Molecular study of the presence and transcriptional activity of HPV in semen.

PURPOSE: Human Papillomavirus (HPV) in semen represents a controversial topic. Recent evidence suggests a correlation with poor semen quality, but its detection is still unstandardized in this biological fluid. Thus, the aims of this study were to verify the ability of nested PCR to reveal HPV-DNA in semen; to evaluate association of seminal HPV with sperm parameters and risk factors for infection; to investigate the rate of HPV-DNA positivity in patients with and without risk factors; to assess HPV transcriptional activity. METHODS: We enrolled sexually active men and collected clinical and anamnestic data during andrological and sexually transmitted infections (STIs) evaluation. For each patient, we performed semen analysis and nested PCR to detect HPV-DNA in semen. In positive semen samples, we proceeded with genotyping and RNA quantification to detect HPV transcriptional activity. RESULTS: We enrolled 185 men (36.0 ± 8.3 years), of which 85 with (Group A) and 100 without HPV risk factors (Group B). Nested PCR was able to reveal HPV-DNA in semen, discovering a prevalence of 8.6% (11.8% in Group A and 6% in Group B, respectively). We observed no correlation between sperm quality and seminal HPV. Genital warts and previous anogenital infection were significantly associated with the risk of HPV positivity in semen. Moreover, no viral transcriptional activity was detected in positive semen samples. CONCLUSIONS: Our study suggests that searching for seminal HPV could be important in patients both with and without risk factors, especially in assisted reproduction where the risk of injecting sperm carrying HPV-DNA is possible.

Human genital tracts microbiota: dysbiosis crucial for infertility.

Human body is colonized by trillions of microbes, influenced by several factors, both endogenous, as hormones and circadian regulation, and exogenous as, life-style habits and nutrition. The alteration of such factors can lead to microbial dysbiosis, a phenomenon which, in turn, represents a risk factor in many different pathologies including cancer, diabetes, autoimmune and cardiovascular disease, and infertility. Female microbiota dysbiosis (vaginal, endometrial, placental) and male microbiota dysbiosis (seminal fluid) can influence the fertility, determining a detrimental impact on various conditions, as pre-term birth, neonatal illnesses, and macroscopic sperm parameters impairments. Furthermore, unprotected sexual intercourse creates a bacterial exchange between partners, and, in addition, each partner can influence the microbiota composition of partner's reproductive tracts. This comprehensive overview of the effects of bacterial dysbiosis in both sexes and how partners might influence each other will allow for better personalization of infertility management.

Incidence of Y chromosome microdeletions in patients with Klinefelter syndrome.

PURPOSE: The aim of this study was to study the incidence of Y chromosome microdeletions in a Caucasian population of Klinefelter syndrome (KS) patients and to investigate the possible association between Y chromosome microdeletions and KS. MATERIALS AND METHODS: We conducted a retrospective study on 118 KS patients, 429 patients with non-obstructive azoospermia (NOA), and 155 normozoospermic men. Eight of the 118 KS patients had undergone testicular sperm extraction (TESE). All patients underwent semen examination and Y chromosome microdeletions evaluated by PCR, using specific sequence tagged site (STS) primer sets, which spanned the azoospermia factor AZFa, AZFb, and AZFc regions of the Y chromosome. RESULTS: Semen analysis of the KS group revealed: 1 patient with oligozoospermia, 1 with severe oligoasthenoteratozoospermia, 2 with cryptozoospermia, and 114 with azoospermia. Eight of the 114 azoospermic KS patients underwent TESE, and spermatozoa were recovered from three of these, all of whom had non-mosaic karyotype 47, XXY. 10.7% of the NOA patients presented AZF microdeletions. In 429 cases with NOA, 8 cases had AZFa + b + c deletion, 6 cases had AZF b + c deletion, 4 cases had AZFa microdeletion, 8 cases had AZFb microdeletion, and 20 cases had AZFc microdeletion. Just one KS patient (0.8%) presented microdeletion in the AZFc region. CONCLUSION: The percentage of microdeletions in KS patients was lower than in NOA patients, suggesting that AZF microdeletions and KS do not have a causal relationship and that Y chromosome microdeletions are not a genetic factor linked to KS.

Characterization of circulating immune cells and correlation with Tie2/Angiopoietins level in well differentiated neuroendocrine gastroenteropancreatic tumors: a cross-sectional analysis.

PURPOSE: The immune environment represents a new, but little explored, tool for understanding neuroendocrine neoplasms (NENs) behavior. An immunosuppressed microenvironment is hypothesized to promote NENs progression. A missing profiling of circulating leukocyte and peripheral blood mononuclear cells (PBMCs) subpopulations would open new perspectives in the still limited diagnostic-therapeutic management of NENs. METHODS: A cross-sectional case-control pilot study was performed recruiting 30 consecutive subjects: 15 patients naïve to treatment, with histologically proven gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) and 15 healthy controls, matched for age and sex. PBMCs subpopulations were studied by flow cytometry. Soluble Tie2 (sTie2), Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) were evaluated by ELISA. RESULTS: Immune cell profiling revealed a significant lower CD3-CD56+ natural killer (NK) cell count in NETs vs controls (p = 0.04). NK subset analysis showed a reduced relative count of CD56+CD16+ NK cells (p =0.002) in NETs vs controls. Patients with NET showed a higher percentage of CD14+CD16++ non-classical monocytes (p = 0.01), and a lower percentage of CD14+CD16+ intermediate monocytes (p = 0.04). A decrease in percentage (p = 0.004) of CD4+ T-helper lymphocytes was found in NET patients. Evaluation of cellular and serum angiopoietin pathway mediators revealed in NET patients a higher relative count of Tie2-expressing monocytes (TEMs) (p < 0.001), and high levels of Ang-1 (p = 0.003) and Ang-2 (p = 0.002). CONCLUSIONS: Patients with GEP-NET presented an immunosuppressed environment characterized by a low count of cytotoxic NK cells, a high count of anti-inflammatory non-classical monocytes, and a low count of T-helper lymphocytes. Higher levels of TEMs and angiopoietins suggest a crosstalk between innate immunity and angiogenic pathways in NETs.

Ovarian 17-ketosteroid reductase deficiency as a possible cause of polycystic ovarian disease.

The deficiency of ovarian 17-ketosteroid reductase (17-KSR) was recently discovered to be a possible cause of polycystic ovarian disease (PCOD) in hirsute women. Forty three patients with PCOD (age range, 18-38 yr) were reevaluated to search for a hormonal pattern that might suggest an ovarian 17-KSR deficiency. Androstenedione, testosterone, FSH, LH, 17-hydroxyprogesterone, and dehydroepiandrosterone sulfate were evaluated basally on the day 17 of the menstrual cycle, when present, and after dynamic tests (ACTH stimulation, 1 mg im for 2 consecutive days; dexamethasone inhibition, 0.5 mg four times a day for 14 days; and cyproterone acetate treatment, 50 mg each day for 14 days) in three successive menstrual cycles or at 30-day intervals. All patients studied presented with hyperestronemia, abnormal gonadotropin pattern, and hyperandrogenism, but showed different responses of androstenedione and testosterone to dynamic tests. In two patients the hormonal pattern suggested an ovarian 17-KSR deficiency: in fact they showed plasma values of androstenedione (22 and 31.3 nmol/L, respectively) and estrone (628 and 849 pmol/L, respectively) that were greatly increased compared with other patients and with controls. Androstenedione did not increase after ACTH stimulation (21.5 and 32.1 nmol/L, respectively) and did not decrease after dexamethasone inhibition (21 and 29 nmol/L, respectively), but only decreased after cyproterone acetate treatment (8 and 10.8 nmol/L, respectively). An hCG test, performed during dexamethasone suppression, confirmed the diagnosis of ovarian 17-KSR defect in one of these two patients (patient 1). Two of three brothers of patient 1 (aged 25 and 34 yr) presented with persistent important pubertal gynecomastia; one brother also had severe oligospermia. These clinical findings and the high values of androstenedione/testosterone (0.85) and estrone/estradiol (4.1) ratios of baseline plasma levels compared with controls (0.18 and 2.1, respectively) suggested a partial testicular 17-KSR deficiency. Five other patients showed PCOD secondary to nonclassic 21-hydroxylase defect diagnosed on the basis of high 17-hydroxyprogesterone plasma values and highly responsive to ACTH. The remaining 36 patients showed increased values of androstenedione and testosterone after ACTH stimulation and a decrease of these two parameters after both dexamethasone inhibition and cyproterone acetate treatment. The discovery of the 17-KSR deficiency in men and women in the same family demonstrates genetic control of this enzyme similar in both sexes, confirming the hypothesis that this disorder is inherited as an autosomal recessive character. Finally, it is strongly supported that ovarian 17-KSR defect may cause a syndrome closely resembling PCOD.

Response to a single dose of human chorionic gonadotropin in prepubertal boys.

In normal men a single dose of hCG induces an increase in plasma testosterone (T) and 17 alpha-hydroxyprogesterone (17 alpha-OHP) 2-4 h after the injection; after 24-36 h a maximum increase in plasma 17 beta-estradiol (E2) and 17 alpha-OHP occurs followed by a second surge in T after 48-96 h. The present investigation focuses on the effect of a single dose of hCG (3500 IU/m2 body surface) on testicular steroid production in 12 boys aged 13 months to 12 yr. Plasma hCG, 17 alpha-OHP, androstenedione (A), T, dihydrotestosterone, and E2 were measured basally and 2, 4, 24, 48, 72, and 96 h after hCG administration. Plasma hCG was measured using a double antibody RIA technique and steroids by RIA after celite chromatography. The results show that hCG peaked 2 h after administration of the hormone and high levels persisted for up until 72 h. Plasma T and dihydrotestosterone increased after 48 h and remained significantly high for another 48 h; 17 alpha-OHP, A, and E2 did not change. These findings show that hCG stimulation in prepubertal boys induces significant production of T without affecting the precursors or aromatization product, in contrast to observation in the adult man, where 17 alpha-OHP, A, and E2 increase significantly. A response comparable to that observed in children has been recorded in adult males with hypogonadotropic hypogonadism.

Diurnal 18-hydroxy-11-deoxycorticosterone pattern in human stable hypertension.

Diurnal 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) pattern was studied with RIA technique in 33 hypertensive patients in supine position and on normal sodium diet. The compound was evaluated every 2 h from 0800-2000 h. Simultaneously, plasma aldosterone and cortisol were measured. Abnormal 18-OH-DOC behavior was observed in only 2 out of 4 patients with Cushing's disease, while sporadic and slight elevations, synchronous with F, were seen in 5 out of 24 stable essential hypertensive patients [1 with normal plasma renin activity (PRA), 1 with low PRA, and 3 with high PRA]. 18-OH-DOC was normal in 2 cases of hypertension due to renal artery stenosis, in 1 patient with nephrosclerosis, and in 1 patient with horseshoe kidney. From these results, 18-OH-DOC does not seem to play an important pathogenetic role in stable essential hypertension, considering also the low mineralocorticoid activity of the compound.

Regional variations of insulin-like growth factor I (IGF-I), IGF-II, and receptor type I in benign prostatic hyperplasia tissue and their correlation with intraprostatic androgens.

Benign prostatic hyperplasia (BPH) is an androgen-dependent disease; it originates exclusively in the inner prostate, which includes tissue surrounding the urethra. Stromal-epithelial interaction has a pivotal role in the regulation of the development and growth of the prostate, and locally produced peptide growth factors are considered important mediators of this interaction. Insulin-like growth factor I (IGF-I) and IGF-II, acting mainly through type 1 IGF receptor (IGFR1), have mitogenic and antiapoptotic effects on epithelial and stromal prostatic cells. In this study the expression of IGF-I, IGF-II, and IGFR1 messenger ribonucleic acid (mRNA), the immunoreactive content of IGF-I (irIGF-I) and IGF-II (irIGF-II) were determined in periurethral, intermediate, and subcapsular regions of BPH tissue to verify their possible regional variation; a correlation to the tissue levels of dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha Diol) was also determined to verify their possible androgen dependence. Prostates were removed by suprapubic prostatectomy from 14 BPH patients and sectioned in the periurethral, intermediate, and subcapsular regions. Gene expression of IGF-I, IGF-II, and IGFR1 was evaluated by semiquantitative RT-PCR, using beta-actin as a control. irIGF-I was measured by RIA, and irIGF-II was measured by IRMA after acidification and chromatography on Sep-Pak C(18) cartridges. DHT and 3 alpha Diol concentrations were evaluated by RIA after extraction and purification on Celite microcolumns. IGF-II and IGFR1, but not IGF-I, mRNA was higher in the periurethral than in the intermediate (P < 0.05) and subcapsular (P < 0.01) region. Also, prostatic levels of irIGF-II, expressed as picomoles per g tissue, were higher in the periurethral (20.84 +/- 1.84) than in the intermediate (14.81 +/- 2.11; P < 0.05) and subcapsular (10.88 +/- 1.21; P < 0.001) region. No significant differences were found in irIGF-I content. Considering prostatic androgen levels, DHT and 3alphaDiol presented a regional variation, with the highest concentrations in the periurethral region. IGF-II mRNA and irIGF-II levels were positively correlated with both DHT and 3 alpha Diol content. These results demonstrate that in BPH tissue a greater IGF-II activity is present in the periurethral region, the site of origin of BPH. Moreover, we can hypothesize that the tissue androgen content may modulate prostatic production of IGF-II, acting at the transcriptional and probably the posttranscriptional level. Therefore, even though further studies will need to confirm this hypothesis, DHT may increase IGF-II activity, mainly in the periurethral region, which, in turn, induces, through IGFR1, benign proliferation of both epithelial and stromal cells, characteristic of BPH.

Is hirsutism an evolving syndrome?

The possibility that hirsutism is an evolving syndrome rather than a static condition involving only one gland has been considered. To assess this proposal 60 untreated hirsute patients aged 12-32 years were divided into five groups according to the duration of the hirsutism (less than 1, 1-2, 2-3, 3-5 and greater than 5 years). Peripheral plasma concentrations of LH and FSH, androstenedione, dehydroepiandrosterone sulphate, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, cortisol, oestradiol-17 beta and oestrone were determined by radioimmunoassay. When the values obtained were compared with those from normal menstruating women, the results showed that in group I there was a significant increase only in the mean plasma 5 alpha-androstane-3 alpha, 17 beta-diol concentration. The mean concentration of this steroid was also raised in all other groups. In groups II and III mean basal levels of plasma dehydroepiandrosterone sulphate were also significantly increased and showed a marked increase after ACTH stimulation (1 mg tetracosactide acetate, i.m.) as did the concentrations of androstenedione and 17 alpha-hydroxyprogesterone. Finally, in groups IV and V, a significant increase in mean plasma concentrations of LH, androstenedione, oestrone and testosterone was found in the basal condition. The clinical picture also became gradually more severe from group I to group V. These data suggest that hirsutism could be an evolving syndrome progressively involving peripheral androgen metabolism, the adrenal gland and finally the ovary possibly through alterations of hypothalamic-pituitary function.

Epidermal growth factor binding sites in human pituitary macroadenomas.

The number of epidermal growth factor (EGF) binding sites was determined by competitive binding assays in a series of 46 pituitary macroadenomas. A single concentration of 125I-EGF (1 nM) was used for all experiments. In four cases, a displacement curve was obtained by adding increasing concentrations of cold EGF, and Scatchard analysis showed the presence of two classes of EGF binding sites, with Kd1 = 0.62 +/- 0.23 nM and Kd2 = 53.8 +/- 8.2 nM for the high- and low-affinity binding sites respectively. The distribution of EGF binding sites was studied in 42 cases by a single-point assay, in the presence and in the absence of a 100-fold cold EGF excess. A non-parametric distribution of EGF binding sites was observed (median 10.2 fmol/mg membrane protein, range 0.0-332.0). EGF-receptor positivity, defined as EGF binding > or = 10.0 fmol/mg protein, was observed in 23 samples (54.8%), especially in prolactinomas (76.5%, P < 0.05 vs other tumors taken together) and in gonadotrope adenomas (62.5%). EGF binding was higher in invasive than in non-invasive adenomas (median: 12.8 vs 0.0 fmol/mg membrane protein, P = 0.047), and especially in adenomas invading the sphenoid sinus (median 26.7 fmol/mg membrane protein, P = 0.008 vs other adenomas). EGF binding also tended to increase with the grade of supra/extrasellar extension according to Wilson (P = 0.15). Sex steroid receptors (SSRs) were simultaneously determined in both cytosolic and nuclear fractions of 31 pituitary adenomas. Estrogen and progesterone receptors were determined by an enzyme-linked immunoassay and androgen receptors by a competitive binding assay with [3H]methyltrienolone. No correlation could be found between EGF binding and either the gender and gonadal status of the patients, or the expression of SSRs by the adenomas. We conclude that the EGF family of growth factors may play a role in the evolution of a significant subset of human pituitary adenomas, especially in their invasiveness, and that a high EGF binding capacity may represent an additional marker of aggressiveness for these tumors. Sex steroids do not appear to have a significant role in the regulation of EGF binding in vivo in these tumors.

Antiandrogens: clinical applications.

Antiandrogens, preventing androgen action at target tissue level, are used in the treatment of various androgen-dependent diseases. Pharmacologically these substances have either a steroidal structure, like cyproterone acetate (CPA) and spironolactone (SPL), or a non-steroidal structure, like flutamide (FLU). In women with hyperandrogenism (PCO syndrome, idiopathic hirsutism, acne), clinical benefit may be obtained with CPA, which also displays a progestational activity and an antigonadotropic effect. CPA (25-50 mg/day) is used in combination with ethinyl-estradiol (EE) (20-30 micrograms/day) in reversed sequential regimen. SPL, less effective than CPA may be employed in moderate hirsutism and acne at dosages of 100-200 mg/day. During SPL treatment menstrual irregularities are frequent: in this case an association with oral contraceptives is indicated. SPL + bromocriptine (2.5-5 mg/day) has been experienced with success in PCO syndrome. The pure antiandrogen FLU, inducing progressive increase in LH and testosterone secretion, may be used only in combination with oral contraceptives. In men antiandrogens have been tested in BPH and prostatic carcinoma. In BPH the decrease in nuclear receptors and DHT nuclear content during CPA or FLU may represent the rational base of the medical treatment. An improvement in urinary obstructive manifestation has been observed with CPA alone or associated with tamoxifen (100 mg + 100 mg day). In advanced prostatic carcinoma antiandrogens represent a good alternative to estrogen therapy with less side effects and in combination with surgical or medical castration (LH-RH analogues) achieve a complete androgen blockade. An increase in the percentage of remissions and survival has been reported.

Steroidal and non-steroidal factors in plasma sex hormone binding globulin regulation.

Sex hormone binding globulin (SHBG) is a specific steroid-binding plasma glycoprotein regulated by several factors. Sex steroids are currently considered to be the main physiological regulators of this protein. SHBG levels, in fact, increase during estrogen treatment and decrease after androgen administration. It is well known, however, that in many physiological and pathological conditions SHBG concentrations cannot be explained only on the basis of steroidal control mechanisms. The regulation of SHBG levels is in fact more complex and other factors can also affect its plasma values. Between the steroidal factors our attention was focused on the role of androgens, of glandular and peripheral origin, in their capacity to lower SHBG plasma levels. We studied hyperandrogenic conditions in prepubertal (65 subjects with precocious adrenarche and 16 girls with prepubertal hypertrichosis, aged between 4 and 8 years) and in adult age (51 hirsute patients aged between 14-35 years and 51 acneic patients aged between 15-40 years). The effects of dexamethasone and ACTH administration on SHBG plasma levels were also evaluated. The results obtained showed that in adult hyperandrogenic patients SHBG levels, significantly lower than in controls, were not always inversely correlated with androgen levels, which, on the contrary, were higher than in controls. In patients with precocious adrenarche we found an inverse correlation only between SHBG, which was significantly lower than normal, and body mass index or bone age but not with androgens, suggesting that in this condition other factors may be more relevant than steroids in SHBG regulation. Between the non-steroidal factors our attention focused on insulin. We studied 40 non-obese hyperandrogenic patients with or without ultrasonographic evidence of polycystic ovaries, aged 18-39 years, and 35 obese patients, aged 19-37 years, with or without hyperandrogenism or evidence of PCO. Low levels of SHBG were found not only in hyperandrogenic obese patients but also in obese patients with normal androgens. It is possible to conclude that (1) several factors (calorie intake, energy balance and growth factors), other than steroids, may be involved in the regulation of SHBG levels in plasma; and (2) each regulating factor may act to a different extent depending on the various periods of the life cycle.

Immunoreactive EGF in human benign prostatic hyperplasia: relationships with androgen and estrogen receptors.

Benign prostatic hyperplasia (BPH) is a sex steroid dependent disease. Estrogens and androgens can modulate in different mammalian tissues epidermal growth factor (EGF) production and/or secretion. In order to clarify the relationships between estrogen and androgen receptor concentrations and those of immunoreactive EGF (irEGF), we have evaluated these parameters in 14 human BPH samples, by means of a dextran-coated charcoal method and radioimmunoassay, respectively. Cytosolic steroid receptors did not seem to correlate with irEGF. A linear significative relationship was evident between nuclear androgen receptor (ARn) levels and endogenous irEGF but not between nuclear estrogen receptors and irEGF: in ARn negative BPH samples, irEGF levels were lower than in ARn positive ones. Therefore, it is possible that androgens act at prostatic tissue level, through their own receptors, by modulating EGF production and/or secretion.

Relationship between epidermal growth factor and its receptor in human benign prostatic hyperplasia.

Human benign prostatic hyperplasia (BPH) samples were analyzed to evaluate the presence of immunoreactive epidermal growth factor (irEGF) and EGF receptor (EGFR). In all BPH samples examined both peptide and its receptor were present. Scatchard analysis of binding data of [125I]EGF showed two classes of binding sites with high and low affinity. Intratissular irEGF concentrations showed a significant inverse linear correlation with EGFR levels. Two groups of samples can be identified: the first showing high irEGF concentrations and low levels of EGF binding sites; the second low irEGF and high concentrations of EGFR. The simultaneous presence of EGF and its receptor in BPH samples indicates that this growth factor may act in an autocrine/paracrine manner in human prostatic tissue. The inverse relationship between EGF and the two sites of EGFR lead one to hypothesize that EGF itself could play a central role in determining receptor cell surface availability.

Sex steroids and epidermal growth factor in benign prostatic hyperplasia (BPH).

Androgens provide the primary signal for the onset of DNA synthesis and cell division in normal prostate and benign prostatic hyperplasia (BPH). It is possible, however, that androgen mitogenic activity is in part indirect and mediated by peptide growth factors. In LNCaP cell lines, R1881 added to DCC-FCS medium increases DNA, epidermal growth factor (EGF) and EGF receptor (EGFR) levels: the antiandrogen hydroxy-flutamide prevents the increase of the growth factor and increases its receptor. In BPH tissue removed by transvesical prostatectomy, DHT, testosterone, 3 alpha-androstanediol and nuclear androgen receptors (AR) show a positive linear correlation with EGF: treatment with flutamide decreases significantly the EGF production. Androgens, therefore, represent important modulatory factors of prostatic EGF release. Moreover, androgens and EGF downregulate EGFR, which is probably internalized into the cell and degraded by lisosomes: in fact, a negative linear correlation between EGF, nuclear AR and the high- and low-affinity binding of EGFR is observed. These findings support the hypothesis that the growth-promoting effects of androgens in the prostate are in part mediated by peptide growth factors. The inhibitory effect of antiandrogens on prostatic cell proliferation may be the result of the decreased androgenic support and decreased EGF release and expression.

Androgen concentrations and their receptors in the periurethral region are higher than those of the subcapsular zone in benign prostatic hyperplasia (BPH).

Benign prostatic hyperplasia (BPH) is an androgen-dependent disease that initially develops in the inner prostate, where the highest concentrations of testosterone (T) and dihydrotestosterone (DHT) are found. In this study, we have evaluated the cytosolic androgen receptors (ARc), the nuclear androgen receptors (ARn), and the concentrations of T, DHT, and 3alpha-androstanediol (3alphaDiol) in BPH tissue to verify the existence of a possible correlation between androgens and their receptor concentrations. Prostatic samples, removed by suprapubic prostatectomy in 15 untreated patients, were sectioned in periurethral, intermediate, and subcapsular zones. Testosterone, DHT, and 3alphaDiol were evaluated by radioimmunoassay after extraction and purification on celite microcolumns, and ARc and ARn were evaluated by means of dextran-coated charcoal method. In total tissue, mean levels of DHT, T, and 3alphaDiol were 2,531+/-308, 260+/-36, and 403+/-35 pg/mg of DNA (mean+/-SE), respectively. Cytosolic androgen receptors, detectable in all cases, were 16+/-2.8 fmol/mg of protein (mean+/-SE), and ARn, detectable in 12 cases, were 108+/-15 fmol/mg of DNA (mean+/-SE). A linear correlation between DHT and 3alphaDiol, T and DHT, and 3alphaDiol and ARn was found. If the different regions are considered, the periurethral zone, site of the primitive BPH nodule, presents the highest levels of androgens and ARn with respect to the other regions. This relative hyperandrogenism may be responsible for the growth-promoting processes of this area, leading to urinary obstruction.

Effects of triptorelin, a gonadotropin-releasing hormone agonist, on the human prostatic cell lines PC3 and LNCaP.

Some analogues of gonadotropin-releasing hormone (GnRH) influence the in vitro proliferation of cultured human cells by complex interactions that are only partially understood. This study explored the effect of Triptorelin, a GnRH agonist, on the LNCaP and PC3 prostatic cell lines, which are, respectively, responsive and unresponsive to androgen stimulation. The toxicity and cell cycle modifications induced by the drug were investigated by FACScan analysis; the effect on cell proliferation in different culture conditions was determined by counting in a Burker chamber; and the expression of binding sites for 125I-Triptorelin was revealed by displacement experiments. PC3 cell growth was completely unaffected by Triptorelin. The drug caused a double stimulatory-inhibitory action on the growth of actively proliferating LNCaP cells, depending upon the dose and environment. A significant inhibitory effect on proliferation, ranging from 25% to 65% compared with controls, was observed at a high dose (10(-4) M) according to the culture conditions; and a proliferative effect (42% compared with controls) was observed at a lower dose (10(-7) M) only in fetal bovine serum-supplemented medium. Displacement experiments revealed the expression of moderately high affinity and low affinity binding sites in LNCaP cells (Kd = 2.6 x 10(-8) and 7.7 x 10(-6) M) but only low affinity binding sites in PC3 cells (Kd = 2.7 x 10(-6) M), which suggests that the expression of binding sites with different affinity could be associated with a biological response to the drug. Proliferation studies in the presence of Cetrorelix, a GnRH antagonist, confirmed the different sensitivity of the 2 cell lines to GnRH analogues and showed that the proliferative effect of Triptorelin on LNCaP cells can be inhibited by the antagonist. Data confirm the cell specificity of Triptorelin's action and the peculiarity of its effects on prostatic cell proliferation in our experimental conditions.

Prevalent decrease of the EGF content in the periurethral zone of BPH tissue induced by treatment with finasteride or flutamide.

The aim of the present investigation is to verify whether treatment with Finasteride or Flutamide influences the regional distribution of testosterone (T), dihydrotestosterone (DHT), and epidermal growth factor (EGF) in benign prostatic hyperplasia (BPH) tissue. Thirty seven BPH patients were studied: 15 untreated, 9 treated with Flutamide (750 mg/day for 2 months), and 13 treated with Finasteride (5 mg/day for 3 months). Testosterone and DHT were evaluated by radioimmunoassay (RIA) after purification of the extracts on celite columns, and EGF was evaluated by RIA after purification on Sep-pak C18 cartridges in total tissue and in periurethral, subcapsular, and intermediate zone. In the untreated group, T, DHT, and EGF of the periurethral region are higher than those of the subcapsular zone (P < 0.01 for T and P < 0.001 for DHT and EGF). In the Flutamide group, DHT is not modified, T is increased (P = 0.045), and EGF is decreased in total tissue (P < 0.02) and in the periurethral zone (P < 0.01). In the Finasteride group, T is increased (P < 0.001), and DHT and EGF are decreased (P < 0.001), particularly in the periurethral zone. A positive linear correlation between DHT and EGF is observed in the Finasteride and in the untreated groups. In conclusion, in BPH the production of EGF is a DHT-dependent receptor-mediated function. The reduction of this growth factor during both treatments, associated with a fall of DHT in only the Finasteride group, is particularly evident in the periurethral zone. Since Finasteride reduces prostatic volume, mainly of the periurethral zone, we can speculate that DHT is responsible, either directly or indirectly through growth factors such as EGF for the enlargement of this region and thus responsible for urinary obstruction.

Hyperandrogenism in the adolescent female.

Hirsutism in adolescent girls commonly starts as an esthetic problem in young women and is later complicated by the development of infertility and polycystic ovary syndrome, which are frequent consequences of prolonged hyperandrogenism. To ascertain whether particular prepubertal clinical manifestations may predict the development of adolescent hirsutism, we followed 70 girls with precocious pubarche (PP) with or without prepubertal hypertrichosis (PH) until 3 years (mean age 14.8 +/- 0.9 years) after menarche. Similar follow-up was carried out in six girls with PP secondary to 21 hydroxylase deficiency (NC-CAH), treated with hydrocortisone. In addition, a retrospective study on the incidence of precocious pubarche was performed in 139 hirsute teenagers (mean age 17 +/- 1.8 years). Testosterone, androstenedione, dehydroepiandrosterone sulphate, 17 alpha-hydroxyprogesterone (basal and after ACTH), luteinizing hormone and follicle-stimulating hormone were evaluated by radioimmunoassay or immunoradio metric assay in the early follicular phase, in cycling subjects. Pelvic ultrasonography was also performed. In the 139 hirsute teenagers, 29 had a history of PP (21% vs. 0.6% in the general Italian population). Of these 139 patients, NC-CAH was diagnosed in 8 (6%), 5 of whom (63%) had PP. Of the 70 girls with PP, hirsutism was present in 44 (63%). PH was present in 37 of 44 patients (84%) with hirsutism, but only in 9 of 26 (35%) without hirsutism. Our results showed that 1) PP represents a risk factor for the development of postpubertal hirsutism; 2) the association with PH seems to increase the risk probability; and 3) patients with hirsutism due to NC-CAH have a higher incidence of PP compared with other hirsute patients, but glucocorticoid treatment in such patients prevents the development of hirsutism. Whether early treatment in the other PP patients may prevent the development of hirsutism remains to be established.

Insulin-like growth factor-I and -II in human benign prostatic hyperplasia: relationship with binding proteins 2 and 3 and androgens.

In prostatic tissue, androgen action may be mediated by growth factors such as insulin-like growth factor-I (IGF-I) and II (IGF-II), which are mitogenic for prostatic cells and modulate the stroma-epithelium interaction. IGF-binding proteins (IGFBPs) have an autocrine and/or paracrine role in regulating the local actions of the IGFs. In this study, testosterone, dihydrotestosterone (DHT), 3 alpha androstanediol (3 alpha diol), IGF-I, IGF-II, IGFBP2, and IGFBP3 concentrations were evaluated in human benign prostatic hyperplasia (BPH) tissue. Samples of prostate tissue were removed by suprapubic prostatectomy from twelve BPH patients. Androgen tissue levels were determined by radioimmunoassay after purification on celite microcolumns. IGF-I, IGFBP2, and IGFBP3 were measured by radioimmunoassay, and IGF-II by immunoradio metric assay, after acidification and chromatography on Sep-pak C18 Cartridges for IGF-I and IGF-II. Androgen concentrations, expressed in ng/g tissue (mean +/- SE), were 0.51 +/- 0.05 for testosterone, 5.3 +/- 0.16 for DHT, and 1.1 +/- 0.07 for 3 alpha diol. IGF-I, IGF-II, IGFBP2, and IGFBP3 levels were 24 +/- 3.7, 121 +/- 14 ng/g tissue and 0.44 +/- 0.05 and 1.2 +/- 0.17 micrograms/g tissue, respectively. No correlation between IGF-I, androgens, and IGFBPs was found. IGF-II was positively correlated with DHT (r = 0.78; p = 0.003) and 3 alpha diol (r = 0.66; p = 0.021) but not with IGFBPs. These data suggest that in BPH, DHT modulates the IGF system by increasing IGF-II without modifying IGFBPs. Therefore, the stroma-epithelium interaction, which plays an important role in prostatic growth, may be regulated by DHT through IGF-II.