NS 3 (2.5)
Comparison of positive rates for various antiphospholipid antibodies in the mid to late pregnancy loss gourp (n=26)
Mid to late pregnancy
loss group (n=26) Control women
n (%) Odds ratio 95%CI p n (%)
aPE, IgG 3 (11.5) 2.52 0.59-10.82 NS 6 (4.9)
aPE, IgM 5 (19.2) 7.02 1.74-28.32 0.008 4 (3.3)
aCL, IgG 6 (23.1) 4.28 1.34-13.64 0.02 8 (6.6)
aCL, IgM 3 (11.5) 3.85 0.81-18.35 NS 4 (3.3)
aCL-b2GPI, IgG 3 (11.5) 5.17 0.98-27.3 NS 3 (2.5)
Associations have been reported between aPL, mainly anticardiolipin antibodies and/or the lupus anticoagulant, and RPL.1,2 Relatively few studies describing aPE have been published.10-18 Recently, we reported that certain aPE are not specific for PE per se, but are directed to PE-binding plasma proteins, for example, HK, LK and the HK-binding plasma proteins factor XI and prekallikrein.4,5 We reported that 90.5% of PE-binding protein dependent aPE from RPL patients was kininogen-dependent.17 Since kininogens appear in mammalian reproductive tissues, we screened pregnancy loss patients for kininogen dependent aPE by ELISA. We already reported a stronger association between RPL and aPE than between RPL and aPL to the anionic phospholipids during early gestational losses.17 We now report an association between mid to late pregnancy loss and IgM aPE as well as IgG aCL.
Many RPL patients who fit a clinical profile compatible with antiphospholipid syndrome are negative for aPL when tested by ELISA. Explanations for negative findings are manifold, but often can be attributed to assay variations. For example, some commercial aCL detection kits (e.g. Yamasa kit) are designed to use purified or recombinant b2GPI as the patient sample diluent. In this situation, patients with aPL dependent upon the presence of prothrombin would appear as negative. In this study, IgG and IgM aCL were measured by Mesacup kit. This kit contains 10% ABS in its dilution buffer which should contain CL-binding proteins other than b2GPI.
Negative results in the aPE ELISA can result from using a patient sample diluent that is low or deficient in the kininogens; fetal calf serum (FCS) and newborn calf serum (NBCS) contain notably low concentrations of HK and LK22,23 and should be avoided. Few studies have appeared implicating aPE in RPL and thrombosis patients.10-12, 14-16 In these studies, FCS or NBCS were utilized as the patient sample diluents. This suggests that many kininogen dependent aPE may be negative in these studies. Moreover, prolonged storage of ABS and ABP at 4°C can result in decreasing kininogen activity, (T. Sugi, unpublished observations). We have also found that each lot of ABS must be screened before use as an aPE testing reagent because some lots of ABS contain low concentrations of intact kininogens. To avoid this problem, ABP has proven to be a better source of PL-binding plasma proteins for aPE detection. For optimal activity the ABS or ABP should be divided into single-use aliquots, stored frozen and thawed each day the aPE ELISA is performed.
In this study, we reported the association between IgM aPE and mid to late pregnancy loss at or beyond the 10th week of pregnancy. This is concordant to recent published data by Gris et al. showing that only IgM aPE, IgG anti-b2-glycoprotein I antibodies, IgG anti-annexin V antibodies and lupus anticoagulant were found to be independent retrospective risk factors for unexplained early fetal loss from 10th to 24th week pregnancy.18 The higher retrospective risk for fetal loss was associated with positive IgM aPE.
Evidence for the presence of a kallikrein-kinin system in fetoplacental vessels has accumulated in several studies.24, 25 Mutoh et al.25 indicated that the kinin generating activity of the kallikrein-kinin system is localized within the uteroplacental unit. Hermann et al.7 reported that kininogen and plasma prekallikrein/plasma kallikrein were present in the endothelial cells of the placental villous capillaries. In larger placental blood vessels and the umbilical cord, neither kininogens nor kallikreins were detected. The co-localization of kininogen and plasma prekallikrein/plasma kallikrein suggests that kinins could be generated locally in the placental capillaries. The functional spectrum of biologically active kinins, such as vasodilation, vasoconstriction, smooth muscle contraction and relaxation, could influence placental blood flow regulation. Moreover, kinins could also have anti-thrombotic/profibrinolytic activities.26,27 Kinins which are released within the placenta may play a role in regulating the placental blood flow and transplacental transport of substrates and metabolites.7 To effectively influence the placental circulation and nutrient supply to the fetus, components of the kallikrein-kinin system should be situated either within or close to the placental vasculature.
Both HK and LK bind to platelets.28-30 Domain 3 (D3) of kininogens was found to non-competitively inhibit thrombin from binding to the platelet thrombin receptor. By using specific monoclonal antibodies, Jiang et al.31 showed the D3 region to be responsible for the inhibition of thrombin binding to platelets. Kunapuli et al.32 found that recombinant D3 inhibited thrombin-induced platelet aggregation. We thus hypothesized that the aPE can bind to kininogens on the platelet. When bound by aPE, the platelet-kininogen complex no longer renders the platelet refractory to thrombin activation, thus predisposing to aggregation and thrombosis. Our recent in vitro data support these observations as we demonstrated that kininogen-dependent IgG-aPE purified from several aPE-positive patient plasmas caused a marked augmentation of thrombin-induced platelet aggregation, while not affecting adenosine diphosphate (ADP)-induced platelet aggregation.33 Moreover, kininogen-independent IgG-aPE did not affect the thrombin-induced platelet aggregation. For this to occur, it is possible that aPE may recognize the D3 region of kininogens subsequent to their binding to platelet PE.
Our recent in vitro data suggest that aPE may recognize the domain 3 (D3) region of kininogens.34 In this study, we used synthetic peptides that span the D3 of kininogens in inhibition and direct binding studies to identify epitopes that are sites for binding aPE. Our data demonstrated that among 24 RPL patients who were positive for kininogen-dependent IgG aPE, 17 patients (70.8%) recognized the LDC27 peptide. We mapped the aPE binding region on D3 using plasma from a RPL patient, who had a high titer of IgG aPE which recognized LDC27. aPE of this patient recognized a 13-residue segment in LDC27, named CNA13. Leu331-Met357 (LDC27) and Cys333-Lys345 (CNA13) are located on the carboxyl-terminal portion of kininogen D3 which is known as the major kininogen heavy chain cell attachment site where it overlaps its cysteine protease inhibitory region. Because aPE interferes with the balance of hemostasis in vitro, aPE may therefore induce a similar condition in patients thereby causing thrombosis and RPL.
In addition to the authors, the Japan Antiphospholipid Project Group comprised the following individuals: Mayumi Sugiura Ogasawara (Nagoya City University Medical School, Nagoya, Japan), Seiichiro Fujimoto (Hokkaido University, Graduate School of Medicine, Sapporo, Japan), Fumiki Hirahara (Yokohama City University, School of Medicine, Yokohama, Japan), Hitoshi Okamura (Kumamoto University School of Medicine, Kumamoto, Japan), Yasunori Yoshimura (Keio University School of Medicine, Tokyo, Japan), Kunihiro Okamura (Tohoku University, Graduate School of Medicine and School of Medicine, Sendai, Japan), Tatsuo Yamamoto (Nihon University, School of Medicine, Tokyo, Japan), Yuji Murata (Faculty of Medicine, Osaka University, Graduate School of Medicine, Osaska, Japan)
We thank the gynecologists and obstetricians who referred their patients to the SRL laboratory. We express our thanks to all patients and control subjects who participated in this study. We thank Dr. S. Shimano for providing a part of normal control sera. We thank the technicians of the SRL laboratory for their excellent assistance in laboratory analysis.
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