The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Scrotal ultrasound reference ranges and associations with clinical, seminal, and biochemical characteristics.

BACKGROUND: Scrotal color Doppler ultrasound (CDUS) still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study to assess the CDUS characteristics of healthy fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report and discuss the scrotal organs CDUS reference ranges and characteristics in HFM and their associations with clinical, seminal, and biochemical parameters. METHODS: A cohort of 248 HFM (35.3 ± 5.9years) was studied, evaluating, on the same day, clinical, biochemical, seminal, and scrotal CDUS following Standard Operating Procedures. RESULTS: The CDUS reference range and characteristics of the scrotal organs of HFM are reported here. CDUS showed a higher accuracy than physical examination in detecting scrotal abnormalities. Prader orchidometer (PO)- and US-measured testicular volume (TV) were closely related. The US-assessed TV with the ellipsoid formula showed the best correlation with the PO-TV. The mean TV of HFM was ~ 17 ml. The lowest reference limit for right and left testis was 12 and 11 ml, thresholds defining testicular hypotrophy. The highest reference limit for epididymal head, tail, and vas deferens was 12, 6, and 4.5 mm, respectively. Mean TV was associated positively with sperm concentration and total count and negatively with gonadotropins levels and pulse pressure. Subjects with testicular inhomogeneity or calcifications showed lower sperm vitality and concentration, respectively, than the rest of the sample. Sperm normal morphology and progressive motility were positively associated with epididymal head size/vascularization and vas deferens size, respectively. Increased epididymis and vas deferens sizes were associated with MAR test positivity. Decreased epididymal tail homogeneity/vascularization were positively associated with waistline, which was negatively associated with intratesticular vascularization. CDUS varicocele was detected in 37.2% of men and was not associated with seminal or hormonal parameters. Scrotal CDUS parameters were not associated with time to pregnancy, number of children, history of miscarriage. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology, improving its management.

[Changed levels of substance P and somatostatin in HIV-positive children]. / Alterati livelli di sostanza P e somatostatina nei bambini HIV positivi.

Substance P (sP) and Somatostatin (SOM), so as other neuropeptides can modulate neurologic and immunologic functions. sP has been described to enhance both in vitro and in vivo immunoglobulin synthesis. On the contrary, SOM has an inhibitory effect on the same activity. The modulating effect is more evident on IgA isotype. Hypergammaglobulinemia and in particular high levels of IgA is a common finding in pediatric AIDS and an imbalance among regulatory effects of neuropeptides might be suggested. In order to evaluate the plasma levels of sP in pediatric AIDS we studied 15 children with HIV infection (status P2), 10 seronegative children born to HIV positive mothers and 10 healthy children of the same age. All the HIV positive children had high plasma levels of IgG and IgA. The plasma level of sP was extremely higher in HIV positive children while no significant difference was found between seronegative children born to HIV positive mothers and healthy children. SOM was decreased in HIV positive children when compared to control groups but a significant difference was not reached. It might be supposed that HIV infection, through a dysregulation among neuropeptides interferes on immune functions and in particular on IgA synthesis. On the other hand it might be suggested that the imbalance between sP and SOM depends on the viral infection of immune cells since it has been demonstrated that SOM and other neuropeptide are synthesized by lymphoid tissue. Further studied relevance of neuropeptide disorders in pediatric AIDS.

VIP restores natural killer cell activity depressed by hepatitis B surface antigen.

Vasoactive intestinal peptide (VIP) has recently been shown to bind to human lymphocytes and modulate immune functions. The ability of VIP in restoring natural killer (NK) cell activity depressed by hepatitis B surface antigen (HBsAg) has been investigated in the present research. Human lymphocytes were incubated with HBsAg and, after washing, a 4-hr cytotoxicity assay was performed. VIP was coincubated with lymphocytes during the preincubation with HBsAg or, alternatively, throughout the cytotoxicity assay. The study revealed that VIP, either preincubated or coincubated in the 4-hr assay, strongly restores NK cell activity depressed by viral antigen. This is noteworthy considering that a number of lymphocyte modulators such as interferons fail in restoring viral-dependent NK cell activity depression. In contrast with previous reports, even when coincubated in the 4-hr assay, VIP is a strong activator of NK cell activity. Further studies will be required to understand which mechanisms are involved in the interrelation between VIP and NK cells during viral infections.

Measurement of seminal LDH-X and transferrin in normal and infertile men.

LDH-X, an isoenzyme of lactate dehydrogenase specific for germinal epithelium activity, has been measured in the seminal plasma of infertile subjects whose infertility had different origins. In the same samples, seminal transferrin, an index of Sertoli cell function, was also measured. In this investigation, seminal LDH-X was not detectable in the vasectomized subjects, in patients with azoospermia due to seminiferous tubular damage, nor in patients who showed a marked decrease in sperm concentration (less than 1 X 10(6)/ml). In oligozoospermic patients (sperm concentration less than 20 X 10(6)/ml) seminal LDH-X levels were reduced to about one-third of those found in normal controls. Seminal LDH-X levels correlated (r = 0.7237) with total sperm count better than seminal transferrin levels (r = 0.5511), while no correlation was found between these two biochemical parameters and sperm motility, viability and morphology. To study their spontaneous variations with time, LDH-X and transferrin were also measured in semen specimens collected monthly from five healthy men, over 1 year. In these samples (N = 60), sperm count variability (43.1%, calculated in terms of the coefficient of variation), was similar to that of LDH-X (40.4%) and higher than for transferrin (23.0%).

Immunoassayable somatomedin C in seminal plasma of azoospermic men.

Somatomedin C is a Sertoli cell peptide and since measurements of other Sertoli cell products in semen have provided a useful indices of testicular function, it was considered pertinent to measure the semen levels of Somatomedin C. Somatomedin C was measured by RIA in seminal plasma of vasectomized subjects (n = 18), subjects with agenesis of the seminal vesicles and vasa deferentia (n = 6) and subjects with azoospermia resulting from seminiferous tubule damage without obstruction (n = 23). Normal fertile subjects (24 men with a sperm concentration greater than 20 X 10(6)/ml) were used as controls. In all subjects, seminal levels of transferrin were also measured as an index of Sertoli cell function. The majority of seminal Somatomedin C appears to derive from the testis and/or epididymis. However, in several normal controls seminal levels of Somatomedin C (median = 3.52; range = 1.10-15.67 U/ejaculate) were found to be within the range for vasectomized subjects (median = 0.78; range = 0.46-4.20 U/ejaculate). In subjects with azoospermia the seminal levels of Somatomedin C (median = 2.06; range = 0.60-10.12 U/ejaculate) were significantly lower (P less than 0.02) than in fertile controls. However, values for these two groups overlapped. It is concluded that Somatomedin C in semen is not a reliable index of seminiferous tubule function and does not appear to be of diagnostic value in male infertility.

Simultaneous measurement of seminal L-carnitine, alpha,1-4-glucosidase, and glycerylphosphorylcholine in azoospermic and oligozoospermic patients.

L-carnitine, alpha,1-4-glucosidase, and glycerylphosphorylcholine were measured in seminal plasma of a selected group of azoospermic men and in an unselected group of oligozoospermic men. In vasectomized subjects the epididymal indices (mean +/- standard error: L-carnitine, 276.9 +/- 27.5 nmol/ejaculate; alpha-glucosidase, 1.2 +/- 0.1 U/ejaculate; and glycerylphosphorylcholine, 1.5 +/- 0.2 mumol/ejaculate) were always below the normal range of fertile subjects (1757.4 +/- 76.7 nmol/ejaculate; 16.4 +/- 0.9 U/ejaculate; and 17.3 +/- 0.7 mumol/ejaculate, respectively). On the contrary in a large number of patients affected by azoospermia because of seminiferous tubular damage (750.4 +/- 83.6 nmol/ejaculate; 6.8 +/- 0.9 U/ejaculate; and 6.1 +/- 0.6 mumol/ejaculate; respectively) and in a few oligozoospermic subjects (1193.7 +/- 72.3 nmol/ejaculate; 10.3 +/- 0.7 U/ejaculate; and 10.8 +/- 0.7 mumol/ejaculate; respectively) the epididymal indices were found in the range of vasectomized subjects, showing an association between seminiferous tubular lesion and epididymal dysfunction. In conclusion, in spite of the low levels of epididymal indices found in patients with obstructive azoospermia, the presence of a large number of subjects with seminiferous tubular lesions without obstruction with similar low values of L-carnitine, alpha-glucosidase, and glycerylphosphorylcholine reduces the usefulness of these indices in differential diagnosis of azoospermia.

Measurement of transferrin in human seminal plasma by a chemiluminescent method.

We describe a luminescence immunoassay for measuring transferrin in human seminal plasma. Human transferrin conjugated with 7-[(N-4-aminobutyl)-N-ethylamino]naphthalene-1,2-dicarboxylic acid hydrazide was used to monitor the immunological reaction. The conjugate was stable for at least one year. The sensitivity (2 ng per tube) of the assay allows measurement of this protein in diluted seminal plasma. Results by this method (y) correlated well (r = .9681) with those by a conventional RIA method (x): y = 1.000x + 1.646 micrograms per ejaculate. Seminal transferrin concentrations are reported for normal control subjects, vasectomized subjects, and infertile patients. The method described appears suitable for measurement of seminal transferrin as an index of Sertoli cell function in male infertility.

Ceruloplasmin and transferrin in human seminal plasma: are they an index of seminiferous tubular function?

Transferrin and ceruloplasmin have been measured by a solid-phase chemiluminescent method in seminal fluid and circulating blood of normal and vasectomized subjects (1 year after operation). This study has confirmed that approximately 80% of seminal transferrin comes from the testis, while seminal ceruloplasmin was not found different in the two groups. In patients affected by azoospermia due to seminiferous tubular damage (n = 15) in whom an obstruction was previously excluded, seminal transferrin was always below the normal range. On the contrary, seminal ceruloplasmin was always in the normal range, and circulating follicle-stimulating hormone was found above the normal range only in nine cases. No correlation was found between seminal transferrin and circulating follicle-stimulating hormone in such groups. In an unselected group of infertile patients with decreased sperm concentration and/or sperm motility, seminal transferrin was found correlated with the sperm count. These studies seem to suggest that seminal transferrin is a reliable index of seminiferous tubular function.

Luminescent immunoassay (LIA) methods for steroid hormones.

The application of luminescence to the development of non-isotopic immunoassay methods for steroids and urinary steroid metabolites is reviewed. On-line computer analysis of the light emission is particularly useful, as it reveals the interfering effects of biological compounds on the reaction and this can improve the quality of luminescence immunoassay (LIA) methods. The main characteristics of chemiluminescent tracers are stability, safety, speed and sensitivity of detection, and reliability. Homogeneous methods, not requiring phase separation, have also been reported and validated. Heterogeneous methods which use dextran-coated charcoal or solid-phase techniques can be used for direct determination of urinary steroids in unextracted samples.

Homogeneous luminescence immunoassay for total estrogens in urine.

We describe an homogeneous luminescence immunoassay for "total" estrogens in enzymically hydrolyzed urine from nonpregnant women. The antiserum, raised against estriol-16,17- dihemisuccinate conjugated to bovine serum albumin, specifically bound the C-19 steroids carrying the estrogen-characteristic phenolic group. 17 beta-Estradiol conjugated with aminobutylethyl isoluminol was used to monitor the immunological reaction; this conjugate was stable for at least two years. Because binding to the antiserum markedly enhances the light-producing efficiency of the tracer, no separation of bound and free antigen is necessary. Results (microgram/24 h) by this method (y) correlated well (r = 0.958) with those by a conventional fluorometric (x) method (y = 2. 51x - 2.83). The sensitivity (detection limit) is 4 micrograms/L and the precision compares well with that of commonly used RIA methods. The method appears suited to large numbers of samples, as in menstrual cycle monitoring.

Preparation and evaluation of steroid chemiluminescent tracers.

Six different isoluminol derivatives have been synthesized and evaluated as potential labels for conjugation to steroid molecules. The resulting steroid-chemiluminescent tracers have been investigated in terms of detection limit and affinity for the homologous antibody. The conjugation to the steroid molecule did not significantly affect the quantum yield of the chemiluminescent label. Consequently the detection limit was very similar using tracers with the same chemiluminescent label and different steroids and it was always at picomolar levels. The affinity of the chemiluminescent tracer versus the homologous antibody was evaluated using tritiated steroids as reference. The bridge and the chemiluminescent molecule could influence the affinity of the chemiluminescent tracer which could be higher, similar or lower than that of the tritiated steroid. In conclusion some isoluminol derivatives (i.e. ABEI or AEEI) can be considered as universal labelling compounds for steroids and their metabolites, because they possess a high light efficiency (similar to luminal itself) and favourable reaction conditions. Moreover the conjugation to the steroid molecule does not modify their chemiluminescent properties.

ATP and ADP content of human ejaculated spermatozoa. II. Time-dependent changes after ejaculation.

ATP and ADP content of ejaculated spermatozoa of 7 donors were measured by a bioluminescent method within 24 h. The semen samples were kept at room temperature and at prefixed time were tested for motility and viability and extracted for ATP and ADP measurement. ATP pattern in relation to the time was studied with a mathematical model and the resulting curve showed a very high significance (P less than 0.001). ADP decrease with time was not significant and was variable among our subjects. The relationship between ATP decrease (%) and the decrease (%) of viability and motility was not linear, and till now no mathematical model has been found to express this phenomenon.

ATP and ADP content of human ejaculated spermatozoa. I. Relationship with semen physical parameters in normal donors and oligozoospermic patients.

ATP and ADP were measured by a bioluminescent method in human ejaculated spermatozoa from 31 normal donors and oligozoospermic patients. ATP and ADP expressed for the sperm total number were significantly correlated (P less than 0.001). ATP snd ADP levels were found significantly correlated with the total motile spermatozoa and with the total viable cells (respectively P less than 0.001 and P less than 0.001 for ATP and P less than 0.001 and P less than 0.001 for ADP). ATP/ADP ratio was found significantly lower in subjects with sperm motility less than 50% (P = 0.024) and total viable cells less than 80% (P = 0.026).

Lipid content of amniotic fluid cells.

The lipid content of fetal cells was determined in 45 samples of human amniotic fluid. Free and total cholesterol were estimated using a gas chromatographic method, and glycerides were evaluated through the enzymatic assay of their glycerol content. The number of orange cells was estimated after staining with Nile Blue sulphate. The chemically measured lipid content appeared closely related to the number of orange cells. Total cholesterol and glycerides showed a sharp increase after the 37th week of pregnancy. These tests seem to assess fetal maturity successfully, providing a further useful aid in the management of high-risk pregnancies. The chemical determination of cell-associated lipids showed good accuracy and reliability and, when compared with the histochemical method, allowed a better evaluation of progressive lipid accumulation within the amniotic fluid cells.

Immuno-enzymatic assay for the detection of Schistosoma mansoni antigens in serum of mice harbouring bisexual or unisexual light worm infections.

Enzyme-linked immunosorbent assay (ELISA) double antibody technique was standardized for detecting S. mansoni polysaccharide and protein antigens in serum of infected mice. Anti-sera specific for either worm components were obtained in sheep and peroxidase conjugates prepared from each serum. The immunoenzimatic test for protein could detect as little as 6 micrograms/ml antigens, and the test for polysaccharides 3 micrograms/ml. Both bisexual and unisexual male worm low infections were produced, and studied for as long as 27 weeks post-exposure. Worm components were found in serum from both types of infections and in progressively higher percentages of animals until the end of the 27 weeks observation period. For unisexual male infections this percentage reached from about 50% to 60% of mice, and 100% for bisexual infections. Significantly higher antigen concentrations in serum were found at 27 weeks for bisexual infections, no antigen increase being detected in relation to starting egg secretion, which occurred at 5 week infections.