The silent variants of pituitary tumors: demographic, radiological and molecular characteristics.

INTRODUCTION: Tumors of the anterior pituitary gland (PTs) are mostly benign tumors with a low prevalence, which has nevertheless increased with advances in brain radiology techniques. Nearly half of PTs are not associated with a clinical endocrine syndrome. These tumors have been indistinctly named non-functioning pituitary adenomas (NFPAs) or silent pituitary tumors (SPTs) and the mechanisms of silencing are not fully known. AIM: To study the frequency and characterize the silent variant of PTs in a large local series, and to assess their pituitary adenohypophyseal gene expression. METHODS: This observational, cross-sectional study was performed in a Pituitary Tumor Center of Excellence and involved 268 PTs. After identifying the different subtypes according to the immunohistochemical (IHC) expression of adenohypophyseal hormones, we studied their gene expression by RT-qPCR. RESULTS: We found that silent tumors were larger and more invasive, but not more proliferative than their functional counterparts. The RT-qPCR complements the IHC typification of PTs, reducing the proportion of null-cell subtype. Finally, some silent PT subtype variants showed lower specific adenohypophyseal hormone gene expression than their functional counterparts, which may contribute to the absence of endocrine manifestations. CONCLUSIONS: This paper highlights the importance of identifying the silent variant of the PTs subtypes. As expected, silent tumors were larger and more invasive than their functioning counterparts. However, there was no difference in the proliferation activity between them. Finally, the lower specific gene expression in the silent than in the functioning counterparts of some PTs subtypes gives insights into the silencing mechanisms of PTs.

Is Somatostatin Receptor and Dopamine Receptor profiling useful in the management of silent somatotroph tumors?

Silent somatotroph tumors (sSTs) are pituitary neuroendocrine tumors (PitNETs) which do not give rise to the clinical syndrome of acromegaly. Differently to their functioning counterparts, the adjuvant medical treatment with somatostatin analogues (SSAs) or dopamine receptors agonists (DAs) has been scarcely addressed in these tumors. As preliminary results of an ongoing research on silencing mechanisms involved in the pathogenesis of sSTs, we have characterized by qRT-PCR the expression of SSTRs and DRDs in a large series of 18 silent and 68 functioning STs. Although the expression of SSTR2 and SSTR5 was lower in sSTs than in functioning ones, we found a negative correlation between SSTR2 and the tumor size of the sSTs. Additionally, levels of expression of DRD2 were similar between the two subtypes suggesting a possible basis for the treatment of these tumors with SSAs and DAs.

Monitoring the John Cunningham virus throughout natalizumab treatment in multiple sclerosis patients.

BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) cases have arisen amongst multiple sclerosis patients treated with natalizumab. Our objective was to gain a better understanding of the mechanisms that underlie the John Cunningham virus (JCV) infection which causes PML. METHODS: A study was made of (i) the quarterly JCV DNA levels in peripheral blood mononuclear cells (PBMCs), serum and urine samples in 100 multiple sclerosis patients during their natalizumab treatment (3-39 months), (ii) the association between human leukocyte antigen (HLA) class II and the previous viral detection and (iii) the identification of the JCV variants in those patients suspected of having PML. RESULTS: (i) JCV DNA in PBMCs and/or serum was detected in 23% of our cohort. Patients with an intermittent JCV excretion in urine had a significant increase of the viral load and prevalence in this compartment during natalizumab treatment. (ii) The frequency of the DRB1*07/DQA1*02:01/DQB1*02:02 haplotype tended to be higher in patients with detectable versus undetectable JCV DNA in PBMCs (P(corrected) = 0.108). (iii) The variants in PBMCs and serum of the non-PML patient matched the archetype. In the patient with non-fatal PML, the archetype and the same neurotropic variant in PBMCs, serum and cerebrospinal fluid was identified at the time PML was diagnosed, whereas in the patient with a worse PML prognosis, four neurotropic variants in the three previous compartments were found by the PML diagnosis. CONCLUSIONS: The detection of the neurotropic variant in blood during natalizumab treatment could be critical in the prevention of the development of severe PML, since this variant appears simultaneously with the clinical symptoms of PML and mutates quickly.

Toll-like receptor-9 in Spanish multiple sclerosis patients: an association with the gender.

BACKGROUND AND PURPOSE: Toll-like receptor-9 (TLR9) is a potent inducer of innate immune system triggered by infection with viruses, some of them previously related to multiple sclerosis (MS). The aim of this study was to analyze the possible association of two TLR9 single nucleotide polymorphisms (SNPs; rs352162 and rs187084) with susceptibility to MS. METHODS: Two independent cohorts of MS patients and controls were included: 574 clinically definite relapsing-remitting MS patients (367 females) and 807 healthy controls (418 females) for the first cohort; and 366 relapsing-remitting MS patients (238 females) and 224 healthy controls (160 females) for the second cohort. Genotyping was performed by TaqMan assays. RESULTS: The AT haplotype was found to be significantly higher in women than in men (P = 0.013 and P = 0.044). CONCLUSIONS: Here two possible genetic markers are proposed that could be also associated with the differences observed in the clinical course of MS in both genders. Further studies with larger cohorts should be performed to confirm these results.

Association between socioeconomic status and hospitalisation requirement in older patients attended at the emergency department: A retrospective cohort study.

INTRODUCTION AND OBJECTIVE: A low socioeconomic status (SES) has been associated with poor health results. The present study aimed to investigate if SES of older patients attending the emergency department is associated with the use of healthcare resources and outcomes. PATIENTS AND METHODS: Observational, retrospective study including consecutive patients 65 years or older admitted to the emergency department. Variables at baseline, index episode, and follow-up were recorded. SES was measured using an indirect theoretical index and patients were categorised into two groups according to whether they lived in a neighbourhood with a low or high SES. Primary outcomes included hospitalisation after the emergency department visit and prolonged hospitalisation (>7 days) at index episode. Secondary outcomes included emergency department re-consultant and hospital admission in the following 3 months after the index episode, and all-cause mortality after long-term follow-up. Logistic regression and cumulative hazards regression models were used to investigate associations between SES and outcomes. RESULTS: The cohort included 553 patients (80 years [73-85], 50.5% female, 55.9% with low SES). After the emergency department visit, 234 patients (42.3%) required hospital admission. A low SES was inversely associated with hospitalisation with an adjusted odds ratio=0.654 (95% CI 0.441-0.970). Among hospitalised patients, a low SES was associated with prolonged hospitalisation (adjusted odds ratio=2.739; 95% CI 1.470-5.104). Follow-up outcomes, including all-cause mortality, were not associated with SES. CONCLUSIONS: Older patients living in more deprived urban areas were hospitalised less often after emergency department care, but hospital stays were longer. Understanding the effect of social determinants in healthcare use is mandatory to tailor resources to patient needs.

Multidisciplinary intervention and cognitive remediation therapy for adults with obesity: A study protocol for a randomized controlled clinical trial.

Background: Cognitive Remediation Therapy (CRT) is a psychological treatment which aims to improve the neurocognitive processes that interfere with the daily functioning of individuals and has proven to be useful in the treatment of obesity. This therapy has been implemented in some countries as a co-adjuvant treatment for people with obesity, but it has not been tested in Mexico, where obesity is one of the main public health problems, so it is essential to implement more studies of this type to obtain effective treatments to control weight. Objective: To describe the research procedure of a multidisciplinary intervention protocol for adults with obesity in a randomized controlled clinical trial. Method: Participants will be adults from 19 to 60 years of age with obesity, who will be randomly assigned to experimental and control groups. The control group will receive intervention only after the experimental group has completed the intervention program. Measurements of body composition, nutritional state, psycho-physiological and physical activities of the participants will be obtained before and after the intervention, with a three-month follow-up after the intervention has concluded. Conclusion: Results of this study will provide useful evidence for the implementation and follow-up of a multidisciplinary intervention with CRT to promote a better efficacy in the treatment and control of obesity.

A variant in GRN of Spanish origin presenting with heterogeneous phenotypes.

INTRODUCTION: The variant c.1414-1G>T in the GRN gene has previously been reported as probably pathogenic in subjects of Hispanic origin in the American continent. METHODS: We report 5 families of Spanish origin carrying this variant, including the clinical, neuroimaging, and laboratory findings. RESULTS: Phenotypes were strikingly different, including cases presenting with behavioral variant frontotemporal dementia, semantic variant primary progressive aphasia, rapidly progressive motor neuron disease (pathologically documented), and tremor-dominant parkinsonism. Retinal degeneration has been found in homozygous carriers only. Ex vivo splicing assays confirmed that the mutation c.1414-1G>T affects the splicing of the exon, causing a loss of 20 amino acids in exon 11. CONCLUSIONS: We conclude that variant c.1414-1G>T of the GRN gene is pathogenic, can lead to a variety of clinical presentations and to gene dosage effect, and probably has a Spanish founder effect.

Increased expression of the endothelin system in arterial lesions from patients with giant-cell arteritis: association between elevated plasma endothelin levels and the development of ischaemic events.

OBJECTIVE: Approximately 15-20% of patients with giant-cell arteritis (GCA) develop ischaemic complications often preceded by transient ischaemia. The expression of the endothelin (ET) system in GCA lesions was investigated to assess its relationship with the development of ischaemic complications. METHODS: Plasma ET-1 was quantified by immunoassay in 61 patients with biopsy-confirmed GCA and 16 healthy donors. ET-1, endothelin-converting enzyme (ECE-1) and endothelin receptor (ET(A)R and ET(B)R) messenger RNA were measured by real-time quantitative reverse transcriptase-PCR in temporal arteries from 35 of these patients and 19 control arteries. Proteins were measured by immunoassay and Western blot. RESULTS: ET-1 concentration was increased at the protein level in temporal artery samples from GCA patients compared with controls (0.98 (SEM 0.32) vs 0.28 (SEM 0.098) fmol/mg, p = 0.028). ECE-1, ET(A)R and ET(B)R/actin ratios (Western blot) were also significantly higher in GCA patients. Intriguingly, mRNA expression of ET-1, ECE-1 and both receptors was significantly reduced in GCA lesions compared with control arteries. When investigating mechanisms underlying these results, platelet-derived growth factor and IL-1beta, present in GCA lesions, were found to downregulate ET-1 mRNA in cultured human temporal artery-derived smooth muscle cells. Glucocorticoid treatment for 8 days did not result in significantly decreased endothelin tissue concentration (0.87 (SEM 0.2) vs 0.52 (SEM 0.08); p = 0.6). Plasma endothelin concentrations were higher in patients with ischaemic complications (1.049 (SEM 0.48) vs 1.205 (SEM 0.63) pg/ml, p = 0.032). CONCLUSIONS: The endothelin system is increased at the protein level in GCA lesions creating a microenvironment prone to the development of ischaemic complications. Recovery induced by glucocorticoids is delayed, indicating persistent exposure to endothelin during initial treatment.

Neutralizing antibodies, MxA expression and MMP-9/TIMP-1 ratio as markers of bioavailability of interferon-beta treatment in multiple sclerosis patients: a two-year follow-up study.

BACKGROUND: The objective of this study was to correlate the detection of neutralizing antibodies (NAbs) by the cytopathic effect (CPE) assay, with the expression of myxovirus resistance protein A (MxA), and the ratio between matrix metalloproteinase 9 (MMP-9) and its tissular inhibitor (TIMP-1), in order to evaluate their usefulness as markers of interferon beta (IFN-beta) bioavailability. METHODS: Pairs of blood and serum samples were collected from 50 patients with multiple sclerosis (MS) during 2 years of IFN-beta treatment. Expression of MxA, MMP-9 and TIMP-1 were analysed by quantitative PCR, and NAbs were measured by CPE assay. RESULTS: During the study, 60% of patients presented NAbs. The number of serum samples that were NAbs+ was significantly increased amongst patients with relapses (41/92 vs. 33/108, P = 0.04). With one serum sample and with a NAb titre >100 tenfold reduction unit (TRU), 66.7% of patients with MS suffered from relapses, 41.7% suffered from progression, and 75% was not an optimal clinical responder. We did not find any significant difference in MxA. We found that 62.5% of patients with MS patients whose ratio was increased twofold after 2 years suffered from relapses, 37.5% suffered from progression, and 68.7% was not an optimal clinical responder. CONCLUSION: The early detection of NAbs by CPE assay and the finding of only one serum sample with a NAb titre >100 TRU seem to be markers of low bioavailability of IFN-beta, whilst a twofold decrease in the MMP-9/TIMP-1 ratio by quantitative PCR assay seems to be a marker of high bioavailability of IFN-beta.

Why don't corticotroph tumors always produce Cushing's disease?

OBJECTIVE: Silent corticotroph tumors are a pituitary neuroendocrine tumor subtype of corticotroph lineage that do not clinically express Cushing's disease. The silencing of this type of tumor is not fully understood. The aim of the present study was to delve into the lack of secretory activity, studying the post-transcriptional and post-translational regulation of POMC/ACTH in a series of molecularly identified functioning and silent corticotroph tumors. DESIGN: We analyzed 24 silent corticotroph, 23 functioning corticotroph and 25 silent gonadotroph tumors. METHODS: We used Sanger sequencing, quantitative real-time PCR and Western blot to analyze genetic alterations in POMC, gene expression of TBX19, NEUROD1, POMC, PCSK1, PCSK2, CPE and PAM and protein expression of POMC, PC1/3, PC2, CPE and PAM. RESULTS: We found different polymorphisms in the POMC gene of corticotroph tumors, some of them related to deficiency of proopiomelanocortin. Silent corticotroph tumors showed lower PC1/3 gene and protein expression than functioning ones, especially compared to micro-functioning corticotroph tumors (all P < 0.05). Moreover, we found a positive correlation between PC2 and CPE gene and protein expression (rho ≥ 0.670, P < 0.009) in silent corticotroph tumors compared with functioning ones. CONCLUSIONS: By studying the post-transcriptional and post-translational processing of POMC and ACTH, respectively, in a large series of silent and functioning corticotroph tumors, we found that the lack of secretory activity of these tumors is related to an impaired processing of POMC and a high degradation of ACTH, with the macro-functioning corticotroph tumor behaving as an intermediate state between micro-functioning and silent corticotroph tumors.

Imatinib mesylate inhibits in vitro and ex vivo biological responses related to vascular occlusion in giant cell arteritis.

OBJECTIVES: Ischaemic complications occur in 15-20% of patients with giant cell arteritis (GCA). The aim of our study was to explore the effect of mesenchymal growth factors expressed in GCA lesions on myointimal cell responses related to the development of intimal hyperplasia and vessel occlusion. METHODS: We developed a method to obtain primary human temporal artery derived myointimal cells (HTAMCs) based on the culture of temporal artery sections on Matrigel. RESULTS: Among the factors tested (platelet-derived growth factor (PDGF)-AB, fibroblast growth factor (FGF)-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor (TGF)beta, chemokine (C-C motif) ligand (CCL)2, interleukin (IL)6 and IL1beta), PDGF exhibited the strongest activity in inducing HTAMC proliferation and migration. As assessed by protein array, immunoassay and quantitative real-time reverse transcriptase (RT)-PCR, PDGF stimulated matrix proteins (collagen I, collagen III and fibronectin) as well as CCL2 and angiogenin production by HTAMCs. Imatinib mesylate inhibited PDGF-mediated activation of signalling pathways (Src, extracellular signal-regulated kinase (ERK) and Akt phosphorylation) related to cell motility and survival, efficiently resulting in inhibition of PDGF-induced HTAMC responses. Myointimal cell outgrowth from cultured temporal artery sections from patients with GCA, where multiple interactions take place, was also efficiently reduced by imatinib. CONCLUSION: Among several mediators produced in GCA, PDGF has the highest vaso-occlusive potential. PDGF may also contribute to disease perpetuation by stimulating the production of angiogenic factors (angiogenin) and chemoattractants (CCL2). Imatinib mesylate strongly inhibits PDGF-mediated responses, suggesting a therapeutic potential to limit vascular occlusion and ischaemic complications in large vessel vasculitis.

Effects of different green manures on soil biological properties and maize yield.

The utilization of green manures as alternatives to reduce the use of mineral fertilizers is considered a good agricultural practice. However, the effect of each green manure on soil properties and crop yield depends upon its chemical composition. The main objective of this work was to study the effect of incorporating three green manures originating from residues of Trifolium pratense, L. (TP), Brassica napus, L. (BN), and the mixture of TP+BN at rates of 5384 and 8973 kg C ha(-1), on soil biological properties (soil microbial biomass-C, soil respiration and soil enzymatic activities), nutrition (leaf N, P and K concentration, pigments and soluble carbohydrate concentrations) and yield parameters of maize (Zea mays cv. Tundra) crop for four years on an Typic Xerofluvent located near Sevilla (Guadalquivir Valley, Andalusia, Spain). All green manures had a positive effect on the soil biological properties, plant nutrition an crop yield parameters, although at the end of the experimental period and at the high organic matter rate, the soil microbial biomass and dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase activities increased more significantly in the TP amended soils (79.2%, 92.1%, 93.9%, 99.3%, 87.9% and 96%, respectively) respect to the control soil, followed by TP+BN amended soils (77.3%, 90.9%, 92.8%, 99.1%, 84.4% and 95.7%, respectively) and BN amended soils (76%, 90.1%, 91.7%, 99%, 83.2% and 95.2%, respectively). Since these soil enzymatic activities measured are responsible for important cycles such as C, N, P and S, an increase of leaf N, P an K contents and pigments and soluble carbohydrate contents were highest in TP amended soils, followed by TP+BN and BN treatments. The application of TP in soils at high doses increased the grain protein concentration, number of grains corncob(-1) and crop yield 44.6%, 6.3% and 22.1%, respectively, compared with the control soil, followed by TP+BN treatment (41.7%, 5.7% and 20.8%, respectively) and BN treatment (39%, 5.3% and 20%, respectively). The explanation of these results can be a consequence to the different chemical composition of the green manures applied to the soils and its mineralization, aspect controlled by the soil C/N ratio.

Application of a green manure and green manure composted with beet vinasse on soil restoration: effects on soil properties.

Beet vinasse (BV), a green manure constituted by Trifolium pratense L. uncomposted (TP) and composted with beet vinasse (at 1:1 rate, (TP+BV)1, and 2:1 rate, (TP+BV)2) at 10t organic matter ha(-1) rate were applied during a period of four years for purpose of restoration of a Xelloric Calciorthid located near Seville (Guadalquivir Valley, Andalusia, Spain). The effect on the plant cover, soil physical (structural stability and bulk density), chemical (exchangeable sodium percentage), and biological properties (microbial biomass, soil respiration and enzymatic activities such as dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase) were determined. The application of BV had a detrimental impact on soil physical (structural stability decreased 16.5% and bulk density increased 18.7% respect to the control soil), chemical (exchangeable sodium percentage increased 87.3% respect to the control soil), and biological properties (microbial biomass, soil respiration, and dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase activities decreased by 53.5%, 24.5%, 27.8%, 15%, 39.7%, 42.7%, and 65.6%, respectively with respect to the control soil), probably because high quantities of monovalent cations (Na principally) were introduced into the soil by the vinasse, thus destabilizing its structure. The application of TP had a positive impact on soil physical (structural stability increased 5.9% and bulk density decreased 6.1% respect to the control soil), and biological properties (microbial biomass, soil respiration, and dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase activities increased by 66.3%, 45.6%, 97.7%, 98.9%, 97.7%, 87.2%, and 89.4%, respectively with respect to the control soil). However, when BV was co-composted with a green manure, principally at a 2:1 rate, the resulting compost had a positive effect on soil physical (structural stability increased 10.5% and bulk density decreased 13.5% respect to the control soil), and biological properties (microbial biomass, soil respiration, and dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase activities increased by 68.9%, 46.2%, 97.5%, 98.4%, 99.1%, 90.5% and 91.6%, respectively with respect to the control soil). After four years, the percentage of plant cover decreased 64.3% in the BV-amended plots respect to the control soil, whereas increased 82.8%, 81.6% and 81% in the (TP+BV)2, (TP+BV)1 and TP treatments, respectively. While the application of BV deteriorates the soil and therefore does not contribute to its restoration, the application of TP, and BV composted with TP protects the soil and will contribute to its restoration.

Production of a carob enzymatic extract: potential use as a biofertilizer.

In this paper, we describe a biological process that converts carob germ (CG), a proteinic vegetable by-product, into a water-soluble enzymatic hydrolyzate extract (CGHE). The chemical and physical properties are also described. The conversion is done using a proteolytic enzyme mixture. The main component of CGHE extracted by the enzymatic process is protein (68%), in the form of peptides and free amino acids, having a high content of glutamine and arginine, and a minor component of phytohormones, which are also extracted and solubilized from the CG. We have also compared its potential fertilizer/biostimulant capacity on growth, flowering, and fruiting of tomato plants (Licopericon pimpinellifolium cv. Momotaro) with that of an animal enzymatic protein hydrolyzate. CGHE had a significantly beneficial impact, most notably regarding the greater plant height, number of flowers per plant, and number of fruits per plant. This could be due primarily to its phytohormonal action.

[Adequacy of clinical interventions in patients with advanced and complex disease. Proposal of a decision making algorithm]. / Adecuación de las intervenciones clínicas en pacientes con enfermedad avanzada y compleja. Propuesta de algoritmo para la toma de decisiones.

Decision making in the patient with chronic advanced disease is especially complex. Health professionals are obliged to prevent avoidable suffering and not to add any more damage to that of the disease itself. The adequacy of the clinical interventions consists of only offering those diagnostic and therapeutic procedures appropriate to the clinical situation of the patient and to perform only those allowed by the patient or representative. In this article, the use of an algorithm is proposed that should serve to help health professionals in this decision making process.

Synthesis of substituted 1-norbornylamines with antiviral activity.

The reaction of (+/-)-camphor (7) with triflic anhydride (Tf2O) yields the bridgehead triflate 8. The Nametkin rearrangement of 8 to 3 was realized by treatment with triflic acid (TfOH). The solvolysis of the bridgehead triflates 3 and 8 in acetonitrile affords the N-acetyl-1-norbornylamines 4 and 9. The Pd(0)-catalyzed hydrogenation of 4 and 9 gives the amides 5 and 10. The corresponding 1-norbornylamines 2 and 13 and the N-ethyl derivatives 1, 6, 11, and 12 were obtained by basic hydrolysis or reduction with LiAlH4, respectively, of the amides 4, 5, 9, and 10. The antiviral activity of the hydrochlorides of some of the obtained 1-norbornylamines was evaluated against influenza A, herpes simplex 2, and African swine fever virus. Particularly noticeable is the activity of the hydrochlorides of 1 and 11 against influenza A virus (SI (selectivity index) = 1000).

Electron impact fragmentation patterns of 3,3-dimethyl-1, 2-norbornane derivatives

The electron impact mass spectra of several 3,3-dimethyl-1, 2-norbornanediols, diamines, amino alcohols and related derivatives have been studied and their fragmentation pathways discussed. Different fragmentation patterns were observed, depending not only on the nature of the substituents, but also on their relative positions on the norbornane framework. In general, the dominant peaks in the spectra of these compounds originate from initial C1-C2 bond cleavage (alpha-cleavage) with charge retention on the heteroatom (oxygen or nitrogen) attached at the bridgehead position, followed by hydride shift and loss of the C2-C3 fragment by homolytic cleavage of the C3-C4 bond. This fragmentation pathway leads to a highly stabilized cyclopentenylimmonium or cyclopentenyloxonium ion, which constitutes the base peak in the spectra in most of the studied compounds. Copyright 1999 John Wiley & Sons, Ltd.

[General analysis of 500 couples with sterility or infertility]. / Análisis general de 500 parejas con esterilidad o infertilidad.

PIP: Sterility is the lack of fertility, the failure to reproduce, and it is often short-term. It often occurs at puberty, before menopause, during lactation, and during gestation. 500 couples were examined, generally meeting these criteria: under 30 and married at least 2 years, or 31-35 and married at least 1 year. 64.8% were 21-30 years old and 22.2% were 31-35. Primary sterility was present in 66.6%, secondary sterility in 33.4%, and infertile couples, 4.2%. In each case, a complete 3-month study was done to determine the causes of sterility or fertility, considering vaginal, cervical, uterine, tubal, ovarian, and thyroid factors. A tubal factor was implicated in 23.3%, cervical in 20.3%, ovarian masculine in 12.1%, uterine in 6.96%, and vaginal in 8.5%. 3256 studies were done on 500 couples; 546 treatments were carried out in 319 couples; 110 pregnancies were obtained.^ieng