Zdenko Sonicki

Comparison of protective media and freezing techniques for cryopreservation of human semen

OBJECTIVE To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology. STUDY DESIGN 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ¿zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]¿ and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen. RESULTS Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software. CONCLUSIONS Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

Public perceptions of cardiovascular risk factors in Croatia: the PERCRO survey.

OBJECTIVE Since there are almost no data about the perceptions and attitudes of the general public concerning risk factors for cardiovascular diseases (CVD) this survey was performed. METHOD Face-to-face interviews were conducted based upon a questionnaire with 883 members of the general population from different parts of Croatia in October-November 2008. RESULTS 36.1% of them correctly identified CVD as the leading cause of death but the most feared disease was cancer (40.0%). CVD was on the second place with only 21.6%. 30.9% was aware that elevated LDL-cholesterol increases CVD risk but 49.0% knew that high HDL-cholesterol is beneficial. 43.3% knew their total cholesterol (TC) value and 74.4% knew their blood pressure (BP). 30.9% knew what their target TC should be and 33.9% knew what their target BP should be. The knowledge about TC and BP target values was positively associated with higher educational level. 53.2% of the general public reported that they have never discussed any CVD risk factor with their physician. CONCLUSION These data suggest insufficient awareness of CVD risk factors in general population and a need for improved promotion of CVD prevention. To achieve this people and physicians should be motivated by the health authorities and/or insurance companies.

The dose-response of nitrous oxide in postoperative nausea in patients undergoing gynecologic laparoscopic surgery: a preliminary study.

BACKGROUND:Whether nitrous oxide (N2O) increases the incidence of postoperative nausea and vomiting (PONV) after laparoscopic gynecologic surgery is still controversial, which may be due to the administration of different concentrations of inspired N2O. We investigated whether N2O results in a dose–response increase in PONV. METHODS:Patients undergoing gynecologic laparoscopic surgery were randomized to receive 30% oxygen with air (G0, n = 46), 50% N2O with oxygen (G50, n = 46), or 70% N2O with oxygen (G70, n = 45). A standardized general anesthetic was used with no PONV prophylaxis. Known risk factors for PONV were controlled. Metoclopramide was used as a rescue antiemetic. The incidence of nausea, vomiting, use of rescue antiemetic, and pain visual analog scale (VAS) score was measured at 2 and 24 h postoperatively. RESULTS:Patient demographics were comparable, and there were no differences among groups regarding factors that may influence PONV. The incidence of PONV at 24 h was 33% (15 of 46) in the G0 group, 46% (21 of 46) in the G50 group, and 62% (28 of 45) in the G70 group (P = 0.018). Subgroup analysis revealed a difference between G0 versus G70 groups (P = 0.018), but no significant difference between G0 versus G50 groups and G50 versus G70 groups. The incidence of nausea showed a similar difference (G0 = 26%, G50 = 35%, and G70 = 56%; P = 0.012), but the incidence of vomiting was not different among the groups although there was a trend (G0 = 28%, G50 = 35%, and G70 = 42%; P = 0.377). The severity of nausea (measured by VAS 100 mm) was significantly increased with increasing N2O concentration (G0 = 10.9, G50 = 12.7, and G70 = 20.5; P = 0.027). The highest VAS score during 24 h was used for the analysis. There was no difference in the use of a rescue antiemetic among groups. Pain VAS scores and opioids consumption were not different among groups (at 2 and 24 h after surgery). CONCLUSIONS:N2O increases the incidence of postoperative nausea after gynecologic laparoscopic surgery. This preliminary finding indicates that N2O may increase PONV in a dose-dependent fashion. A study with a sample size of >400 patients in each group would be necessary to demonstrate a statistically significant difference among each of these three groups. We do not recommend using a high concentration of N2O in this clinical setting.

The perception and knowledge of cardiovascular risk factors among medical students

Aim To assess perceptions, knowledge, and awareness of cardiovascular disease (CVD) risk factors among medical students (freshmen and graduating students). Methods A descriptive cross-sectional survey based on an anonymous self-administered questionnaire was conducted in 2008 on 443 medical students – 228 freshmen on their enrollment day and 214 students on the day of their final exam at the University of Zagreb School of Medicine, Croatia. Results The perception and knowledge of some CVD risk factors, eg, dyslipidemia, arterial hypertension, and metabolic syndrome as well as of lipid-lowering therapy important for CVD prevention was significantly better among graduating students but was still not sufficient. Only 66% of graduating students reported that they would prescribe lipid-lowering therapy to high risk patients. Disappointingly, many graduating students were smoking (30.4%) and had low-awareness of obesity as an important CVD risk factor. Conclusion These results suggest an urgent need to improve medical students’ knowledge of obesity and low physical activity as important CVD risk factors and of the methods for increasing low high-density lipoprotein-cholesterol and for smoking cessation. All this provides a rationale for modifying the university core curriculum to include more information concerning these issues.

CD43 Expression Is an Adverse Prognostic Factor in Diffuse Large B-Cell Lymphoma

BACKGROUND CD43 is a transmembrane glycoprotein expressed in different hematopoietic cells, including some subsets of B lymphocytes. About a quarter of diffuse large B-cell lymphomas (DLBCLs) express CD43, but its prognostic significance is unknown. PATIENTS AND METHODS We analyzed the prognostic effect of immunohistochemically determined CD43 expression in 119 patients with newly diagnosed DLBCL. All were treated with CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone)-like chemotherapy, 57 without and 62 with rituximab. RESULTS A total of 31 DLBCL cases (26%) expressed CD43. Patients with CD43+ and CD43- lymphomas did not differ regarding sex, International Prognostic Index (IPI) factors and score, rituximab treatment, presence of bulky disease, or germinal center subtype. Median follow-up was 45 months. Patients with CD43+ DLBCL had significantly lower complete response rates (59% vs. 80%; P = .019), 2-year event-free survival (EFS) rates (34% vs. 64%; P = .003), and overall survival (OS) rates (45% vs. 76%; P = .002). The prognostic significance of CD43 expression was retained in multivariate analysis (relative risk [RR] 2.04; P = .013 for EFS; RR 2.17; P = .016 for OS). In subgroup analysis, the effect of CD43 expression was significant in patients treated with rituximab and those with low IPI, whereas it was not reached in patients treated without rituximab. The effect was not observed in patients with high IPI. CONCLUSION These results indicate that CD43 expression is an important independent adverse prognostic factor in DLBCL.

The Role of Classic Risk Factors and Prothrombotic Factor Gene Mutations in Ischemic Stroke Risk Development in Young and Middle-Aged Individuals

BACKGROUND In young individuals, a genetically predisposing hypercoagulability and classic modifying risk factors can act synergistically on the ischemic stroke risk development. The aim of the study was to compare the prevalence of classic vascular risk factors and polymorphisms of the G20210A coagulation factor II (prothrombin), Arg506Glu coagulation factor V Leiden, C677T methylenetetrahydrofolate reductase (MTHFR), and 4G/5G plasminogen activator inhibitor-1 (PAI-1) and the impact of these gene mutations and classic vascular risk factors on the overall stroke risk in individuals aged 55 years or younger. METHODS The study included 155 stroke patients aged 55 years or younger and 150 control subjects. Stroke prevalence and odds ratio (OR) were assessed for the following parameters: G20210A prothrombin, Arg506Glu factor V Leiden, C677T MTHFR, and 4G/5G PAI-1 polymorphisms; total number of study polymorphisms in a particular subject (genetic sum); and classic vascular risk factors of hypertension, obesity, diabetes mellitus, cigarette smoking, hypercholesterolemia, hypertriglyceridemia, and elevated levels of low-density lipoprotein (LDL) cholesterol and very low-density lipoprotein cholesterol. RESULTS The prevalence of hypertension (P < .001), smoking (P < .001), decreased HDL cholesterol levels (P < .001), obesity (P = .001), elevated LDL cholesterol (P = .036), C677T MTHFR polymorphism (P < .001), and genetic sum was significantly higher in the group of stroke patients. The following parameters were found to act as independent risk factors for ischemic stroke: decreased HDL cholesterol level (P < .001; OR 4.618; 95% confidence interval [CI] 2.381-8.957); hypertension (P = .001; OR 2.839; 95% CI 1.519-5.305); obesity (P = .040; OR 2.148; 95% CI 1.036-4.457); smoking (P = .001; OR 2.502; 95% CI 1.436-4.359); and genetic sum as a continuous variable (P < .01; OR 2.307; 95% CI 1.638-3.250). CONCLUSIONS Gene mutations of the procoagulable and proatherosclerotic factors investigated exerted a synergistic action in the development of overall risk of ischemic stroke in young and middle-aged individuals.

The triple-marker test in predicting fetal aneuploidy: a compromise between sensitivity and specificity

OBJECTIVE to review the contribution of unconjugated estriol in Downs syndrome detection, and influence of maternal age, cut-off choice, and population specificity on the balance between triple-marker test sensitivity and specificity. STUDY DESIGN Prenatal karyotyping was performed in 2833 pregnant women, 73% of them over the age of 34. Duration of gestation was determined by ultrasound in 98% of women. Prior to amniocentesis, the serum levels of alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol were evaluated and corrected for weight. The risk of trisomy 21 was calculated for the first 986 subjects using default medians, and for 1847 by our population-specific medians. The cut-off was initially 1:300 at term, but the 1:100 and 1:200 risks were also tested. Down syndrome risk was calculated with alpha-fetoprotein and human chorionic gonadotropin combination as well. Linear logistic regression model was performed to test the ability of aneuploidy markers to classify patients into normal and trisomic groups. RESULTS There were twelve cases of Downs syndrome, seven of trisomy 18, four of trisomy 13, and one trisomy 22. Four cases of aneuploidy (16.7%) referred to women younger than 35. With the cut-off risk of 1:300, detection rate was 87.5% and specificity 63.3%, and with 1:100, 66.7% and 79.5%, respectively. The sensitivity for Downs syndrome was from 75% for cut-off=1:100 to 92% for cut-off=1:300, while detection of other trisomies was less successful (58% and 83%, respectively). Exclusion of unconjugated estriol MoM from the risk calculations reduced detection rate by 33% and improved specificity by 4% independently of cut-off choice. Linear logistic regression analysis showed that only unconjugated estriol was able to correctly classify patients between normal and trisomy 21 (p=0.011, odds ratio=1.445), and normal and trisomy 18 (p=0.0023, odds ratio=1.96) groups. CONCLUSIONS The unconjugated estriol significantly contributes in Downs syndrome detection with cost of slightly reduced specificity. The 1:300 risk caused an unfavorable compromise between sensitivity and specificity. A higher cut-off, 1:100, would indicate performance of amniocentesis in women aged 39 years and older, and in those aged 35-39 only after the screen-positive result.

A clinical decision rule for triage of children under 5 years of age with hydrocarbon (kerosene) aspiration in developing countries

Background. Unintended hydrocarbon ingestion is a common reason for pediatric hospitalization in the developing world. Objective. To derive a clinical decision rule, to identify patients likely to require a higher level facility (resource-requiring cases), that can be used at primary health care facilities with limited diagnostic and therapeutic resources. Methods. A prospective study of children 2 to 59 months old presenting to a poison treatment facility within 2 hours of oral hydrocarbon exposure. History and objective signs were recorded at admission and at 6, 12, 24 and, if present, 48 hours. Inclusion in the resource-requiring outcome group required: oxygen saturation <94%; any CNS depression; any treatment with (salbutamol); any care in the ICU; or death. Results. 256 met the inclusion criteria and completed the study. Of these, 170 had a course requiring resources unavailable at most primary health care facilities, and 86 did not. The presence of wheezing, any alteration in consciousness (lethargy or any restlessness), or a rapid respiratory rate for age (RR ≥50/min if age < 12mo, ≥ 40/min if age ≥ 12 mo) at presentation identified 167 of 170 of these patients (sensitivity 0.98). Thirty-six of 86 patients classified as non-resource requiring were correctly identified (specificity 0.42). No combination of clinical symptoms provided better discrimination while preserving sensitivity. Conclusions. This study suggests a triage decision rule based on the presence of wheezing, altered consciousness, or a rapid respiratory rate within 2 hours of hydrocarbon exposure. Such a rule requires validation in other settings.

Prevalence and socioeconomic aspects of epilepsy in the Croatian county of Sibenik-Knin: community-based survey.

The aim of the survey was to estimate the prevalence rate of epilepsy, as well as seizure types, frequency, etiology, and comorbidity, by gender, age, and socioeconomic status among people with epilepsy in the Croatian county of Sibenik-Knin. The survey revealed that of 112,871 inhabitants, 1228 were identified as having active epilepsy. The crude prevalence rate for Sibenik-Knin County was 10.9 per 1000 inhabitants. Prevalence rates (per 1000) by age and gender were: 6.9 (ages 0-19); 10.6 (ages 20-59); 15.1 (ages 60+), 10.1 (females), and 11.7 (males). A significant number of subjects had different comorbid disorders. Although the literature suggests that Dalmatia is a region with a low prevalence of epilepsy, our results showed that Sibenik-Knin County has a higher prevalence of epilepsy than expected for European populations. The most common comorbid disorders, such as mental retardation, psychotic episodes, and substance addiction, highly influence socioeconomic status and quality of life.

A Clinical Decision Aid for Triage of Children Younger Than 5 Years and With Organophosphate or Carbamate Insecticide Exposure in Developing Countries

STUDY OBJECTIVE Unintentional pediatric exposure to insecticides is common in developing countries. A clinical decision aid could guide early triage decisionmaking. METHODS Study design was prospective observational data collection in a specialty poisoning hospital in Cairo, Egypt. Patients were children 2 months to 59 months of age, without pretreatment, presenting within 2 hours of an exposure to an organophosphate or carbamate insecticide. A resource-requiring course was defined as any occurrence of hypoxia, use of atropine or obidoxime, use of ICU care, or death. The goal of analysis was derivation of a clinical decision aid to predict a resource-requiring course with 100% sensitivity. RESULTS During the 21-month study, 197 children 2 months to 59 months of age exposed to an organophosphate or carbamate insecticide were treated at the center. One hundred two of these children met the study inclusion criteria: 95 had parental consent and completed the study observation period of which 65 used resources (4 died). All patients who ultimately met resource-requiring criteria initially did so at arrival. Pinpoint pupil alone identified 63 of 65 of these patients yet wrongly identified only 5 of 30 minimally ill patients. Pinpoint pupil or diarrhea identified 65 of 65 patients with a resource-requiring course while identifying 7 of 30 patients with a non-resource-requiring course (sensitivity 1.00; 95% confidence interval 0.95 to 1.00; specificity 0.77; 95% confidence interval 0.58 to 0.90). CONCLUSION Using 2 features, pinpoint pupils and diarrhea, we identified at presentation all patients who ultimately had a course using medications or advanced resources. According to this preliminary study, symptoms occur rapidly, so using an early triage aid may be feasible. A validation study is necessary.