抗磷脂抗体与体外受精-胚胎移植结局的关系

目的探讨IVF—ET反复失败与抗磷脂抗体（anti—phospholipid antibodies，APA），包括抗心磷脂抗体（anti—cardiolipin antibodies，ACA）和狼疮抗凝抗体（lupus anticoagulant antibodies，LA）的关系。方法188例种植失败和种植后妊娠丢失的患者225个周期（研究组）和75例同期接受IVF—ET治疗受孕的患者89个周期（对照组），分别采用酶联免疫吸附法和活性部分凝血酶原时间法，测定静脉血清中的APA。其中，研究组分为种植后妊娠丢失组（26周期）、一次种植失败组（126周期）和反复种植失败组（73周期）。结果研究组中APA、ACA、LA的阳性百分比均高于对照组，与对照组相比，差异均有显著性（P〈0．01）。种植后妊娠丢失组和反复种植失败组此3项指标的阳性百分比分别与对照组比较，差异有显著性（P〈0．01），一次种植失败组与对照组相比，差异无显著性（P〉0．05）。结论APA与IVF—ET种植失败有关，尤其是种植后妊娠丢失和多次种植失败。因此，应对预进行体外受精-胚胎移植患者常规筛查APA，以利于尽早干预治疗以期提高IVF临床妊娠率，降低妊娠丢失率。     

Different Antiphospholipid Antibody Specificities are Found in Association with Early Repeated Pregnancy Loss Versus Recurrent IVF-Failure Patients

PROBLEM: Patients having in vitro fertilization and embryo transfer (IVF-ET) failures show an increased incidence of antiphospholipid (aPL) antibodies; but controversy exists whether aPL can induce IVF-failure. This study was designed to compare aPL specificities between recurrent IVF-failure patients versus repeated early pregnancy loss (RPL) patients. METHOD OF STUDY: Anticardiolipin (aCL), lupus anticoagulant (LA), antiphosphatidylserine (aPS), antiphosphatidylethanolamine (aPE), and antinuclear antibodies (ANA) were measured in 74 recurrent IVF-ET failure patients and compared with 273 early RPL patients ( < 10 weeks). RESULTS: An increased incidence of IgG-aPE and ANA was observed for both groups in comparison with controls. Patients with recurrent IVF-ET failure showed a significantly higher prevalence of IgG-aPS (P = 0.02) and IgG-aCL (P = 0.02) when compared with early RPL patients or controls. CONCLUSIONS: IgG-aPS and IgG-aCL may be responsible for some IVF-failures. Additional studies are needed to clarify the pathogenic role of IgG-aPS and IgG-aCL on IVF-ET failure.     

The possible role of antiovary antibodies in repeated in vitro fertilization failures

PROBLEM: The study was conducted to investigate the possible role of circulating ovarian autoantibodies (ov-ab) in patients with repeated in vitro fertilization embryo transfer (IVF-ET) failure and to evaluate the effectiveness of immunosuppression treatment in these patients. METHOD OF STUDY: The study group comprised 80 IVF patients who had five or more failed treatment cycles (mean 10.2; range 7-22). The presence of ov-ab was compared between these women and 1) 50 IVF patients who conceived during the first three treatment cycles; 2) 50 healthy nulligravidae. All participants were seronegative to nonorgan-specific and antithyroid autoantibodies. Patients in the study group who were positive for ov-ab were treated with 10 mg/day prednisone starting 1 month before ovulation induction. Embryo grading was compared in the IVF cycles before and after treatment. RESULTS: Ov-ab were found in ten patients (12.5%) in the study group, compared to none in the control groups (P = 0.01). Nine of the patients positive for ov-ab were treated with prednisone for their following cycle. A statistically significant improvement in embryo grading was noted. Three patients conceived after treatment (33%), with a take-home baby rate of 22%, compared to only six patients (8.6%) who conceived among the rest of the seronegative study group, with a take-home baby rate of 7.1% (P = 0.05). CONCLUSIONS: Ov-ab are a possible marker of an autoimmune disorder that may be one of the causes of repeated IVF failures. Immunosuppression treatment may prove efficient in ov-ab seropositive patients with repeated IVF failures by improving embryo grading and pregnancy rate.     

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

Citation Velayuthaprabhu S, Matsubayashi H, Sugi T, Nakamura M, Ohnishi Y, Ogura T, Tomiyama T, Archunan G. A unique preliminary study on placental apoptosis in mice with passive immunization of anti‐phosphatidylethanolamine antibodies and anti‐factor XII antibodies. Am J Reprod Immunol 2011; 66: 373–384 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.     

Factor XII activity and antigen concentrations in patients suffering from recurrent thrombosis

A group of 107 patients with recurrent venous thrombosis was tested for factor XII coagulant activity and antigen. Among these patients eleven were identified with Hageman factor (FXII) levels below our normal mean value minus two standard deviations (SD). A control group of 55 individuals without thrombosic problems was studied simultaneously. The factor XII levels of these persons were all within the normal range (normal mean ± 2 SD). The I I patients with reduced levels of factor XII belonged to seven families, 5 with positive history of thrombosis. On average, they experienced their first thrombosis at a younger age than the other patients, which supports the theory of a hereditary defect. Thus, reduced levels of factor XII should be considered as a risk factor in the development of thrombosis.     

Phenotypic consequences of genetic variation at hemizygous alleles: Sotos syndrome is a contiguous gene syndrome incorporating coagulation factor twelve (FXII) deficiency.

PURPOSE: We tested the hypothesis that Sotos syndrome (SoS) due to the common deletion is a contiguous gene syndrome incorporating plasma coagulation factor twelve (FXII) deficiency. The relationship between FXII activity and the genotype at a functional polymorphism of the FXII gene was investigated. METHODS: A total of 21 patients including those with the common deletion, smaller deletions, and point mutations, and four control individuals were analyzed. We examined FXII activity in patients and controls, and analyzed their FXII 46C/T genotype using direct DNA sequencing. RESULTS: Among 10 common deletion patients, seven patients had lower FXII activity with the 46T allele of the FXII gene, whereas three patients had normal FXII activity with the 46C allele. Two patients with smaller deletions, whose FXII gene is not deleted had low FXII activity, but one patient with a smaller deletion had normal FXII. Four point mutation patients and controls all had FXII activities within the normal range. CONCLUSION: FXII activity in SoS patients with the common deletion is predominantly determined by the functional polymorphism of the remaining hemizygous FXII allele. Thus, Sotos syndrome is a contiguous gene syndrome incorporating coagulation factor twelve (FXII) deficiency.     

Association of C46T genetic polymorphism of coagulation factor XII with deep venous thrombosis: a cohort study on Egyptian patients

Deep vein thrombosis (DVT) is a common multi-factorial disease, with serious short- and long-term complications, and a potential fatal outcome. Many genes are involved in determining the interindividual variation in traits that define the onset and progression of disease, as well as the response to treatment. Several association studies have designed the relationship between factor XII C46T polymorphism and the risk of arterial and venous thrombosis. Some studies reported that FXII gene polymorphism is not associated with venous thrombosis, whereas other studies found an increased risk of venous thrombosis in carriers of a FXII-T variant. We constructed an age–gender–ethnic–matched case–control study including 52 DVT patients and 100 healthy volunteers. C46T polymorphism of the coagulation factor XII was carried out using allelic discrimination assay by real-time polymerase chain reaction for patients and controls, while plasma factor XII activity was detected by one-step clotting assay. FXII C46T genotyping in DVT patients revealed that 34.6% were heterozygous harboring the FXII-CT heterotype and 3.85% were homozygous; FXII-TT homotype, with no statistically significant difference in the distribution of the mutant genotypes between DVT patients and the control group. FXII activity was significantly reduced in DVT patients harboring the mutant genotypes. In the present study, FXII C46T gene polymorphism was not associated with increased risk of deep venous thrombosis.     

Influence of antiphospholipid antibodies on pregnancy outcome in women undergoing in vitro fertilization and embryo transfer

PROBLEM: Antiphospholipid antibodies (APA) are thought to be involved in recurrent pregnancy loss. Therefore, we investigated the impact of APA on pregnancy outcome in women undergoing in vitro fertilization and embryo transfer (IVF-ET). METHOD OF STUDY: Blood samples taken from 54 Korean women referred for IVF were tested for the presence of APA, anticardiolipin antibody IgG and IgM and lupus anticoagulant. The standard gonadotropin-releasing hormone agonist long protocol was used for ovarian stimulation. RESULTS: Nine patients (16.7%) were positive and 45 (83.3%) were negative for APA. There were no significant differences between the two groups in clinical characteristics such as age, infertility duration, and response to controlled ovarian hyperstimulation. However, pregnancy outcome significantly differed between the two groups (p < 0.05). The APA positive group and APA negative group had abortion rates of 62.5% and 20.0%, respectively and delivery rates of 37.5% and 80.0%, respectively. CONCLUSION: The presence of APA in women undergoing IVF-ET was associated with a poor pregnancy outcome.     

Thromboelastography, whole-blood haemostasis and recurrent miscarriage

BACKGROUND: Some cases of recurrent miscarriage have a thrombotic basis. Thromboelastography is a rapid, reproducible test of whole-blood haemostasis. METHODS: Thromboelastography was performed in 494 consecutive, non-pregnant women (median age 35 years; range 21-48) with a history of miscarriages at <12 weeks gestation (median 4; range 3-12) and 55 parous women (median age 33 years; range 20-41) with no history of pregnancy loss. The prospective outcome of untreated pregnancies amongst 108 women with recurrent miscarriage was studied. RESULTS: The maximum clot amplitude (MA) (median 66.0 mm; range 48.0-76.0) was significantly higher and the rate of clot lysis (LY30) (median 2.5%; range 0.5-7.8) significantly lower amongst women with recurrent miscarriage compared with controls (MA 61.5 mm; range 50.0-67.0; P = 0.01; LY30 4.9%; range 2.9-9.7; P = 0.01). The pre-pregnancy MA was significantly higher amongst women who subsequently miscarried (median 66.0 mm; range 54.0-73.0) compared with those whose had a live birth (median 61.7 mm; 48.0-71.5; P < 0.01). A pre-pregnancy MA >or=64 mm has a sensitivity of 68% and specificity of 82% to predict miscarriage. CONCLUSIONS: Thromboelastography identifies a subgroup of women with recurrent miscarriage to be in a prothrombotic state outside of pregnancy. Women in such a state are at increased risk of miscarriage in future untreated pregnancies.     

Prednisone and aspirin improve pregnancy rate in patients with reproductive failure and autoimmune antibodies: a prospective study

PROBLEM: The study was conducted to investigate the efficacy of prednisone and aspirin in autoantibody seropositive patients with repeated in vitro fertilization embryo transfer (IVF ET) failure. METHODS OF STUDY: The study group comprised 52 consecutive patients seropositive for non-organ-specific autoantibodies, i.e., anti-cardiolipin antibodies (ACA), anti-nuclear antibodies (ANA), anti-double-stranded (ds) DNA, rheumatoid factor (RF), and lupus anti-coagulant (LAC). These patients were treated with prednisone, 10 mg per day, and aspirin, 100 mg per day, starting 4 weeks before induction of ovulation in 52 IVF cycles. RESULTS: The clinical pregnancy rate per cycle was 32.7% (17/52). No increased incidence of pregnancy complications, including premature labor, gestational diabetes mellitus, and pregnancy-induced hypertension, were found. CONCLUSIONS: Combined treatment of prednisone for immunosuppression and aspirin as an anti-thrombotic agent, starting before ovulation induction, may improve pregnancy rate in autoantibody seropositive patients who have had repeated IVF-ET failures.     

Analysis of intra-uterine cytokine concentration and matrix-metalloproteinase activity in women with recurrent failed embryo transfer

BACKGROUND: In all IVF programmes, some patients fail to achieve an ongoing pregnancy, even after numerous embryo transfer procedures. An unfavourable environment within the uterus might be a contributory factor to such recurrent implantation failure. This question was addressed by measuring cytokine concentrations and matrix metalloproteinase activities in fluid derived from uterine irrigation of such patients. METHODS AND RESULTS: The uterine cavities of 22 patients who had previously undergone embryo transfer of at least 10 embryos without ongoing pregnancy were irrigated during the luteal phase. The resultant fluid was assayed for the concentration of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, leukaemia inhibitory factor (LIF), IL-10 and matrix metalloproteinase (MMP) -2 and -9 activity. The results were compared with those of a control population of women known to be previously fertile (n = 16) and also with women with recurrent spontaneous abortion (n = 13). In the recurrent implantation failure group, the MMP score and IL-1beta concentration were significantly higher than those in the control group, whereas concentrations of IFN-gamma and IL-10 were significantly lower. CONCLUSIONS: In IVF patients with recurrent implantation failure, an altered pattern of intra-uterine cytokine concentration and MMP activity was observed.     

Antinuclear antibody reduces the pregnancy rate in the first IVF-ET treatment cycle but not the cumulative pregnancy rate without specific medication

PROBLEM: It has been shown that the presence of antinuclear antibody (ANA) might reduce pregnancy rates after in vitro fertilization-embryo transfer (IVF-ET). However, the mechanism of implantation failure by ANA has not yet been clarified. This study was performed to investigate the impact of ANA on pregnancy rates after IVF-ET, and the necessity of specific medication for infertile women who have ANA in their sera. METHOD OF STUDY: A total of 108 infertile women were treated by IVF-ET or intracytoplasmic sperm injection (ICSI)-ET. ANA was examined by an indirect fluorescent antibody procedure. Data from women under 40 years old were analyzed retrospectively. RESULTS: The implantation rates per embryo transferred in the first treatment cycles were 14.8% (eight of 54) and 32.4% (33 of 102), in women with and without ANA, respectively. There was a significant difference in the implantation rates between the two groups (P < 0.05). The pregnancy rates per ET in the first treatment cycles were 28% (seven of 25) and 54.2% (26 of 48), respectively. There was also a significant difference in the pregnancy rates between the two groups (P < 0.05). Afterwards, treatments with IVF-ET or ICSI-ET were repeatedly performed for unsuccessful patients, without any specific medication for ANA. The average ET cycles were 1.80 +/- 1.13 and 1.27 +/- 0.54, in women with and without ANA, respectively. The cumulative pregnancy rates per patient were 68% (17 of 25) and 55.6% (35 of 63), respectively. There was no significant difference in the overall pregnancy rates between the two groups. CONCLUSIONS: These findings suggest that ANA might have an impact on implantation failure in women treated by IVF-ET or ICSI-ET. ANA reduced the pregnancy rates in the first IVF-ET or ICSI-ET cycles but not the cumulative pregnancy rates without medication. This indicates that the mechanisms of implantation failure by ANA could be solved, and effective and safe medication should be developed for better implantation rates, especially in the first treatment cycle.     

Endometrial protein PP14 and CA-125 in recurrent miscarriage patients; correlation with pregnancy outcome

The concentrations of endometrial proteins PP14 and CA-125 were measured in uterine flushings taken on days LH+10 and LH+12 (10 and 12 days after luteinizing hormone surge) of the menstrual cycle from 15 normal, fertile women and 49 women who suffered recurrent miscarriage. The concentration of PP14 was significantly lower in the flushings from the recurrent miscarriage patients than in those from fertile controls on both day LH+10 (median: 1300, range: 3-10 300 ng/ml versus median: 13 933, range: 2174-40 404 ng/ml; P < 0.01) and LH+12 (median: 1560, range: 820-12 100 ng/ml versus median: 14 047, range 1402-62 108 ng/ml; P < 0.05). Similarly concentrations of CA-125 were significantly lower in flushings from recurrent miscarriage women compared to controls on both day LH + 10 (median: 1555, range: 47-6710 U/ml versus median: 6385.5, range 2884-27 731 U/ml, P < 0.01) and LH+12 (median: 2892, range: 956-9974 U/ml versus median: 7127.5, range: 1591-21 343 U/ml; P < 0.05). In contrast there was no significant difference in the concentration of PP14 in plasma samples taken on the same days as the flushings from recurrent miscarriage patients and fertile controls. The concentrations of PP14 in uterine flushings obtained on day LH + 10 or LH + 12 from recurrent miscarriage women during a pre-pregnancy investigative cycle were significantly lower (P < 0.05) in patients who went on to miscarry (median: 1000, range: 9-2900 ng/ml) than those who went on to have a live birth (median: 1440, range: 4-12 100 ng/ml) during a subsequent pregnancy. In contrast there was no significant difference in uterine CA-125 or plasma PP14 concentrations between these two groups of recurrent miscarriage patients. The results suggest that measurements of uterine PP14 and CA-125 may be useful in the assessment of endometrial development in recurrent miscarriage patients and suggest the importance of PP14 in preparing the endometrium for embryo implantation. In addition pre-pregnancy uterine PP14 measurements may be useful in predicting subsequent pregnancy outcome.      

Recurrent pregnancy loss and its relation to FV Leiden, FII G20210A and polymorphisms of plasminogen activator and plasminogen activator inhibitor

Thrombophilic disorders and hypofibrinolysis were demonstrated to be risk factors in a majority of women with recurrent pregnancy loss (RPL) and infertility. We investigated the association of FV G1691A mutation, F II G20210A gene polymorphism (PM), 4G/5G PAI-1 and Alu I/D tPA PM in 32 women with infertility and 49 women with at least 2 unexplained early abortions. FV Leiden mutation was significantly more common in women with RPL (10%, p = 0.02) and infertility (19%, p = 0.0005) compared with controls (2%). PAI-1 4G PM and t-PA Alu I PM, alone or in combination, were not associated with RPL or infertility. 9/49 women with RPL showed coagulation disorders with heterozygous FV Leiden mutation (5), FXII (1), protein C (1) or protein S (2) deficiency. However, due to the small number of patients studied, no definite conclusion can be drawn.     

自身抗体与不孕及自发性流产关系的探讨

目的检测抗心磷脂抗体(ACA)和抗精子抗体(AsAb)两种自身抗体在不孕及自发性流产患者中存在的情况,并观察应用阿司匹林治疗ACA阳性反复流产患者的临床效果。方法应用酶联免疫吸附(ELISA)法检测150例原发或继发不孕患者(不孕组)、198例自发性流产或有胚胎停育史患者(流产组)及40例正常对照组血清中的ACA及AsAb抗体。对其中53例ACA阳性反复流产患者在孕前一个月或孕早期采用低剂量阿司匹林治疗。结果不孕组及流产组ACA总阳性率分别为48.00%和50.51%,与对照组(7.50%)相比有非常显著性差异(P<0.001);不孕组及流产组AsAb阳性率分别为31.33%和25.25%,与对照组(10.00%)比较亦有显著性差异(P<0.05)。53例经治疗患者活产婴儿48例,妊娠成功率为90.57%。结论ACA和AsAb等自身抗体是导致不孕及自发性流产的免疫学因素之一,应用低剂量阿司匹林治疗ACA阳性反复流产患者是保证其妊娠成功的有效方法。     

Idiotypic and anti-idiotypic elastin autoantibodies: Implications for IVIg and pregnancy loss

PROBLEM: The aim of this study was to investigate anti-elastin and anti-anti-elastin autoantibodies in intravenous immunoglobulin (IVIg) lots as an attempt to further explain the effect of IVIg in recurrent pregnancy loss (RPL). METHOD OF STUDY: Serum samples of 10 female patients with RPL and 10 healthy subjects were tested for anti-elastin autoantibodies and used in competitive inhibition studies. A total of 44 IVIg lots (ZLB Behring, Switzerland) were tested for anti-elastin and anti-anti-elastin idiotypes. One way analysis of variance (ANOVA) and Least Significant Difference (LSD method) were used for statistical analysis of differences between the lots. RESULTS: Serum anti-elastin IgG autoantibodies were significantly higher in the study group, compared to the controls. In all lots anti-elastin IgG antibodies were identified. All lots (except two of them) showed similar dose-dependent inhibition of serum anti-elastin activity by anti-elastin anti-idiotypes in IVIg. CONCLUSIONS: Anti-elastin IgG autoantibodies were increased in patients with RPL - a finding which needs further explanation. Anti-elastin and anti-anti-elastin idiotypes were identified in different IVIg lots. The presence in IVIg of anti-idiotypes against anti-elastin autoantibodies from patients' sera could be an additional mechanism of the beneficial effect of IVIg in reproductive failure.     

Detectable levels of interleukin-18 in uterine luminal secretions at oocyte retrieval predict failure of the embryo transfer

BACKGROUND: Most implantation failures after successful in vitro fertilization-embryo transfer (IVF-ET) result from inadequate uterine receptivity. There is currently no way to predict this receptivity. METHODS: We investigated whether the detection of interleukin-(IL)18 by ELISA in uterine luminal secretions might predict implantation failure. Secretions of 133 patients enrolled in our IVF-ET program were sampled by uterine flushing immediately before oocyte retrieval. We assessed the following outcomes: pregnancy rate, multiple pregnancy rate, and implantation rate per embryo transferred. RESULTS: Interleukin-18 was detected in the flushing fluid of 38 patients (28.6%). Although the two groups were comparable for all other characteristics (age, etiology, ovarian reserve, number of embryos transferred, quality of embryos), all outcome variables differed significantly. The pregnancy rate was 37.9% in the IL-18 - ve group and 15% in the IL-18 + ve group, the multiple pregnancy rate 27.7% and 0%, and the implantation rate per embryo transferred 19.4% and 6.7% (all comparisons, P=0.02). Only embryos meeting good quality criteria were transferred to 65 patients: 50 IL-18 - ve and 15 IL-18 + ve. The pregnancy rate was 51% for the IL-18 - ve group and 20% for the IL-18 + ve group, the multiple pregnancy rate 36% and 0.0%, respectively, and the implantation rate 29% and 8.3% (P = 0.02). CONCLUSION: This non-invasive and simple method predicted inadequate uterine receptivity, independent of embryo quality.     

Missense mutation Thr309Lys in the coagulation factor XII gene in a Spanish family with hereditary angioedema type III

Background:  A new type of hereditary angioedema (type III) affecting mainly women with normal C1-inhibitor level and function has been described. Exposition to estrogens is an important precipitating factor. Recently, a missense mutation in the gene of the blood coagulation factor XII (Hageman factor) has been reported in a few families with this type of hereditary angioedema.
Aim:  To study a patient and her family with recurrent swelling attacks during pregnancy.
Methods:  Complement factors C3 and C4 as well as C1-inhibitor level and function were determined. Genomic DNA was isolated from venous blood samples and screened for mutations in the coagulation factor XII gene.
Results:  C3 and C4 levels as well as C1-inhibitor level and function were normal. A missense mutation Thr309Lys was identified in factor XII gene with a heterozygotic pattern. This mutation was also identified in the mother of the patient, her daughter and her son.
Conclusion:  These results support that the mentioned mutation in factor XII gene causes hereditary angioedema type III.     

Fetal rejection: infertility and immunity

Defective reaction toward fetal alloantigens could result in both recurrent spontaneous abortions (RSAs) and recurrent early pregnancy failures (REPFs), the latter existing in couples with unexplained infertility and multiple failures of implantation after in vitro fertilization embryo transfer. Immunological mechanisms leading to RSA and REPF seem to be different, although both syndromes probably have a genetic background that has not been identified so far. Despite the fact that antiphospholipid syndrome is a well-established cause of repeated pregnancy loss, the role of different autoantibodies existing in RSA and REPF patients needs to be elucidated. Immunotherapy is believed to correct the detrimental immune reactions; however, its real effectiveness and safety for the treatment of distinct forms of pregnancy loss need to be reconsidered.