Preventable Hazards from in Vitro Fertilization - A Case Series of CF Patients from Bulgaria.

Pre-implantation genetic diagnosis (PGD) is not often performed when donor gametes are used, due to its high cost. This is with the presumption that the donors are healthy. We report on five cases of babies with confirmed cystic fibrosis (CF), being the result from in vitro fertilization (IVF) with donor (4 cases) or own gametes (one case). There has been no family history for CF in any of the families affected. The clinical presentation in the children ranged from meconium ileus to recurrent respiratory infections and severe nasal polyposis. The age of diagnosis also varied from birth until 9 years. Since one of the presented cases was discovered in a very renowned private IVF clinic, the clinic changed their own protocol, and currently they test every donor for CF carriership. The percentage of CF carriers in the donor population is roughly the same as the one predicted in the general population of Bulgaria - 1/33. Although PGD is costly, the costs for proper care for a CF patient are currently much higher. The more economical option would to screen every donor for CF carriership. IVF requires a lot of physical and psychological stamina. The couples that go through this procedure also require a great deal of hope. It is essential to be more preconscious for possible congenital diseases. We advocate every IVF center to test the donors for CF carriership or to provide PGD for their clients.

Pontocerebellar hypoplasia type 1 for the neuropediatrician: Genotype-phenotype correlations and diagnostic guidelines based on new cases and overview of the literature.

Pontocerebellar hypoplasia type 1 (PCH1) is a major cause of non-5q spinal muscular atrophy (SMA). We screened 128 SMN1-negative SMA patients from Bulgaria for a frequent mutation -p.G31A in EXOSC3, and performed a literature review of all genetically verified PCH1 cases. Homozygous p.G31A/EXOSC3 mutation was identified in 14 Roma patients, representing three fourths of all our SMN1-negative Roma SMA cases. The phenotype of the p.G31A/EXOSC3 homozygotes was compared to the clinical presentation of all reported to date genetically verified PCH1 cases. Signs of antenatal onset of disease present at birth were common in all PCH1 sub-types except in the homozygous p.D132A/EXOSC3 patients. The PCH1sub-types with early death (between ages 1 day and 17 months), seen in patients with p.G31A/EXOSC3 or SLC25A46 mutations have a SMA type 1-like clinical presentation but with global developmental delay, visual and hearing impairment, with or without microcephaly, nystagmus and optic atrophy. Mutations with milder presentation (homozygous p.D132A/EXOSC3 or VRK1) may display additionally signs of upper motor neuron impairment, dystonia or ataxia and die at age between 5 and 18 years. Other EXOSC3 mutations and EXOSC8 cases are intermediate - SMA type 1-like presentation, spasticity (mostly in EXOSC8) and death between 3 months and 5 years. There is no correlation between neurological onset and duration of life. We add marble-like skin and congenital laryngeal stridor as features of PCH1. We show that imaging signs of cerebellar and pontine hypoplasia may be missing early in infancy. EMG signs of anterior horn neuronopathy may be missing in PCH1 patients with SLC25A46 mutations. Thus, there is considerable phenotypic variability in PCH1, with some cases being more SMA-like, than PCH-like. Detailed clinical evaluation and ethnicity background may guide genetic testing and subsequent genetic counseling.

Inherited Thrombophilias Could Influence the Reproductive Outcome in Women with Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease associated with different reproductive complications in the affected women. Inherited thrombophilias are genetic factors increasing the risk for thromboembolism and recurrent pregnancy loss, but their influence on other reproductive disturbances in SLE patients has not been completely clarified. Two hundred and twenty-three Caucasian women (112 with SLE and 111 controls) were included in the study. Complete reproductive history of all SLE patients was carefully obtained. Genotyping for the FVLeiden, FIIG20210A, and MTHFRC677T polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences in the prevalence of the FVLeiden, FIIG20210A, and MTHFRC677T polymorphisms between patients and controls were established. Patients with FVLeiden had fewer pregnancies (0.57 ± 0.98 vs. 2.18 ± 1.58; p = 0.007) than the others, while no significant differences in the reproductive history of FIIG20210A carriers and non-carriers were observed (p >0.05). In the SLE group, 41.67% of women with the MTHFRC677T TT genotype had at least one miscarriage in comparison to only 14.00% of the other female patients (p = 0.030). While the prevalence of the investigated thrombophilias was similar in patients with SLE and healthy women, a substantial influence of the inherited prothrombotic factors on the reproductive history of patients was revealed. The investigations of the FVLeiden and MTHFRC677T polymorphisms in SLE patients could help to identify women at highest risk for reproductive failure and thus, further studies in other ethnic groups would be of strong clinical importance.

TLR4 polymorphisms seem not to be associated with prediabetes and type 2 diabetes but predispose to diabetic retinopathy; TLR4 polymorphisms in glucose continuum.

OBJECTIVES: Compared to type 1 diabetes, the role of the immune and autoimmune pathogenetic mechanisms is much less studied in the type 2 diabetes. Toll-like receptors 4 (TLR4) have a leading role in inflammation, insulin resistance, and vascular damage. This study aimed to analyze the relationship between the polymorphisms in TLR4 gene and different stages in the glucose continuum from prediabetes to the type 2 diabetes and chronic microvascular complications. MATERIALS AND METHODS: The study included 113 patients with the type 2 diabetes, 29 participants with prediabetes, and 28 controls. Polymerase chain reaction (PCR) was used for genotyping Asp299Gly and Thr399Ile polymorphism, followed by restriction analysis. RESULTS: The difference in the genotype frequency for both polymorphisms in patients with the type 2 diabetes or prediabetes compared to that in controls was not significant. Patients with heterozygous genotype of Asp299Gly polymorphism had a higher prevalence of diabetic retinopathy (42.9%) than participants with homozygous genotype (9.0%) (OR [95%CI]=7.61 [1.41-41.08]; p=0.018). No association was established for diabetic polyneuropathy and nephropathy. Prevalence of chronic diabetes complications was not related to Thr399Ile polymorphism. CONCLUSION: Our study demonstrates that Asp299Gly and Thr399Ile polymorphisms seem not to be associated with the type 2 diabetes and prediabetes but Asp299Gly may contribute to diabetic retinopathy predisposition.

Melatonin receptor 1b polymorphisms in women with Systemic Lupus Erythematosus.

AIM: The pineal hormone melatonin could exert an important influence on the immune system and autoimmunity. Its effect on the immunocompetent cells might be mediated at least partially through specific melatonin receptors. However, the role of melatonin - melatonin receptor 1B (MTNR1B) interrelations in human autoimmune diseases is still unknown. Therefore, the present study aimed to investigate the possible influence of the MTNR1B gene polymorphisms for the development and clinical expression of systemic lupus erythematosus (SLE). METHODS: 109 female SLE patients and 101 healthy women were genotyped for the MTNR1B rs1562444, rs10830962 and rs10830963 polymorphisms. RESULTS: No genotype distribution differences were found between patients and controls. The presence of MTNR1B rs10830963 C/C genotype was related to increased prevalence of leucopenia compared to genotypes C/G and G/G after Bonferroni correction for multiple comparisons [36.5% vs. 14.5%, p=0.014]. Moreover, the rs10830963 G/G carriers had lower number of lupus criteria in comparison to patients with C/C genotype. CONCLUSIONS: The present data suggested that MTNR1B polymorphisms could influence the clinical features in lupus patients, and especially the susceptibility to leucopenia.

[INDICATIONS FOR ANTITHROMBOTIC MEDICATION DURING PREGNANCY--ANALYSIS OF CURRENT PRACTICES IN BULGARIA].

AIM: To analyze current practices in Bulgaria regarding antithrombotic medication (AM) during pregnancy, and to compare them with the ones recommended in literature. MATERIALS AND METHODS: In 84 pregnant women who were low dose aspirin (LDA) or/and low molecular weight heparin (LMWH) or unfractionated heparin (UH), data about AM were collected and analyzed. A descriptive analysis was performed of the indications for AM, its type, the applied doses and therapeutic regimens. RESULTS: 39/84 pregnant women (46.4%) had indications for AM. In 18/84 cases (21.4%) the type of AM was precisely selected according to the indications. Of them 12 were on LDA alone (8--history of early preeclampsia/IUGR, 2--diabetes, 1--autoimmune disease, 1--chronic hypertension), 4--on LMWH/UF alone (2--venous thromboembolism, 2--essential thrombocytemia) and 2 received both LMH and LDA (1--antiphospholipid syndrome, 1--phlebothrombosis and stillbirth). Another 21/84 cases (25%) had indications for AM but its type was not appropriately selected. This subgroup included cases with high risk inherited thrombophylia (IT), history of placental mediated disease (PMD) in previous pregnancies and/or accompanying medical disorders. These patients had indications for either LDA or LMA administration, but were on combined medication. In 45/84 cases (53.6%) with uneventful past obstetric history or early pregnancy losses (before 10 w.g.) but no PMD there were no indications for AM. Among them, 17/84 (20.2%) had low risk IT in 18/84 (21.4%) IT was ruled out and 10/84 (12%) were not tested for IT at all. In total, 64/84 patients (76.2%) were on LDA--alone (25/84 - 29.8%), or in combination with LMWH (39/84 - 46.4%). Treatment with LDA was indicated in 45/64 (70.3%) cases--12/25 (48%) of the ones who were on LDA alone and 23/39 (59%) of those on LDA and LMW In 19/64 cases (29.7%) LDA administration was not indicated. 59/84 (70.2%) of the patients were on LMW/UH- alone (20/84 - 23.8%) orin combination with LDA (39/84 - 46.4%). This therapy was indicated in only 6/59 cases (10.2%), treated with LMWH/UH - 4/20 (20%) of the ones on LMWH/UH alone and 2/39 (5.2%) of the ones on LDA plus LMWH. In 53/59 cases (89.8%) the administration of LMWH was not indicated. LDA was started preconceptionally, in the Ist or in the lind trimester in 12/64 (18.7%), 32/64 (50%) and 6/64 (9.4%) of the cases respectively. LMWN/UH was started preconceptionally, in the Ist orin the lind trimester in 5/59 (8.6%), 33/59 (56.9%) and 5/59 (8.6%) of the cases respectively. The information when AM was started was not reliable in 14/64 (21.9%) cases on LDA and in 16/59 (27.1%) - on LMWH. In 12/84 cases (14.3%) LDA and LMWH were administered every other day. This referred to 5/64 (7.8%) cases on LDA and to 7/58 (12.1%) - on LMWH. LDA and LMWH were administered in PAI 4G/4G polymorphism despite the fact that in'these cases fibrinolysis but not coagulation was affected. CONCLUSIONS: AM was administered according to strict indications and with appropriately selected preparations in only 21.4% of the studied cases. In another 25% there were indications for AM, but its type was not precisely selected. 53.6% of the patients had no indications for AM. LDA administration (alone or in combination with LMWH) was indicated in 70.9% of the cases; the same referred to only 10.2% of the ones who were on LMWH. Application/administration of AM every other day was inappropriate given the pharmacokinetics of the preparations. AM was also applied in IT with decreased fibrinolysis but not increased coagulability.

[Types of screening for congenital diseases - results in Bulgaria, conclusions and future objectives].

UNLABELLED: Bulgarian statistics of recent years (2008-2012) indicate weak knowledge and use of the various screenings . Low percentage of pregnant passed screening tests and improper interpretation of the calculated risks, has led on the one hand to the high percentage of invasive diagnostics in the surveyed population (6% instead of 3%) to diagnose 90% of pregnancies with trisomy 21, and at the same time low percentage diagnosed pregnancies with Down's syndrome in the population of all pregnant women (tested and untested). We are performing invasive procedures of almost all pregnant women over 35 years old, and instead of the screening in the 1st trimester, we still use the screening in the 2nd trimester which is with limited application in Europe because of the high rate of false positive (false positive rate) and the unsatisfactory rate of recognized pregnancies with chromosomal defects (detedtion rate 70%). OBJECTIVE: To make the analysis of screening data, in the 1st and 2nd trimester for the period 2008-2012 in the Hospital "Maichin Dom" and our Center for Prenatal Diagnosis (respectively 93956 and 5046 pregnant women observed), the percentage of invasive diagnostic and percent diagnosed with Down syndr. in the total number of pregnancies, 99002 versus the number born with the disease in the country according to data of the National Statistical Institute. METHODS AND MATERIALS Used was the algorytm of FMF London to assess the effectiveness of the various screenings (Prof Nikolaides et al.) more specific for each of them - Detection rate and False positive rate, and statistical meta-analyses of H. Cuckle and D. Wright, for the estimated number of pregnancies with Down syndr.and the expected percentage of invasive diagnostics for locating over 90% of them in a population of 100,000 pregnant women. RESULTS: 99002 Surveyed pregnancies for the period 2008-2012 are around 25% of the total population (377,040 births), there are 7645 invasive procedures (i.e. 7,7 % of the expected 3%) and are diagnosed with Down's syndrome pregnancies were 181 from statistically expected about 200 (i.e., 90%) in the same period, 152 babies are born with TRI 21. CONCLUSIONS: In terms of the lack of a National Strategy for screening for congenital diseases, non-compliance with the principles and protocols for consultation of patients about the results obtained and generally poor knowledge of the various screenings have led to the negative attitude of the doctors and patients to screening programs and low percentage of pregnant women targeted or seeking such tests (about 30%) and a high percentage of invasive diagnosis (due to the high percentage of false positive rate and incorrect interpretation of the calculated risks).

MBL2 polymorphisms - manifestations in Bulgarian patients with adult dermatomyositis and systemic lupus erythematosus.

Deficiency in some complement factors is known to cause both systemic lupus erythematosus (SLE) and dermatomyositis (DM). Mannose-binding lectin (MBL) is a recognition molecule of the lectin pathway, and its low levels are reported to influence some autoimmune diseases. Furthermore, MBL2 polymorphisms have been described associated with low MBL serum levels due to impaired MBL structure and function. This is a pilot study to investigate the role of MBL2-550G/C (H/L), -221G/C (Y/X), Arg52Cys (D), Gly54Asp (B), Gly57Glu (C) polymorphisms and MBL serum levels as a risk factor for a development of adult DM and SLE in Bulgarian patients. None of the studied MBL2 polymorphisms appeared associated with the diseases investigated. However, we have found an increased OR of MBL2-221XY genotype in the patients with SLE (OR 1.64, 95%CI 0.77-3.52). MBL2 polymorphisms seemed to affect MBL serum levels and to be associated with the clinical features although none of the associations remained statistically significant after Bonferroni correction. The-550L allele showed an association with electromyography findings in patients with DM. The-221XY genotype was associated with photosensitivity in patients with SLE. The 54AB genotype showed an association with malar rash in patients with SLE, but it appeared decreased among SLE patients with ANA. In conclusion, our results suggest that the MBL2 polymorphisms have rather a disease modifying role and they are not associated with the disease susceptibility in adult DM and SLE among Bulgarian patients.

APC promoter 1B deletion in familial polyposis--implications for mutation-negative families.

Over 1500 adenomatous polyposis coli (APC) gene mutations have already been identified as causative of familial adenomatous polyposis (FAP). However, routine genetic testing fails to detect mutations in about 10% of classic FAP cases. Recently, it has been shown that a proportion of mutation-negative FAP cases bear molecular changes in deep intronic and regulatory sequences. In this study, we used direct sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) of genomic DNA from family members, affected by classic FAP. We first reported the family as mutation negative. With the launch of a new version of MLPA kit, we retested the family and a novel full deletion of promoter 1B was detected. The exact breakpoints of the deletion were determined by array comparative genomic hybridization (CGH) and long range polymerase chain reaction (PCR), followed by direct sequencing. The total APC expression levels were investigated by quantitative polymerase chain reaction (qPCR) assay and allele-specific expression (ASE) analysis. The APC gene expression was highly reduced, which indicates causative relationship. We suggest that there is a significant possibility that APC promoter 1B mutations could be found in mutation-negative FAP patients. In the light of our findings it seems reasonable to consider targeted genetic re-analysis of APC promoter 1B region in a larger cohort of unsolved cases.

[PAL-1 5G/4G polymorphism in patients with systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) is a connective tissue disease affecting predominantly women that has been widely associated with obstetric complications. Inherited thrombophilias are significant risk factors for pregnancy loss, but their role in patients with SLE, and especially in those without concomitant secondary antiphospholipid syndrome (APS) has not been clarified. The aim of the present study was to study PAI-1 5G/4G polymorphism in women with lupus. A total of 103 SLE patients as well as 69 healthy volunteers were genotyped for PAI-1 5G/4G (rs1799889). No significant differences in the PAI-1 5G/4G genotype prevalence between patients and controls were found. After exclusion of the women with secondary APS, the frequency of pregnancies and spontaneous abortions, as well as the number of live births were similar in the studied patients with different PAI-1 genotype (p> 0.05). PAI-1 5G/4G polymorphism was not significantly related to any of the lupus ACR criteria or disease activity (p > 0.05), but it could influence the platelet number in the studied patients (263.52 ± 91.10 [5G/5G genotype] versus 210.12 ± 71.79 [4G/4G genotype], p = 0.023). In conclusion, our results showed that PAI-1 4G/5G polymorphism did not worsen the reproductive outcome in SLE women without secondary APS.

[Genomic study in patients with idiopathic azoospermia and oligoasthenoteratozoospermia].

Genome disbalances are related to the different types of infertility and they play a role in the treatment of human infertility. Comparative genome hybridization (CGH) combined with microchips is a modern high resolution technique for all human chromosomes investigations. We analysed the genome disbalances in 16 blood samples of men with an idiopathic oligoastenozoospermia or azoospermia using CGH and microchips for the whole human genome. Our data showed a few affected loci, including: 3q26.32 deletion accompanied by 14q11.1 deletion, 9q12 deletion, 5q35.1 amplification, 7p22.3 amplification and 17q12-17q21.2 amplification. In this study we have match the deletions: in two patients in the same area in the 8 chromosome, as well as in 5 patients in 14 chromosome. The deleted region contains 25 genes. Two of them (SPAG11B and SPAG11A) are associated with stages of spermatogenesis and in particular formation and maturation of the spermatozoa. These genes play a role during spermatogenesis and fertilization. Loss. chr.14q11.2 (EDDM3A and EDDM3B) affected the proteins that are synthesized and secreted by epididymal epithelial cells that has been found up-regulated in epididimis of nonobstructive azoospermic men. Our results displayed the significance of CGH and microchip analysis as a promising area of research with serious clinical application for resolving the problems of the male infertility and still have an important annex for selecting the most appropriate methods for the treatment in these patients as a perspective scientific field of investigations with a clinical appliance.

CHEK2 I157T and colorectal cancer in Bulgaria.

PURPOSE: Germline variants of the CHEK2 gene have been shown to act as low-penetrance cancer susceptibility alleles for a wide range of human malignancies. CHEK2 I157T has particularly been linked to colorectal cancer (CRC) risk. We aimed at establishing the population frequency and contribution of this variant to colorectal carcinogenesis in Bulgaria. METHODS: We have genotyped 802 population controls and 343 CRC patients from Bulgaria for the CHEK2 I157T variant. RESULTS: Heterozygous were 9 of 343 patients (2.62%, odds ratio/OR=1.0, 95% confidence interval/CI = 0.42 - 2.33, p=0.99% and 21 of 802 controls (2.62%). Higher frequencies were found among patients with multiple polyposis (2/40, 5%, p=0.28) and the rarer mucinous histology (1/11, 9.09%, p= 0.26). CONCLUSION: We conclude that CHEK2 I157T is not relevant for CRC risk in Bulgaria, but studies on a larger scale might help evaluate its possible significance in respect to disease characteristics.

[Impact of inherited thrombophilia on the development of some pregnancy complications].

AIM: To assess the clinical significance of inherited thrombophilia [IT] for the development of some pregnancy complications. MATERIAL AND METHODS: The incidence of the following factors was studied in 97 pregnant women with pregnancy complications and in 103 controls: R506Q mutation encoding Factor V Leiden [FVL] synthesis, Prothrombin G20210A mutation, T677 methylenetetrahydropholate reductase mutation [MTHFR], 4G/4G polymorphism of the plasminogen activator inhibitor [PAI 4G/4G]. Among 97 patients in the group studied 39 had early onset severe preeclampsia [PE], 14--placental abruption [AP] without PE, 18--intrauterine growth restriction [IUGR] without PE, 12--stillbirth [SB] without PE, 14--habitual spontaneous abortions [HSA]. The control group included 103 clinically healthy pregnant women with at least one previous uneventful pregnancy, without history of thromboembolic disorders. In addition, patients with severe PE with and without IT were compared regarding g. a. and birthweight at delivery and intrauterine fetal loss rate. DNA analysis was performed according to internationally accepted standards. Pregnancy outcomes were ascertained from hospital records. Statistical significance (p < 0.05) was assessed by means of Student's t-test. RESULTS: FVL mutation was found in 23.7% (23/97) of the patients from the studied group and in 5.8% (6/103) of the controls. Prothrombin G20210A carriers were 11% (11/97) of the studied and 3.8% (4/103) of the controls, while with PAI 4G/4G polymorphism they were 30.9% (30/97) and 14.5% (15/103) respectively. MTHFR T677 was not more frequent in the studied group (8.2%) compared to the control one (29%). Eight of the patients (9.6%) were carriers of more than one mutation. In 22 cases with early onset severe PE and IT gestational age and birthweight at delivery were lower than in the cases with severe PE without IT while intrauterine fetal loss rate did not differ significantly between the two groups. CONCLUSIONS: Inherited thrombophilia is found more frequently in women with pregnancy complications like PE, IUGR, AP, SB, HSA. The incidence of homozygous MTHFR T677 is not higher in these cases. IT worsens the prognosis of severe PE. The diagnosis of IT is important since anithrombotic therapy has to be considered to protect the mother and the fetus.

[Contemporary low-invasive, videoendoscopic and organ-sparing abdominal operations].

The paper presents the results of low-invasive, videosurgical, and organ-sparing operations, developed and performed by the authors. Original low-invasive techniques of external, external-and-internal and internal bile-duct drainage, and bile-duct endoprosthesis replacement have been performed in 25 patients with benign and malignant bile-duct strictures. Transcutaneous puncture drainage operations, including those combined with an original method of transdrainage sclerotherapy with nitric oxide, have been performed in 93 patients with postnecrotic pancreatic cysts. Endoscopic virsungotomy have been performed as part of the complex treatment of 43 patients with external pancreatic fistulas and 14 patients with polycystic head of pancreas. X-ray endovascular embolization has been performed in 7 patients with internal arterio-pancreatic fistulas. Program laparoscopic abdominal cavity sanation by an original method have been performed in 50 patients with diffuse peritonitis. The results of the study demonstrate high effectiveness of the methods, which in most cases can be applied as an alternative to conventional surgical interventions.

[Sucessful pregnancy outcome following three severe placental abruptions and intrauterine fetal death in the patient--heterozygous carrier of R506Q mutation of factor V (Leiden)]. / Uspeshen izkhod na bremennostta sled tri predshestvuvashti tezhki abruptsii na plantsentata s murtuv plod pri patsientka--kheterozigoten nositel na mutatsiia R506Q na faktor V (Leiden).

A case of successful pregnancy outcome is reported in a patient with 3 preceding severe placental abruptions with intrauterine fetal death and caesarean deliveries. In the course of the current pregnancy heterozygosity for R506Q mutation of factor V (Leiden) was diagnosed in 26 weeks of gestation [w.g.] and low molecular weight heparin [LMWH] therapy initiated. Maternal condition was stable until delivery and all laboratory findings were within normal range. The fetus was followed up by ultrasound biometry and Doppler blood flow studies. From 28 w.g. on NST and biophysical profile were included. An emergency caesarean section was performed in 34 w.g. because of contractions not responding to tocolysis. The newborn was in good condition with weight and length corresponding to the 10th centile for gestational age [g.a]. Histologic study of the placenta showed anemic infarctions and recent haemorrhages in the basal and the chorionic plate. The initiation of LMWH therapy in the case reported was late (26 w.g.). By that moment there was already evidence of impaired fetal growth with fetal biometry corresponding to the 10th centile for g.a. After LMWH therapy was started no further slow down of fetal growth was registered. Successful pregnancy outcome may be related not only to LMWH therapy but also to other factors like active fetal monitoring after 28 w.g. and the emergency caesarian delivery immediately after the onset of uterine contractions. Patients with past obstetric history of severe preeclampsia, placental abruption or fetal growth restriction have to be screened for hereditary or acquired thrombophilia. If a thrombophillic state is present early LMWH therapy has to be considered. It is aimed to prevent anaemic placental infarctions and thrombotic complications.