Diagnostic value of serum chromogranin-A combined with MIBG scintigraphy inpatients with adrenal incidentalomas.

AIM: Adrenal incidentalomas (AI) are defined as asymptomatic adrenal masses occasionally discovered during high-resolution imaging procedures, as computed tomography or magnetic resonance. Pheochromocytoma, a chromaffin tumour, must be excluded before any invasive diagnostic or therapeutic procedure, in order to avoid dangerous acute catecholamines-release into blood stream. Chromogranin-A (CgA) is a member of the granin family contained in secretory vesicles of chromaffin system. This study investigated the performance of serum CgA in detecting or excluding pheochromocytoma among patients with AI. METHODS: We enrolled 348 patients by AI > 20 mm without clinical or biochemical signs for corticosteroids overproduction. Serum CgA was assayed by a specific immunoradiometric method and a [123I] metaiodobenzylguanidine (MIBG) scan was performed in the 39 CgA-positive patients. RESULTS: Eighteen out of these patients showed a positive scan and were submitted to laparoscopic adrenalectomy. Pheochromocytoma was histologically confirmed in all cases . The patients with positive serum CgA, were reassessed 1 year later by clinical examination and serum CgA assay. None of patients developed clinical symptoms of chromaffin-tissue hyperactivity , nor showed a serum CgA increase. Serum levels of CgA were significantly higher inpatients with pheochromocytoma than inpatients without (P<0.0001). CONCLUSION: We concluded that serum CgA assay is effective as a single marker to detect or exclude sporadic pheochromocytoma among patients with AI > 20mm. Particularly, a negative serum CgA assay may be used to rule out [123I] MIBG imaging and /or other diagnostic procedures.

Nature's motility blockers: controlling human sperm motility machinery from the outside. Chemical characterization of a peritoneal fluid lipid that induces sperm immobilization.

A molecule isolated from the peritoneal fluids of women undergoing laparoscopy for in-vitro fertilization techniques has been chemically characterized and identified as 1-palmitic-3-phosphorylcholine (lysophosphatidylcholine, LPC). This lipid is able, at physiological concentrations, to completely inhibit sperm motility in vitro in a dose-dependent way. Synthetic LPC induced rapid and complete arrest of sperm motility when added to sperm suspensions at physiological concentrations without any damage to cell membranes. Taken together, these results suggest that LPC may represent a previously unrecognized in-vivo modulator of human sperm motility.

Preincubation in peritoneal fluid decreases the follicular fluid-induced acrosomal reactivity of human spermatozoa.

The aim of this study was to determine the effects of preincubation in peritoneal fluid on the follicular fluid-induced acrosomal reactivity of human spermatozoa in vitro. Thirty women participating in our IVF-ET program were given a GnRH-analogue, highly purified FSH and hCG in order to induce superovulation. Peritoneal and follicular fluids were aspirated during pick-up laparoscopy, centrifuged, filtered and frozen until use. An aliquot of swim-up suspension from normospermic semen specimens (n = 30) was incubated with peritoneal fluid or HAM-F10 for 30-180 min, and follicular fluid (in volumetric proportion approximately 50/50 with peritoneal fluid) was subsequently added. The percentage of acrosomally-reacted spermatozoa was assessed using the FITC-conjugated Pisum sativum lectin before and after incubation in peritoneal fluid or control medium, as well as after follicular fluid addition. Peritoneal fluid was not able to stimulate acrosomal reactivity; further, preincubation in peritoneal fluid decreased, but not abolished, the follicular fluid-induced acrosomal reactivity. A longer pre-incubation in peritoneal fluid was associated with a lower percentage of reacted spermatozoa in response to the addition of follicular fluid. In conclusion, our data suggest that peritoneal fluid acts maintaining spermatozoa in an unreacted status in the upper female genital tract. After mixing with follicular fluid, a phenomenon that is likely to occur at ovulation, peritoneal fluid reduces, but does not abolish, the stimulating effect of follicular fluid on acrosomal reactivity.

Effect of peritoneal fluid, follicular fluid, and their volumetric mixture on acrosomal reactivity in vitro.

OBJECTIVE: To determine the effects of peritoneal and follicular fluids (PFs, FFs) on sperm acrosomal reaction (AR). DESIGN: Prospective, randomized study. SETTING: Three hospital-based infertility units. PATIENTS: Twenty-three women participating in GIFT programs; 23 men participating in AIH programs. INTERVENTIONS: Hormonal stimulation after buserelin desensitation; laparoscopy 36 hrs after hCG injection. MAIN OUTCOME MEASURE: Percentage of acrosomally-reacted sperm. RESULTS: Compared with Earle's medium (control), moderate but significant increases of ARs were observed as function 1) of the relative content of FF in the incubation medium and 2) as function of time (these increases were constantly lower than those registered for the respective positive control, i.e. 2x frozen/thawed sperm). In contrast, when PF alone was present in the incubation medium, no such effects on AR were registered. CONCLUSIONS: FF and mixtures of PF and FF--but not PF alone--seem to induce some rapid and time-dependent processes which finally lead to an AR. Therefore, and independently on the infertility cause (tubal, male-dependent, unexplained infertility), PF seems able to exert effects on sperm motility (Revelli et al., Fertil. Steril 57, 654-60 [1992]) while maintaining an unreacted sperm status.

Computer-aided semen analysis: sperm concentration assessment by the Strömberg-Mika system.

The scope of this study was to evaluate the accuracy, precision and specificity of the sperm concentration measurements by the Strömberg-Mika Cell Motion Analyser (SM-CMA). Our data show that the instrument generally underscores the sperm concentration and therefore the uncorrected measurements must be corrected by the operator using the 'mouse'-driven option. In terms of precision, the system appears to have an excellent internal precision whereas its repeatability is influenced by the sperm concentration, the sample's homogeneity and the correction of the raw data. In order to increase the system's repeatability, we suggest that sperm counts should be carried out in various fields of the counting chamber, and the mean of the corrected values be taken as representative of the sperm concentration in the ejaculate if the various measurements show a homogeneous (poissonian) distribution. The correction of the raw data with the 'mouse'-driven correction option was also shown to improve the system's reproducibility. Concerning specificity, our data evidenced that, without technical correction, the instrument failed to correctly classify certain spermatozoa as such, thereby grossly underscoring sperm counts. This finding was more evident at low sperm counts. Overall, the SM-CMA requires additional laboratory time but the corrected sperm counts are comparable to manual counts and semi-automated counts with the added option that it provides the andrologists with various motility characteristics not possible with the latter methodologies.

Steroid receptors in human spermatozoa.

The presence of steroid binding sites in (or on) human spermatozoa was first suggested in the late 1970s, by studies showing that some steroids were able to influence sperm function. Subsequently, several effects exerted on spermatozoa by biological fluids, such as follicular fluid, were found to be probably linked to the action of steroids, and among them progesterone. Since the effects of progesterone on spermatozoa were rapid, dose-dependent and not affected by progesterone conjugation with high molecular weight proteins unable to cross the plasma membrane, the existence of a novel class of non-genomic progesterone receptors was strongly suspected. This hypothesis was further supported by the observation that some of the effects of progesterone on human spermatozoa were not abolished by inhibitors of the classical progesterone nuclear receptors, nor mimicked by progesterone genomic receptor agonists. Recently, surface progesterone binding sites were directly identified on the membranes of human spermatozoa, and their mechanism of action partially characterized.

Effect of peritoneal fluid supplemented with exogenous progesterone on sperm motility in vitro.

Since progesterone has been claimed to induce acrosomal reaction and hyperactivated motility of human spermatozoa, the present study was undertaken to determine if its presence at concentrations similar to those of peri-ovulatory follicular fluid could influence the effect of peritoneal fluid on sperm motility in vitro. To this end, 11 sperm samples were incubated at 37 degrees C with five peritoneal fluids with/without exogenous progesterone, and sperm motility was assessed using a computer-assisted analyser at time (t) = 0, 2.5, 5 and 24 h. Overall there was no observable constant trend for enhancement or inhibition of sperm motility. Progesterone generally induced a negative effect on those sperm samples with high velocities in the native peritoneal fluids and a positive effect on those sperm samples demonstrating low motility in the native peritoneal fluids. The incorporation of progesterone into the incubation medium seemed to result in a 'tuning' of sperm velocity to around 30-50 micron/s. However, a given sperm sample reacted differently when incubated with various peritoneal fluids and, reciprocally, different semen samples incubated with the same peritoneal fluid showed very variable motility patterns. The greater variability of the effects exerted by progesterone on sperm motility could arise from the fact that each sperm sample may contain subpopulations of gametes with different sensitivity to progesterone.

Bacteroides ureolyticus in men consulting for infertility.

A screening of 3196 semen analyses performed in our clinic from January 1986 to December 1990 revealed 314 (9.8%) patients whose semen was infected with Bacteroides ureolyticus. Investigating the relationship between the presence of B. ureolyticus, the seminal microflora and the conventional semen parameters, we observed that the presence of this micro-organism in the semen was coupled (1) to an increased presence of Enterococcus species, (2) to an increased number of short-tailed spermatozoa and epithelial cells, and (3) to a decreased total fructose concentration (mg ejaculate-1). These results suggest that B. ureolyticus or its toxins may influence sperm morphology and function by yet unknown mechanisms and may also increase the number of epithelial cells by soft tissue infection in vivo. The decreased fructose levels suggest that this anaerobic micro-organism might specifically colonize the seminal vesicles, while the normal zinc values recorded suggest a normal prostatic function. Overall, our data support the hypothesis that the presence of B. ureolyticus is not associated with nongonococcal urethritis.

Influence of peritoneal fluid from spontaneous and stimulated cycles on sperm motility in vitro.

Peritoneal fluids (PFs) from spontaneous (n = 14) and gonadotrophin-stimulated cycles (n = 20) were obtained during diagnostic laparoscopy and gamete intrafallopian transfer (GIFT) procedures, respectively. The effects of these fluids on the linear component of sperm motility and on the percentage of motile spermatozoa were studied in vitro by objective motility assessments and compared to a control medium (B2-Ménézo). Overall, the two types of PFs were found to have rather similar effects on the motility parameters studied. However, the fluids from hormonally-stimulated cycles sustained motility better (i.e., sperm velocity and percentage of motile sperm) and in a rather constant manner as a function of time (narrower range distributions of the motility measurements). Furthermore, it was observed that under identical experimental conditions motility measurements depended not only on the type of PF used but also on the sperm sample. These results suggest that assisted reproduction procedures in which PF is the medium where the gametes eventually meet and interact, such as direct peritoneal insemination (DIPI) or peritoneal oocyte and sperm transfer (POST), could have different success rates if performed in spontaneous rather than in stimulated cycles. At the same time, our results may help to explain why different pregnancy rates were reported in different studies using DIP or POST.

Purification of a factor from human peritoneal fluid that is able to immobilize spermatozoa.

Human peritoneal fluid has been claimed to influence sperm motility. This report gives evidence for the presence in midcycle peritoneal fluid of a protein-bound, lipidic (hydrophobic) component able to immobilize spermatozoa as a function of time. This component was extracted from molecular weight-sieving and ion-exchange/high pressure liquid chromatography (HPLC)-purified peritoneal fluid fractions by either chloroform/methanol or charcoal treatments; resuspension of the chloroform/methanol extract with BWW-buffer and subsequent testing on spermatozoa resulted in sperm immobilization. Sequential or step-down chromatographic procedures (molecular weight-sieving-->cation-exchange-->anion-exchange HPLC separations of native peritoneal fluid) and extensive dialysis against double distilled water allowed the purification of the sperm immobilizing factor, as evidenced by the shorter incubation times necessary for sperm immobilization. Furthermore, the active fraction was found to immobilize spermatozoa without affecting its viability. Separation of the chloroform/methanol extracted immobilizing fraction on thin layer chromatography under conditions for phospholipid detection allowed the identification of a characteristic band which, after re-extraction, was found to be the sperm immobilizing substance. This factor does not contain choline, ethanolamine or serine. These results suggest that some lipidic peritoneal fluid components may influence sperm motility.

Effect of volumetric mixtures of peritoneal and follicular fluid from the same woman on sperm motility and acrosomal reactivity in vitro.

OBJECTIVE: To study the effect of peritoneal (PF) and follicular fluids (FF from the same woman) as well as of given volumetric combinations thereof on sperm motility and acrosomal reactivity. DESIGN: Prospective. Peritoneal fluid and FF were incubated separately or in given volumetric combinations (PF/FF = 100/0, 75/25, 50/50, 25/75, 0/100; vol/vol) with swim-up sperm suspensions. SETTING: In vitro fertilization and general infertility clinic and laboratories. PATIENTS, PARTICIPANTS: Women participants of the gamete intrafallopian transfer program (motility study, n = 20; acrosomal reaction study, n = 14). Sperm donors of the artificial insemination program and men with given sperm parameters. INTERVENTIONS: Hormonal stimulation. Laparoscopy. MAIN OUTCOME MEASURES: Progressive velocity and percentage of motile gametes measured with multiple-exposure photography. Acrosomal reactivity measured by an immunofluorescent technique. RESULTS: Follicular fluid always influenced progressive motility and also sustained the number of motile gametes, as function of time, better than PF or the PF/FF mixtures (P less than 0.05). The acrosomal reactivity of sperm incubated in the various PF/FF combinations was low; after 5 hours only the FF-sperm suspensions showed a significant enhancement of acrosomally reacted gametes. CONCLUSION: At ovulation, FF transmit positive (motility- and acrosomal reactivity-enhancing) signals to sperm, whereas PF may transmit positive, neutral, or negative signals (noise signals). The volumetric combination of FF and PF in the tubal environment, which may differ from cycle to cycle and from woman to woman, could therefore result in synergic (or antagonistic) effects on the sperm fertility potential.

Etiology of severe asthenozoospermia and fertility prognosis. A screening of 5216 semen analyses.

A review of n = 5216 semen analyses performed in our two Clinics from January 1986 to December 1989 allowed to identify n = 35 patients whose sperm had constantly very low motility (less than 5% progressive motile gametes in three subsequent analyses; necrozoospermia cases were excluded from this study). This apparently rare but severe anomaly was found to be associated not only with ultrastructural anomalies (n = 18), but also with positive seminal bacteriology (n = 8) or the presence of antisperm antibodies (n = 2). In eight cases the cause(s) for this constant asthenozoospermia remained obscure. The fertility potential of the men affected was followed-up and is discussed in relation to their anamnesis, physical exam and seminal characteristics.

Follicular fluid enhances sperm motility and velocity in vitro.

Forty-three follicular fluids (FFs) obtained during laparoscopy were tested in vitro for their effect(s) on sperm motility using gametes obtained by the swim-up procedure. Both the proportion of motile sperm and the velocity distribution patterns were evaluated as function of time by multiple-exposure photography technique. At the various incubation periods considered, all FFs maintained or then enhanced sperm motility as compared with the paired control suspension incubated with a sperm survival medium. The results of the sperm contact test for FFs from women who achieved pregnancy versus FFs from women who remained infertile were not significantly different for both parameters measured. Comparing these with our previously reported results, we may hypothesize that FF released at ovulation into the peritoneal cavity may counteract some sperm-immobilizing effect of peritoneal fluid, thereby increasing the fertility potential of the male gametes.

Removal of sperm-coat from human spermatozoa by interaction with cervical mucus or a capacitating medium.

Human spermatozoa were labelled with cationized ferritin and their interaction with cervical mucus or a capacitating medium enriched with 3% bovine serum albumin (BSA) was investigated by transmission electron microscopy. It was found that the sperm-coat was removed from the surface of spermatozoa after incubation in the BSA-enriched capacitating medium, as well as after crossing a column filled with cervical mucus. These results suggest that both media have a quantitatively similar action in removing the sperm-coat.

Linear and non-linear relationships between the 'swelling test' and conventional semen variables in men suspected of primary infertility.

The relationship and degree of association between the percentage of sperm swelling (HOS-test) and conventional semen variables was investigated in 263 consecutive ejaculates. The semen samples were exclusively obtained from men suspected of primary infertility. It was found that the correlation coefficients (Spearman's rho) followed the order: percentage of progressive motility at 3 h greater than count/ml greater than percentage of total motility at 3 h greater than percentage of normal spermatozoa. Of the three morphology sub-classes considered (sperm head, mid-piece and tail abnormalities), only mid-piece abnormalities correlated with the outcome of the HOS-test (rho = -0.409). Linear relationships between HOS-test results and sperm motility and morphology, but not sperm count, were indicated by LOWESS-smoothing. However, a linear relationship between the HOS-test, sperm count and a 'functional index' combining the conventional semen variables could be demonstrated after normalization of the data. Our findings suggest that the HOS-test may be of value in assessing the functional integrity and viability of spermatozoa; however, its prognostic power for fertility is probably not different from that of conventional semen variables.

Electrophoretical patterns of seminal plasma proteins in patients with cystic fibrosis.

Seminal plasma (n = 12) from cystic fibrosis (CF) patients were analyzed by gel-electrophoresis using seminal plasma and expressed prostatic secretion from fertile men as controls. Heavy precipitation at the entering position of the gel and streaking in the gel matrix was observed, demonstrating a reduced solubility of seminal proteins in CF. Comparison of the protein patterns evidenced that CF-seminal plasma (CF-SP) mainly consisted of prostatic components. Although lactoferrin was undetectable in all samples, trace amounts of low molecular weight proteins were observed in two patients. This latter finding could imply that CF-SP may contain proteolytic fragments of prostatic and/or vesicular proteins or de novo synthesized components.

Analytical biochemistry of seminal vesicle secretion: a challenge to andrological laboratories.

Chemical characterization of seminal vesicle secretion through seminal vesicle proteins would have the following advantages: (1) to judge on the secretory competence of the gland, (2) to recognize atypical secretory patterns, (3) to identify specific molecules and their epitopes for anatomical, diagnostic, therapeutic, anti-fertility and forensic purposes, and (4) to study physiologically active proteins or peptides of seminal plasma. There are different approaches for collection of the specific samples, each of which has peculiar advantages and disadvantages: ejaculate collection in the presence of protease inhibitors, use of split or multi-split ejaculates, utilization of autopsy or surgical material. Liquefied proteins are submitted to different chromatographic and electrophoretic procedures. One must keep in mind, however, that a whole series of biochemical processes can rapidly and irreversibly alter in vivo and in vitro the secretory proteins. The study of the secretion from male accessory sex glands and their interaction with spermatozoa therefore still deserves an absolute research priority.

Semen anomalies due to voiding defects of the ampullo-vesicular tract. Infertility due to ampullo-vesicular voiding defects.

Anatomical (congenital or postinflammatory) or functional anomalies of the uroseminal intersection can induce a voiding dysfunction of the deferential ampullae and seminal vesicles, leading to infertility. In case of azoospermia or OAT-syndrome with poor semen volume and decreased vesicular markers, some clinical history and examination data can cause suspect of one of such anomalies. The transrectal ultrasonographic findings of anechoic area(s) inside the prostate and/or seminal vesicle (even after recent ejaculation), the peculiar vasovesiculographic pictures (especially after ejaculation following the contrast medium injection into the vasa deferentia), plus the evaluation of the "deferential-ampullary sperm reserve", will permit a detailed diagnosis of the voiding dysfunction of the uroseminal crossing. A successful appropriate treatment of these pathologies can be done. Ultrasonically-guided transrectal puncture, or transurethral incision upstream of (or resection of) the ejaculatory duct orifices, or prolonged sexual abstinence, or artificial spermatocele of the vas deferens, or sperm recovering from urine for GIFT or IVF/ET after washing-out of the seminal tract by vas puncture are all methods, which can be selectively used depending on the individual case, to treat these forms of infertility. The authors present some paradigmatic clinical cases, and report that this pathology presumably occurs in up to 9% of non-selected infertile patient population.

Poor reproductive prognosis in severe teratozoospermia with a predominant sperm anomaly.

A retrospective examination of the semen analyses performed in our clinic from January 1979 to September 1986 revealed that approximately 15% of the patients were affected by severe teratozoospermia (greater than 80% abnormal forms and greater than 5 x 10(6) sperm/ml). In approximately 8% of these cases, a single predominant anomaly (same defect in greater than 50% of the sperm) was reported and confirmed by subsequent analyses (n = 37). The types of monomorphic teratozoospermia encountered in this study included round head (n = 6), amorphous head (n = 16), small head (n = 6), tapering head (n = 2), pin head (n = 1) and midpiece anomaly (n = 6). The clinical data suggest that familial genetic factors are probably involved in round head-monomorphic teratozoospermia, whereas testicular factors may be associated with amorphous head-monomorphic teratozoospermia. No matter what type of monomorphic teratozoospermia, the data in vivo (no pregnancies recorded in the follow-up period ranging from 2-8 years) and in vitro (negative SPAs) suggest a poor prognosis for the couples affected by this syndrome.