Advanced molecular approaches in male infertility diagnosis†.

In the recent years a special attention has been given to a major health concern namely to male infertility, defined as the inability to conceive after 12 months of regular unprotected sexual intercourse, taken into account the statistics that highlight that sperm counts have dropped by 50-60% in recent decades. According to the WHO, infertility affects approximately 9% of couples globally, and the male factor is believed to be present in roughly 50% of cases, with exclusive responsibility in 30%. The aim of this article is to present an evidence-based approach for diagnosing male infertility that includes finding new solutions for diagnosis and critical outcomes, retrieving up-to-date studies and existing guidelines. The diverse factors that induce male infertility generated in a vast amount of data that needed to be analyzed by a clinician before a decision could be made for each individual. Modern medicine faces numerous obstacles as a result of the massive amount of data generated by the molecular biology discipline. To address complex clinical problems, vast data must be collected, analyzed, and used, which can be very challenging. The use of artificial intelligence (AI) methods to create a decision support system can help predict the diagnosis and guide treatment for infertile men, based on analysis of different data as environmental and lifestyle, clinical (sperm count, morphology, hormone testing, karyotype, etc.), and "omics" bigdata. Ultimately, the development of AI algorithms will assist clinicians in formulating diagnosis, making treatment decisions, and predicting outcomes for assisted reproduction techniques.

Impact of chronic administration of anabolic androgenic steroids and taurine on blood pressure in rats.

Supraphysiological administration of anabolic androgenic steroids has been linked to increased blood pressure. The widely distributed amino acid taurine seems to be an effective depressor agent in drug-induced hypertension. The purpose of this study was to assess the impact of chronic high dose administration of nandrolone decanoate (DECA) and taurine on blood pressure in rats and to verify the potentially involved mechanisms. The study was conducted in 4 groups of 8 adult male Wistar rats, aged 14 weeks, treated for 12 weeks with: DECA (A group); vehicle (C group); taurine (T group), or with both drugs (AT group). Systolic blood pressure (SBP) was measured at the beginning of the study (SBP1), 2 (SBP2) and 3 months (SBP3) later. Plasma angiotensin-converting enzyme (ACE) activity and plasma end products of nitric oxide metabolism (NOx) were also determined. SBP3 and SBP2 were significantly increased compared to SBP1 only in the A group (P<0.002 for both). SBP2, SBP3 and ACE activity showed a statistically significant increase in the A vs C (P<0.005), andvs AT groups (P<0.05), while NOx was significantly decreased in the A and AT groups vs controls (P=0.01). ACE activity was strongly correlated with SBP3 in the A group (r=0.71, P=0.04). These findings suggest that oral supplementation of taurine may prevent the increase in SBP induced by DECA, an effect potentially mediated by angiotensin-converting enzyme.

Impact of chronic administration of anabolic androgenic steroids and taurine on blood pressure in rats

Supraphysiological administration of anabolic androgenic steroids has been linked to increased blood pressure. The widely distributed amino acid taurine seems to be an effective depressor agent in drug-induced hypertension. The purpose of this study was to assess the impact of chronic high dose administration of nandrolone decanoate (DECA) and taurine on blood pressure in rats and to verify the potentially involved mechanisms. The study was conducted in 4 groups of 8 adult male Wistar rats, aged 14 weeks, treated for 12 weeks with: DECA (A group); vehicle (C group); taurine (T group), or with both drugs (AT group). Systolic blood pressure (SBP) was measured at the beginning of the study (SBP1), 2 (SBP2) and 3 months (SBP3) later. Plasma angiotensin-converting enzyme (ACE) activity and plasma end products of nitric oxide metabolism (NOx) were also determined. SBP3 and SBP2 were significantly increased compared to SBP1 only in the A group (P<0.002 for both). SBP2, SBP3 and ACE activity showed a statistically significant increase in the A vs C (P<0.005), andvs AT groups (P<0.05), while NOx was significantly decreased in the A and AT groups vs controls (P=0.01). ACE activity was strongly correlated with SBP3 in the A group (r=0.71, P=0.04). These findings suggest that oral supplementation of taurine may prevent the increase in SBP induced by DECA, an effect potentially mediated by angiotensin-converting enzyme.

Colocalization methods in pituitary tumorigenesis aged-related in MEN1 KO and wild type mice.

BACKGROUND: Colocalization analysis of confocal fluorescence and electron microscopy (EM) are important tools to detect the expression of multiple anterior pituitary hormones within the same cell. Heterozygous (Men1+/-) mice developed pituitary tumors, mostly reported somatolactotrophinomas and ACTH secreting pituitary adenomas but also nonfunctioning tumors. The aim of the study was to run immunohistochemistry protocols to study colocalization of pituitary hormones in newborn mice in tumoral and non-tumoral tissue in MEN1-KO and wild type control mice. METHODS: Pituitary samples from nine Men1+/- mice, 29-34 days old male mice (n=8) and one year old (n=1) and control group, four new born (1,5 days old) wild type (mus musculus) mice were analyzed by immunofluorescence immunohistochemistry (GH, PRL, gonadotrophs) to find hormonal colocalization in pituitary cell. Moreover, pituitaries were embedded in LRGold for immunogold labeling technique (GH, PRL, gonadotrophs and alpha-SU) also. RESULTS: Pituitary tumors, immunoreactive only for PRL were found in three MEN1 - KO mice. No sign of pituitary hyperplasia was found in MEN1-KO. MEN1-KO non-tumoral pituitary displayed similar immunoreactivity to wild type pituitary. Colocalization studies revealed individual cells PRL-FSH immunoreactive and GH-FSH immunoreactive in the non-tumoral tissue from MEN1-KO mice and in wild type pituitaries respectively but no colocalization in the tumoral tissue. In conclusion, colocalization is a feature of neonate mouse pituitary but not in adults. The MEN1-KO pituitary tumors were prolactinomas and unlike non-tumoral pituitary tissue of MEN1-KO, displayed no PRL-FSH colocalization.

Statins: mechanism of action and effects.

The beneficial effects of statins are the result of their capacity to reduce cholesterol biosyntesis, mainly in the liver, where they are selectively distributed, as well as to the modulation of lipid metabolism, derived from their effect of inhibition upon HMG-CoA reductase. Statins have antiatherosclerotic effects, that positively correlate with the percent decrease in LDL cholesterol. In addition, they can exert antiatherosclerotic effects independently of their hypolipidemic action. Because the mevalonate metabolism generates a series of isoprenoids vital for different cellular functions, from cholesterol synthesis to the control of cell growth and differentiation, HMG-CoA reductase inhibition has beneficial pleiotropic effects. Consequently, statins reduce significantly the incidence of coronary events, both in primary and secondary prevention, being the most efficient hypolipidemic compounds that have reduced the rate of mortality in coronary patients. Independent of their hypolipidemic properties, statins interfere with events involved in bone formation and impede tumor cell growth.

The total peroxyl radical trapping potential in serum - an assay to define the stage of atherosclerosis.

Lipid peroxides were identified among the factors that contribute to the atherosclerotic plaque formation in the arterial wall. We hypothesised that a correlation may exist between the content of antioxidant constituents in the serum and the gravity of atherosclerosis. To this purpose, we have determined the serum total peroxyl radical- trapping potential (TRAP), which is the combined capacity of all antioxidants to neutralize free radicals in serum and followed its variation in hyperlipemic animals in correlation with the stage of atherosclerosis. In addition, we compared TRAP values in the sera of coronary heart disease (CHD) patients, with or without type II diabetes mellitus. Results showed that after 18 weeks of hyperlipemic diet, the mean TRAP values measured in sera isolated from hyperlipemic hamsters exhibited an about 44% decrease, in good agreement with the increase of serum cholesterol and triglycerides. In the 3 groups of CHD patients, TRAP values decreased with about 10% in sera of stable angina patients, 20% in unstable patients, as compared with normal subjects. The lowest TRAP values were detected in the sera of patients with acute myocardial infarction. The results obtained for different experimental animals and for CHD patients sera indicate that the TRAP method, as adapted in our laboratory, is a reliable and reproducible assay, fit to be used in clinical studies as an ex vivo measurable parameter that correlates with the stage of the atherosclerosis.

The hyperlipemic hamster - a model for testing the anti-atherogenic effect of amlodipine.

Male Golden Syrian hamsters were subjected to a hyperlipemic diet. At intervals ranging from 2 to 14 weeks, the animals were examined for changes in serum constituents and structural modifications of lesion-prone areas: the cardiac valves, coronary arteries and aortic arch. Serum was characterized by a gradual increase in cholesterol, triglycerides and a decrease in total peroxyl-radical trapping potential. The sequence of modifications of the endothelial cells, smooth muscle cells, and migrating plasma monocytes as well as of the extracellular matrix were established. Amlodipine treatment of hyperlipemic hamster was assessed. Amlodipine exhibited an athero-protective effect, acting as antioxidant, reducing the LDL uptake by the vessel wall and consequently, limiting the size and extent of lesioned areas. The hyperlipemic hamster is a reliable model to unravel the cellular alterations leading to atheroma formation, and for testing the effect of drugs in this process.

Pathobiology of the heart in experimental diabetes: immunolocalization of lipoproteins, immunoglobulin G, and advanced glycation endproducts proteins in diabetic and/or hyperlipidemic hamster.

Diabetes is known to be accompanied by atherosclerotic disease and general cardiovascular complications. Hamsters were previously shown to develop hyperlipemia-induced atherosclerosis, similar in many respects to the human atherosclerotic process. To study the effect of hyperglycemia on heart vessels and valves, male Golden Syrian hamsters were rendered either diabetic or hyperlipemic and diabetic; controls were age-matched normal hamsters. At time intervals ranging from 2 to 24 weeks, animals were killed; plasma glucose, cholesterol, and lipid peroxides were measured; and the aortic arch and valves, coronary arteries, and heart microvessels were examined for ultrastructural modifications and for the presence of low-density lipoproteins (LDL), immunoglobulin G (IgG), and advanced glycation endproducts (AGE) proteins. Elevation of plasma glucose, peroxides, and cholesterol were observed in both diabetic as well as hyperlipemic and diabetic animals, along with characteristic diabetic changes: microangiopathy of the myocardium (ie, capillary narrowing, hyperplasia of basal lamina, and proliferation of extracellular matrix) and macroangiopathy of the aortic arch, valves, and coronary arteries (ie, intimal proliferation, fatty-streak formation, and calcification). LDL, IgG, and AGE-proteins were immunolocalized in focal deposits, ie, in the shoulder and cap of the plaques; these antigens were distributed diffusely in the extracellular space or within macrophage-derived foam cells and smooth muscle cells. Our findings indicate that hyperglycemia alone induces atherosclerotic lesions in the coronary arteries, aortic arch, and aortic valves as well as alterations of the extracellular matrix of heart microvessels and cardiomyocytes, changes which together may lead to cardiomyopathy, a common and severe complication of diabetes. In addition, the present study suggests that when hyperglycemia is accompanied by hyperlipemia, detectable amounts of modified LDL (possibly oxidized or glycated) and AGE are present in the intima of atherosclerotic arteries; and also that modified lipoproteins can act as immunoactive components of the atheroscerotic process generated by hyperglycemia.

Immunodetection of modified lipoproteins in plasma and arterial walls of patients with coronary heart disease.

Data from literature indicate that immune processes play an important role in atherogenesis. Modified lipoproteins might be immunogenic and generate autoantibodies in plasma. To determine whether the level of such circulating autoantibodies correlates with the extent of atherosclerosis expressed as cholesterol values in plasma (C), very low density (VLDL-C), low density (LDL-C), and high density lipoproteins (HDL-C), we compared the level of plasma autoantibodies of a group of coronary heart disease patients (CHD-P) with that of normal, age-matched donors, with no history of cardiac disease (N). All CHD-P (even normocholesterolemic) were characterized by an LDL-C/HDL-C ratio > 4, while all N (even hypercholesterolemic) had this ratio < 4. A double level of circulating autoantibodies against VLDL and LDL in CHD-P as compared to N group was detected. The anti-LDL antibodies level correlated well with LDL-C level and was negatively correlated with the age of patients. For tissue localization of native and modified LDL (as well as other possibly modified proteins) we used immunohistochemical techniques, employing antihuman LDL, antihydroxynonenal-lysine (HNE-Lys), and antiadvanced glycation end-products (AGE) proteins. Antibodies were applied on consecutive cryosections of the aortic arch, valves and coronary arteries of CHD-P. The immunodetected antigens were colocalized in focal deposits, in the cap and shoulders of the atheroma. Native LDL and modified proteins (AGE, HNE-Lys) were detected either diffuse-extracellularly or associated with macrophage-derived foam cells and smooth muscle cells of the intima. These data indicate the following: a) the existence of an elevated level of circulating autoantibodies against VLDL and LDL, which correlates negatively with the age of CHD patients; b) the presence of LDL (possibly glycated or oxidized) in detectable amounts in the intima of atherosclerosis-affected arteries; c) the modified lipoproteins are immunoactive components in the atherosclerotic process.

Pathobiochemistry of combined diabetes and atherosclerosis studied on a novel animal model. The hyperlipemic-hyperglycemic hamster.

Because accelerated atherosclerosis is the main complication of diabetes, we devised a new animal model that combines these two diseases, and investigated their joint impact on the main plasma components and organs known to be most affected in each disorder. Male Golden Syrian hamsters were subjected to three experimental conditions: streptozotocin-induced diabetes (D), diet-induced hyperlipemia (H), and a combination of hyperlipemia and diabetes (HD). At time intervals ranging from 2 to 24 weeks, the animals were sacrificed, the appropriate plasma constituents were determined, and the ultrastructural modifications of relevant tissues such as the heart, cardiac valves, coronary arteries, aorta, retina, and kidney were examined. The HD hamsters were characterized by marked alternations of plasma components, ie, increase in circulating glucose, cholesterol and lipid peroxide levels, glycation of albumin, and the appearance of irreversibly glycated albumin (AGE-Alb). These humoral changes coexisted with micro- and macroangiopathic lesions characteristic to both diseases, ie, capillary narrowing, hyperplasia of endothelial basal lamina, proliferation of perivascular extracellular matrix (abnormalities reminiscent of type I diabetes), and concomitant intimal accumulation of modified lipoproteins and macrophage-derived foam cells in the aorta, coronaries, and cardiac valves, leading to accelerated formation of atherosclerotic plaques. These changes eventually appeared in the D hamsters also, but at a much slower rate, whereas the H group showed only modifications characteristic for atherosclerosis. Our findings indicate that, overall, 1) diabetes accelerated the early development and progression of atherosclerotic lesions leading to rapid calcification, and 2) hyperlipidemia associated with diabetes accelerated the rate of development of diabetes-induced microvascular disease. The hamster model may be useful to study the impact of various drugs on the diabetes-related vascular complications.

Correlated biochemical modifications of plasma lipoproteins in coronary heart disease: an accelerating pathologic factor.

We investigated the presence of biochemically modified plasma lipoproteins as pathologic factor for coronary heart disease in 15 patients with angina pectoris (CHD-P) vs 20 normal subjects (N). Decreased HDL were the most significant pathological feature present in P over 66 years old, while, P under 66 had, in addition to low HDL-Cholesterol (HDL-C), high levels of plasma cholesterol (C), LDL-Cholesterol (LDL-C), and lipid peroxides (TBARS), together with the presence of desialylated LDL and VLDL. We demonstrated by statistic analysis that these risk factors are correlated: high plasma C with a more pronounced imbalance between LDL and HDL, which, in turn, is associated with high TBARS levels, and also with circulating desialylated VLDL; high plasma TBARS values with desialylated LDL. We detected an increased level of autoantibodies towards autologous LDL and VLDL, in P vs N. The level of autoantibodies anti-LDL correlated with LDL-C level and with LDL desialylation, thus modified circulating LDL being most probably atherogenic. Circulating anti-LDL autoantibodies together with the low level of HDL might contribute to acceleration and aggravation of the atherosclerotic process.

Preliminary clinical and methodologic observations on the determination of serum gamma-glutamyltranspeptidase and of the alpha-amylase clearance in nephropathies.

Serum gamma-glutamyltranspeptidase (GGTP) and alpha-amylase clearance were determined in a total group of 90 patients of whom 60 with renal diseases and 30 with extrarenal diseases. The renal patients were distributed, according to diagnosis in the following groups: acute glomerulonephritis, chronic glomerulonephritis, acute pyelonephritis, chronic pyelonephritis, nephrotic syndrome and manifest chronic renal failure. The 30 controls were hospitalized for different extrarenal diseases such as: pneumonia, gastroduodenal ulcer, arterial hypertension stage I and angina pectoris. Serum GGTP assay was performed in 60 patients (40 renal patients and 20 controls) using Boehringer monotest kits and in 30 patients (20 renal patients and 10 controls) using Romanian kits (I.C.C.F.). No changes suggesting a particular type of nephropathy were observed. The results obtained by using the two types of kits for the serum GGTP assay have proved to be very close. Alpha-amylase clearance was determined in all the patients with Spofa (R.S.C.) tablets concomitantly with the urea and creatinine clearance. Important decreases of alpha-amylase clearance in concordance with decreases of urea and creatinine clearances were observed in all the patients with severe renal failure. More moderate decreases of alpha-amylase clearance were observed in the patients with acute and chronic glomerulonephritis. The utility of this clearance as a test of glomerular filtration and sometimes as a prognostic test, is discussed.

Drug allergy: incidence in terms of age and some drug allergens.

The authors studied the incidence of drug allergy in 3 groups of subjects. In the first group the incidence was established in terms of the subjects' age: 1.8% in children up to 14 years old (323 subjects), 5.8% in middle-aged subjects (389 subjects) and 2.9% in the elderly (243 subjects). In the second partly selected group, drug allergy was studied in patients admitted to the 3rd Medical Clinic--Craiova between 2 Jan. 1981 and 31 Dec. 1982; 103 of the 8,760 patients admitted presented allergic reactions, a relatively high incidence rate (1.01%) as compared to other published reports. The third group of previously selected cases consisted of 197 patients who had come to the allergology service with complaints of present or past sensitivity to drugs. The total number of patients was 336 selected or preselected from about 10,000 subjects, to which 17 patients with a diagnosis of "asthmatic triad" must be added. Drug allergy in terms of the drug used was dominated by penicillin, pyramidon-algocalmin, aspirin, iodine preparations, etc.; foremost among the clinical syndrome were the dermatologic syndromes types I, III and IV; syndromes of the "anaphylactic shock" type, "serum sickeness" bronchial asthma, angioneurotic oedema.

Treatment of bronchial asthma with disodium cromoglycate. Special indications.

Forty patients with various types of bronchial asthma were treated with disodium cromoglycate (DSCG) for various periods of time ranging from 30 days to 90 months. The drug was inhaled through a spinhaler at 8-hour intervals. The symptoms of asthma, the respiratory function and the drug-induced reactions were checked during treatment. DSCG was salutary in about 80% of the patients with extrinsic asthma (even in those with pollen allergy diagnosed within the pollination period). The drug was also effective in patients with exercise induced asthma, asthma associated with pregnancy, asthma with adrenocorticosteroid psychosis. The patients with meal allergy (bakers and millers) treated with DSCG were able to keep their occupational activities. The drug may be used in the intrinsic asthma only in subjects with many positive skin tests and hypereosinophilia.

New studies on the mold spore incidence in the homes of asthmatics.

The incidence of mould spores was studied during 11 months using Petri plates with Czapek medium exposed once a month in 17 homes of asthmatics in the town, "C", situated in an agro-industrial region. Penicullium was the prevalent mould in all season; Aspergillus was dominant in February-September; Cladosporium was found in May and September; Alternaria was absent. It is assumed that some of the asthmatics in this area could have a type III hypersensitivity (asthma with precipitins).