Fertility preservation and management of pregnancy in melanoma patients requiring systemic therapy.

Melanoma is one of the most common cancers in adolescents and adults at fertile age, especially in women. With novel and more effective systemic therapies that began to profoundly change the dismal outcome of melanoma by prolonging overall survival, the wish for fertility preservation or even parenthood has to be considered for a growing portion of melanoma patients-from the patients' as well as from the physicians' perspective. The dual blockade of the mitogen-activated protein kinase pathway by B-Raf proto-oncogene serine/threonine kinase and mitogen-activated protein kinase inhibitors and the immune checkpoint inhibition by anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated protein-4 monoclonal antibodies constitute the current standard systemic approaches to combat locally advanced or metastatic melanoma. Here, the preclinical data and clinical evidence of these systemic therapies are reviewed in terms of their potential gonadotoxicity, teratogenicity, embryotoxicity and fetotoxicity. Recommendations for routine fertility and contraception counseling of melanoma patients at fertile age are provided in line with interdisciplinary recommendations for the diagnostic work-up of these patients and for fertility-protective measures. Differentiated recommendations for the systemic therapy in both the adjuvant and the advanced, metastatic treatment situation are given. In addition, the challenges of pregnancy during systemic melanoma therapy are discussed.

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.

Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPalpha by inhibiting its ubiquitination.

Functional inactivation of transcription factors in hematopoietic stem cell development is involved in the pathogenesis of acute myeloid leukemia (AML). Stem cell regulator C/enhancer binding protein (EBP)alpha is among such transcription factors known to be inactive in AML. This is either due to mutations or inhibition by protein-protein interactions. Here, we applied a mass spectrometry-based proteomic approach to systematically identify putative co-activator proteins interacting with the DNA-binding domain (DBD) of C/EBP transcription factors. In our proteomic screen, we identified c-Jun N-terminal kinase (JNK) 1 among others such as PAK6, MADP-1, calmodulin-like skin proteins and ZNF45 as proteins interacting with DBD of C/EBPs from nuclear extract of myelomonocytic U937 cells. We show that kinase JNK1 physically interacts with DBD of C/EBPalpha in vitro and in vivo. Furthermore, we show that active JNK1 inhibits ubiquitination of C/EBPalpha possibly by phosphorylating in its DBD. Consequently, JNK1 prolongs C/EBPalpha protein half-life leading to its enhanced transactivation and DNA-binding capacity. In certain AML patients, however, the JNK1 mRNA expression and its kinase activity is decreased which suggests a possible reason for C/EBPalpha inactivation in AML. Thus, we report the first proteomic screen of C/EBP-interacting proteins, which identifies JNK1 as positive regulator of C/EBPalpha.

Testosterone treatment of hypogonadal men participating in competitive sports.

Testosterone has a steeply dose-dependent effect on muscle mass and strength irrespective of gonadal status. So, for reasons of fairness, people who engage in competitive sports should not administer exogenous testosterone raising their blood testosterone levels beyond the range of normal. There is a ban on exogenous androgens for men and women in sports, but an exception has been made for men with androgen deficiency due to pituitary or testicular disease. Men who receive testosterone administration for the indication hypogonadism have an interest in the use of testosterone preparations generating blood testosterone levels within the normal range of healthy, eugonadal men. On the grounds of a positive correlation between blood testosterone concentrations muscle and volume/strength, they are best served with a parenteral testosterone preparation, rather than transdermal testosterone, but they should not run the risk of being excluded from competition because of supraphysiological testosterone levels. The latter is a realistic risk with the traditional parenteral testosterone esters. The new parenteral testosterone undecanoate preparation offers much better perspectives. Its pharmacokinetics have been investigated in detail and there is a fair degree of predictability of resulting blood testosterone levels with use of this preparation.

European Academy of Andrology guideline Management of oligo-astheno-teratozoospermia.

BACKGROUND: Oligo-astheno-teratozoospermia is frequently reported in men from infertile couples. Its etiology remains, in the majority of cases, unknown with a variety of factors to contribute to its pathogenesis. The aim of this European Academy of Andrology guideline was to provide an overview of these factors and to discuss available management options. MATERIALS AND METHODS: PubMed was searched for papers in English for articles with search terms: male infertility and oligo-astheno-teratozoospermia. For evidence-based recommendations, the GRADE system was applied. Issues related to urogenital infections/inflammations have not been included in this document as they will be covered by separate guidelines. RESULTS: For men with oligo-astheno-teratozoospermia, the European Academy of Andrology recommends: A general physical examination to assess signs of hypogonadism. A scrotal physical examination to assess (i) the testes and epididymes for volume and consistency, (ii) deferent ducts for total or partial absence, and (iii) occurrence of varicocoele. Performing two semen analyses, according to World Health Organization guidelines to define an oligo-astheno-teratozoospermia. An endocrine evaluation. A scrotal ultrasound as part of routine investigation. Karyotype analysis and assessment of Yq microdeletions in infertile men with a sperm concentration ≤5 × 106 /mL. Cystic fibrosis transmembrane conductance regulator gene evaluation in case of suspicion for incomplete congenital obstruction of the genital tract. Against quitting physical activity to improve the chance of achieving pregnancy. Against androgen replacement therapy to improve the chance of achieving pregnancy. Assisted reproduction techniques to improve the chance of achieving pregnancy, in case other treatment options are not available or not efficient. Androgen replacement therapy in patients with biochemical/clinical signs of hypogonadism, after completion of the fertility treatment. CONCLUSION: These guidelines can be applied in clinical work and indicate future research needs.

Proteomics of acute myeloid leukaemia: Cytogenetic risk groups differ specifically in their proteome, interactome and post-translational protein modifications.

Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of beta-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

Proteomic discovery of Max as a novel interacting partner of C/EBPalpha: a Myc/Max/Mad link.

The transcription factor CCAAT/enhancer binding protein a (C/EBPalpha) is important in the regulation of granulopoiesis and is disrupted in human acute myeloid leukemia. In the present study, we sought to identify novel C/EBPalpha interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPalpha in our screen. We confirmed the in vivo interaction of C/EBPalpha with Max and showed that this interaction involves the basic region of C/EBPalpha. Endogenous C/EBPalpha and Max, but not Myc and Max, colocalize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPalpha on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBPalpha promoter in vivo by Max and Myc under cellular settings and by C/EBPalpha and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPalpha results in granulocytic differentiation of the human hematopoietic CD34(+) cells, as evidenced by CD11b, CD15 and granulocyte colony-stimulating factor receptor expression. Silencing of Max by short hairpin RNA in CD34(+) and U937 cells strongly reduced the differentiation-inducing potential of C/EBPalpha, indicating the importance of C/EBPalpha-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPalpha functions, thereby suggesting a possible link between C/EBPalpha and Myc-Max-Mad network.

Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis).

The role of FSH in spermatogenesis was investigated in nonhuman primates depleted of testosterone by GnRH antagonist treatment. The GnRH antagonist antide (Nal-Lys; [N-acetyl-D-2-naphthyl-Ala1,D-4-chloro-Phe2,D-pyridyl-Ala3, nicotinyl-Lys5,D-nicotinyl-Lys6,isopropyl-Lys8,D-Ala10 ]-GnRH) was used at a daily dose of 450 micrograms/kg to suppress endogeneous gonadotropin and androgen production. Four groups of five cynomolgus monkeys (Macaca fascicularis) were subjected to the following treatment throughout a 16-week period: vehicle (group 1), GnRH antagonist (group 2), and GnRH antagonist plus human FSH (Fertinorm; 2 x 15 IU/day.animal; hFSH) during weeks 0-8 (group 3) or 8-16 (group 4). Testicular biopsies were performed before and after 4, 8, and 16 weeks of treatment. The tissue was analyzed by light microscopy and flow cytometry. Serum testosterone levels were suppressed into the range of orchidectomized animals in all GnRH antagonist-treated groups. In the absence of hFSH, serum inhibin levels were also markedly lowered. Concomitant administration of hFSH attenuated the GnRH antagonist-induced reduction of testicular size, while delayed treatment with hFSH failed to restimulate testicular volume. Numbers of A-dark spermatogonia, the reserve stem cells, were not altered by any of the treatments. hFSH either fully maintained or increased the counts for A-pale spermatogonia (renewing stem cells). The development of pachytene spermatocytes and round and elongated spermatids was markedly reduced or inhibited by the GnRH antagonist within 6-18 weeks. In contrasts, hFSH maintained these cell types at about 50% of baseline for 8 weeks. After 8 weeks of GnRH antagonist administration, hFSH stimulated A-pale spermatogonia and spermatocytes 2- to 3-fold with only minor effects on spermatid numbers. By means of flow cytometry, testicular cells were quantified according to DNA content. Within 8-16 weeks of GnRH antagonist treatment the percentage of 4C (mainly primary spermatocytes), 1C (round spermatids), and 1CC cells (elongated spermatids) had fallen from 65-75% to 5-25%. hFSH completely maintained the relative number of these cells, but failed to significantly restimulate the formation of 1CC cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Testosterone buciclate (20 Aet-1) in hypogonadal men: pharmacokinetics and pharmacodynamics of the new long-acting androgen ester.

Due to unfavorable pharmacokinetics of the available androgen esters for substitution therapy of male hypogonadism, there is a demand for new testosterone (T) preparations producing constant serum levels in the physiological range. To assess the pharmacokinetics and pharmacodynamics of the new ester testosterone buciclate (TB) [20 Aet-1] in hypogonadal men a clinical phase I-study was performed. After two control examinations 8 male patients with primary hypogonadism were randomly assigned to 2 treatment groups (n = 2 x 4) given single doses of either 200 (group I) or 600 mg (group II) TB im. Blood samples were obtained 1, 2, 3, 5, and 7 days post injection and then weekly in the course of 4 months. In group I serum androgen levels did not rise to normal values. However, in group II androgens increased significantly and were maintained in the normal range up to 12 weeks with maximal serum levels of 13.1 +/- 0.9 nmol/L (mean +/- SE) in study week 6. No initial peak release of T was observed in either study group. Pharmacokinetic analysis revealed a terminal elimination t1/2 beta of 29.5 +/- 3.9 days and a mean residence time of 65.0 +/- 9.9 days in group II. In one patient in group II dihydrotestosterone levels slightly exceeded the upper normal limit during the study course. Sex hormone-binding globulin remained unchanged and estradiol serum levels never exceeded the normal range in any patient. In group II gonadotropins were significantly suppressed, whereas no change was seen in group I. A significant increase in body weight, hematological parameters, and libido/potency was observed after TB injection which was more pronounced in the higher dose group. Regardless of the dose administered, no significant change was seen in uroflow, prostate volume measured by transrectal ultrasonography, or prostate specific antigen. No adverse side-effects including changes in clinical chemistry were observed. In conclusion, single injections of 600 mg TB in hypogonadal patients show favorable pharmacokinetics and pharmacodynamics. This new long-acting T ester is a promising new agent for substitution therapy of male hypogonadism and for male contraception.

Depot gonadotropin-releasing hormone agonist blunts the androgen-induced suppression of spermatogenesis in a clinical trial of male contraception.

Thus far, when tested as male contraceptives, GnRH agonists in combination with androgens were not very effective in producing azoospermia. Since in previous studies androgens were always given simultaneously with the GnRH agonist or later, we tested whether GnRH agonist administration after an initial androgen suppression phase might yield better results. After a control period, 3 groups of young healthy men (n = 8/group) received an initial loading dose of 400 mg 19-nortestosterone hexyloxyphenylpropionate (19NT-HPP), followed by 200 mg of the ester every 3 weeks for 24 weeks. One week after the first 19NT-HPP injection, 2 groups were given a single sc implant injection of 3.3 or 6.6 mg of the GnRH agonist buserelin, respectively, whereas a placebo implant was given to the third group. In the group receiving only 19NT-HPP, serum LH and FSH were markedly suppressed and remained low during the treatment phase. In the 16 volunteers receiving the buserelin implant LH and FSH were also suppressed on day 7, followed by a marked increase in the gonadotropins up to 2 weeks after buserelin implant injection. While LH was consistently suppressed for the remaining treatment phase, FSH returned to almost normal values in weeks 9-15. In contrast to the group treated with 19NT-HPP alone, in which sperm concentrations were reduced to oligozoospermia after only 3 weeks of treatment, the first suppressive effect in the 19NT-HPP/buserelin-treated groups was not seen before week 9. After 30 weeks, when the maximal suppression of spermatogenesis was seen, 4 of 8 volunteers in the group treated with 19NT-HPP alone were azoospermic, and the remaining 4 volunteers were oligozoospermic. In the groups treated with 19NT-HPP/buserelin, no more than 4 of 16 volunteers were azoospermic, and no more than 8 of 16 volunteers were oligozoospermic at any time point. It is concluded that GnRH agonist depot preparations have a blunting effect on the suppression of pituitary and testicular function caused by androgens in men participating in contraceptive trials.

PIP: Administration of a depot gonadotropin-releasing hormone agonist (buserelin, Hoechst, Frankfurt am Main, Germany, GnRH) 7 days after full suppression of spermatogenesis with depot androgen, nortestosterone enanthate, reduced the degree of azoospermia compared to treatment with androgen alone. 3 groups of 8 men were given 400 mg 19-nortestosterone hexyloxyphenylpropionate (Anadur, Pharmacia Arzneimittel, Ratigen, Germany, 19NT-HPP) then 200 mg every 3 weeks for 24 weeks. The 3 groups then received a single sc implant of 3.3 or 6.6 mg GnRH or a placebo. In the 19NT-HPP-placebo group, LH and FSH both were fully suppressed throughout treatment, and 1 to 4 men were azoospermic from weeks 6-30. In contrast, in the men given GnRH agonist, LH and FSH were suppressed at first, spiked initially, then FSH rose to a maximum at 12 weeks, declined, and again rose to normal levels after week 27. Testosterone mirrored LH in remaining suppressed throughout. The appearance of azoospermia was noted in 6 weeks in Group 1, in 12 weeks in Group 2, and in 24 weeks in Group 3. Azoospermia lasted longest in Group 1 (median length 21 weeks), shortest in Group 3 (median 6 weeks). Testicular volume, sperm concentration and motility followed similar trends. The only change in clinical findings was increased hemoglobin and hematocrit in all groups. Subjects reported no change in physical symptoms or frequency of coitus, but a few men in each group noted decreased in libido in Week 9. These results corroborate previous studies which showed less inhibition of spermatogenesis in men given GnRH in addition to androgens. Apparently androgens with GnRH antagonists are more effective in male effective in male contraception than GnRH agonists.

High loading and low maintenance doses of a gonadotropin-releasing hormone antagonist effectively suppress serum luteinizing hormone, follicle-stimulating hormone, and testosterone in normal men.

The GnRH antagonist cetrorelix effectively suppresses serum LH, FSH, and testosterone (T) in normal men without major side-effects. However, as with other available GnRH antagonists, relatively high doses of 10 mg/day were required for sustained reduction of T levels during 1-week administration in normal men. Therefore, we investigated whether a suppression of LH, FSH, and T achieved by initial high dose cetrorelix can be maintained by continued low dose injections. Sixteen young male volunteers were randomly assigned to four study groups (n = 4/group). Twelve men were injected s.c. with 10 mg cetrorelix at 0800 h for 5 days, followed by injections of 2 mg/day (group I), 2 x 1 mg/day (group II), and 1 mg/day (group III) up to the end of the 3-week injection period. For the control, group IV was given daily placebo injections for 3 weeks. Morning and evening blood samples were obtained daily for 4 weeks and then at increasing time intervals up to week 13. Initial injections of 10 mg/day cetrorelix suppressed LH, FSH, and T effectively. This initial reduction of serum levels was maintained during the following low dose maintenance injections in all groups. In comparison to the initial suppression, significantly lower levels of LH, FSH, and T near the assay detection limits were measured during study weeks 2 and 3. The results show that compared to previous long term studies, much lower daily doses of the GnRH antagonist are sufficient for effective suppression of LH, FSH, and T after initial high loading dose injections. In addition to competitive receptor blockage, other mechanisms of GnRH antagonist action, such as receptor down-regulation, appear to be involved during long term administration in men.

Long-term effects on testicular function of high-dose testosterone treatment for excessively tall stature.

High-dose testosterone treatment is applied during puberty to reduce the predicted adult height in excessively tall boys. To date it has remained unclear whether this therapy produces any long-term effects on reproductive functions of the patients. To clarify this question, we performed a follow-up study in 47 tall men, determining seminal and hormonal parameters 10.6 +/- 2.5 years (mean +/- SD) after cessation of therapy. The tall men treated were compared with 123 normal men attending the Institute of Reproductive Medicine as volunteers for various clinical studies. Clinical examination revealed a significantly higher prevalence of varicoceles and history of maldescended testes in the testosterone-treated tall men compared with the controls. Semen analysis revealed significantly lower progressive motility in the tall men compared with the normal men (49.2 +/- 13.4 vs. 54.3 +/- 12.8%). A nonsignificant tendency towards lower sperm concentration (43.8 +/- 35.4 vs. 57.8 +/- 45.6 mL/mL), lower total sperm count (184.4 +/- 158.0 vs. 225.4 +/- 277.5 mL/ejaculate), and reduced normal sperm morphology (27.6 +/- 12.5 vs. 30.9 +/- 13.1%) was evident in the testosterone-treated tall men. Although there was no difference in testicular volume and FSH between the groups, testosterone was lower in the testosterone-treated tall men (19.9 +/- 7.4 vs. 23.9 +/- 7.0 nmol/L). Statistical analysis of the subgroups of testosterone-treated tall men and control men without varicocele and cryptorchidism revealed no differences in any ejaculate parameter. The small difference in semen variables may be explained by a higher prevalence of varicocele and maldescended testes in the testosterone-treated tall men.

Potential of testosterone buciclate for male contraception: endocrine differences between responders and nonresponders.

Suppression of serum LH and FSH, by testosterone (T) alone or in combination with other agents, has proved to be the most promising approach to male contraception. T enanthate, the only androgen preparation tested in male contraceptive efficacy trials so far, must be injected every week due to its short terminal elimination half-life of 4.5 days and leads to supraphysiological T serum levels. A new T ester synthesized under WHO and NIH auspices, testosterone buciclate (TB), showed a favorable pharmacokinetic profile, with a terminal half-life of 29.5 days when tested in hypogonadal men. Here we describe the results of the first clinical trial with TB for male contraception. After two control examinations, normal healthy male volunteers were given a single im injection of 600 mg TB (group I; n = 4) and 1200 mg TB (group II; n = 8) on day 0. Follow-up examinations were performed every 2 weeks up to week 32. In both groups mean serum T levels remained in the normal physiological range throughout the study course. Serum levels of dihydrotestosterone (DHT) showed a dose- and time-dependent increase, with serum levels slightly above the normal range in group II for several weeks and a maximal concentration of 3.8 +/- 0.5 nmol/L (mean +/- SE) in week 6. No suppression of spermatogenesis to oligozoospermia was observed in group I. However, in group II, spermatogenesis was suppressed to azoospermia in three of eight volunteers in week 10 that persisted up to weeks 14, 20, and 22, respectively. In these three men, LH and FSH were suppressed by TB injections to the respective assay detection limits, whereas in the other five subjects, mean serum levels were only decreased to values near the lower normal limit for LH and FSH, respectively. In addition, throughout the study course, a significant difference in serum sex hormone-binding globulin was detected between the responders (mean values, 21.2-26.4 nmol/L) and nonresponders (mean values, 36.2-46.3 nmol/L). Serum levels of LH as well as total and free T at baseline and after TB injection were lower in the responders than in the nonresponders. Both subgroups showed similar increases in serum LH and FSH after GnRH stimulation. In a newly introduced GnRH antagonist suppression test, serum LH and T were decreased to significantly lower levels in the responders. These results indicate a different hormonal equilibrium and probably different susceptibility to feedback regulation of the responders compared to the nonresponders.(ABSTRACT TRUNCATED AT 400 WORDS)

PIP: Testosterone alone and the combination of gestagens and gonadotropin-releasing hormone antagonists with testosterone represent the most feasible potential agents for hormonal male contraception. The World Health Organization's Special Program of Research, Development, and Research Training in Human Reproduction has initiated a testosterone ester synthesis program and identified testosterone buciclate (TB) as the most promising approach to suppression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This paper presents the results of the first clinical trial of TB. 12 men were given a single intramuscular injection of 600 mg (group I) or 1200 mg (group II) of TB and followed every 2 weeks for up to 32 weeks. Mean serum testosterone levels in both groups remained in the normal physiological range throughout the study period. Serum levels of dihydrotestosterone showed a time- and dose-dependent increase in group II. Although no suppression of spermatogenesis was observed in the 4 men in group I, azoospermia occurred in 3 of the 8 volunteers in group II beginning at week 10 and persisting to weeks 14, 20, and 22, respectively. In the remaining 5 men in group II, mean serum LH and FSH values were depressed to values near the lower limit of normal. Serum sex hormone-binding globulin and free testosterone were lower in responders than non-responders to TB. The range of responses recorded in group II suggests that men have a different hormonal equilibrium or different set-point for regulation of gonadotropin secretion and, thus, susceptibility to exogenous hormonal steroids.

Effective suppression of luteinizing hormone and testosterone by single doses of the new gonadotropin-releasing hormone antagonist cetrorelix (SB-75) in normal men.

In rats and nonhuman primates the new GnRH antagonist cetrorelix (SB-75; [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]GnR H) has been shown to suppress testosterone secretion effectively and persistently. A clinical phase I study was performed to assess the hormonal effects of this highly potent antagonist in normal men. After 2 control examinations 30 young male volunteers were randomly assigned to 6 treatment groups (n = 5/group), and single doses of 0 (placebo), 0.25, 0.5, 1.0, 2.0, and 5.0 mg cetrorelix were administered sc. Blood samples were obtained over the course of 7 days postinjection. After maximal cetrorelix serum levels were achieved 1 h postinjection, the GnRH antagonist serum levels decreased with a terminal t1/2 of 29.8 +/- 4.2 h (mean +/- SE). LH secretion was suppressed dose- and time-dependently; maximal suppression occurred 4-6 h postinjection. Suppression of FSH did not reach statistical significance. Doses of 1.0, 2.0, and 5.0 mg cetrorelix significantly suppressed testosterone secretion compared to that in the placebo group. After the administration of 1.0 mg cetrorelix, maximal suppression was seen 8 h after injection, with testosterone levels of 7.5 +/- 1.1 nmol/L compared to 15.8 +/- 2.2 nmol/L in the placebo group. Maximal testosterone suppression by 2.0 and 5.0 mg cetrorelix occurred 12 h after injection, with testosterone concentrations of 4.9 +/- 0.5 and 2.2 +/- 0.4 nmol/L, respectively, compared to 16.5 +/- 1.7 nmol/L in the placebo group. Twenty-four hours after the injection of 1.0 and 2.0 mg cetrorelix, testosterone values were no longer significantly different from those in the placebo group, whereas in the 5.0-mg dose group testosterone concentrations increased slightly and reached serum concentrations in the lower normal range after 48 h. The only side-effect observed after the administration of cetrorelix was a transient local erythema at the injection site that disappeared within 30 min. No local induration or pruritus, or any adverse systemic side-effect occurred in any volunteer. In conclusion, the new GnRH antagonist cetrorelix effectively decreases serum LH and testosterone concentrations in a dose- and time-dependent manner and, therefore, has potential for treatment of sex hormone-dependent diseases and male contraception.

Long-term effect of testosterone therapy on bone mineral density in hypogonadal men.

In both men and women, a decrease in bone mineral density (BMD) is a major symptom of hypogonadism. Although the effects of estrogens on osteoporosis in women are well documented, comparatively little is known about the effects of long term testosterone substitution on BMD in hypogonadal men. Therefore, we studied BMD in 72 hypogonadal patients (37 men with primary and 35 men with secondary hypogonadism) under testosterone substitution therapy that continued for up to 16 yr. Thirty-two of these men were also seen before initiation of therapy. At annual intervals, trabecular BMD of the lumbar spine was measured by quantitative computed tomography, a true volumetric and reproducible method for long term serial BMD measurements. Serum levels of testosterone increased to the normal range in all androgen-treated hypogonadal men. The most significant increase in BMD was seen during the first year of testosterone treatment in previously untreated patients, when BMD increased from 95.2 +/- 5.9 to 120.0 +/- 6.1 mg/cm3 hydroxyapatite (mean +/- SE). Long term testosterone treatment maintained BMD in the age-dependent reference range in all 72 hypogonadal men, independent of the type of hypogonadism. Transdermal testosterone patches applied to the scrotum were as effective in normalizing BMD as im testosterone enanthate injections. In summary, testosterone therapy increases BMD in hypogonadal men regardless of age. The greatest increase is seen during the first year of treatment in previously untreated patients with low initial BMD. In hypogonadal men, BMD can be normalized and maintained in the normal range by continuous, long term testosterone substitution.

Ovarian response to follicle-stimulating hormone (FSH) stimulation depends on the FSH receptor genotype.

Because the ovarian response to FSH stimulation in assisted reproduction is variable, ranging from hyporesponse to hyperresponse, with the possible complication of ovarian hyperstimulation, it would be of great benefit to predict the response of the patients to FSH. To date, no clear-cut predictors of ovarian responsiveness to FSH have been identified. In this study, we investigated the role of two distinct FSH receptor (FSHR) variants, Thr307/Asn680 and Ala307/Ser680, in the response to FSH in women undergoing controlled ovarian stimulation. The FSHR polymorphism at position 680 was analyzed by restriction-fragment-length polymorphism in 161 ovulatory women below the age of 40 yr. With reference to the couple, infertility has been diagnosed as being attributable to male causes (76%), tubal factor (11%), or both (13%). The distribution was 29% for the Asn/Asn, 45% for the Asn/Ser, and 26% for the Ser/Ser FSHR variant. Peak estradiol levels, number of preovulatory follicles, and number of retrieved oocytes were similar in the 3 groups. However, basal FSH levels were significantly different among the 3 groups (6.4 +/- 0.4 IU/L, 7.9 +/- 0.3 IU/L, and 8.3 +/- 0.6 IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.01). The number of FSH ampoules required for successful stimulation was significantly different among the 3 groups (31.8 +/- 2.4, 40.7 +/- 2.3, and 46.8 +/- 5.0 for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.05). According to multiple linear regression analysis, the number of ampoules needed could be predicted from a linear combination of both the type of polymorphism and basal FSH levels (P < 0.001). These clinical findings demonstrate that the ovarian response to FSH stimulation depends on the FSHR genotype.

Suppression of endogenous testosterone in young men increases serum levels of high density lipoprotein subclass lipoprotein A-I and lipoprotein(a).

We investigated the effect of testosterone suppression on lipoprotein metabolism in men. After a baseline period of 14 days, 12 healthy young men received over a period of 3 weeks daily s.c. injections of Cetrorelix, an antagonist of GnRH. The volunteers were then followed-up for 10 additional weeks. Administration of Cetrorelix suppressed testosterone significantly up to day 35, after which values returned to baseline. Suppression of testosterone was associated with significant and consistent increases in mean serum levels of high density lipoprotein (HDL) cholesterol by 20% (P < 0.0001), apolipoprotein A-I (apoA-I) by 10% (P = 0.0032), apoA-II by 7% (P = 0.0112), HDL subclass lipoprotein A-I (LpA-I) by 23% (P = 0.002), and plasma lecithin:cholesterol acyltransferase by 7% (P < 0.001). Serum levels of HDL subclass LpA-I/LpA-II changed insignificantly. Moreover, suppression of testosterone significantly increased the median of lipoprotein(a) [Lp(a)] levels from 5.5 to 8.5 mg/dL (P < 0.0001). The increase in Lp(a) levels was positively correlated with baseline levels of Lp(a) (r = 0.91; P < 0.001) and amounted to 40-60% in individuals with baseline levels of Lp(a) higher than 3 mg/dL. We conclude that endogenous testosterone is involved in the regulation of HDL cholesterol and Lp(a) levels and may thereby influence cardiovascular risk.

Clinical trial of transdermal testosterone and oral levonorgestrel for male contraception.

Approaches to hormonal male contraception are predominantly based on injectable testosterone (T) application. As most users would prefer an injection-independent modality, this study was designed to develop a self-applicable hormonal male contraceptive regimen by combining transdermal T with an oral gestagen. Eleven healthy men (23-40 yr old) were treated with oral levonorgestrel and transdermal T for 24 weeks. T was applied daily as a transdermal patch to be worn on the trunk. Levonorgestrel was taken orally at a dose of 250 microg daily up to week 12, followed by 500 microg to week 24 in those volunteers who had not become azoospermic by that time. Within 24 weeks, 2 of 11 volunteers had become azoospermic, and 3 of 11 showed sperm concentrations below 3 million/mL. The sperm concentrations of the remaining volunteers declined, but failed to reach the limit considered compatible with contraception by WHO. Treatment resulted in suppression of LH, FSH, and sex hormone-binding globulin, whereby the volunteers with lower sperm concentrations showed more pronounced suppression than the others. Mean T concentrations remained within the lower limit of normal and on occasions were below this level. There were no complaints of hypoandrogenism. Although mean levels of low density lipoprotein cholesterol, apolipoprotein B, as well as basal and postprandial insulin increased, high density lipoprotein cholesterol and apolipoprotein A-I decreased during the treatment phase. Changes in lipid parameters were normalized within 3 weeks after cessation of medication. Although only 5 of 11 volunteers reached the target sperm counts (<3 million/mL), the study shows that a self-applicable hormonal male contraceptive could be developed.

Can testosterone alone maintain the gonadotrophin-releasing hormone antagonist-induced suppression of spermatogenesis in the non-human primate?

The combination of gonadotrophin-releasing hormone (GnRH) antagonist and delayed testosterone substitution provides a promising approach towards male contraception. However, the GnRH antagonists used clinically so far cause side-effects and have to be administered continuously. We therefore used the non-human primate model to see whether the GnRH antagonist cetrorelix (which exhibits a favourable benefit-to-risk ratio in terms of anti-gonadotrophic action in normal men) induces complete and reversible suppression of spermatogenesis and whether GnRH antagonist-induced suppression of spermatogenesis can be maintained by testosterone alone. Four groups of adult cynomolgus monkeys (Macaca fascicularis; five per group) were injected daily with 450 micrograms cetrorelix/kg ([N-acetyl-D-2-naphthyl-Ala1, D-4-chloro-Phe2, D-pyridyl-Ala3, D-Cit6, D-Ala10]-GnRH). Group 1 received the GnRH antagonist for 7 weeks followed by vehicle administration for another 11 weeks; group 2 was treated with GnRH antagonist for the entire 18 weeks with each animal receiving a single testosterone implant during weeks 11-18 to restore the ejaculatory response to electrostimulation; group 3 received the GnRH antagonist for 18 weeks and testosterone buciclate (TB) was injected during week 6 of GnRH antagonist treatment; group 4 was subjected to GnRH antagonist administration for 7 weeks and received TB (200 mg/animal) during week 6. Under GnRH antagonist treatment alone serum concentrations of testosterone were suppressed. TB maintained testosterone levels two- to fourfold above baseline levels in groups 3 and 4 and prevented the recovery of LH secretion for about 20 weeks after GnRH antagonist withdrawal, whereas inhibin levels increased significantly from week 8 onwards. Group 2 animals were azoospermic during weeks 12-18 of GnRH antagonist administration. The TB-replaced groups developed azoospermia or became severely oligozoospermic. Quantitation of cell numbers by flow cytometry during weeks 6 and 18 revealed that TB (groups 3 and 4) had prevented a further decline of germ cell production compared with group 2 but had maintained the spermatogenic status present at week 6 (onset of TB substitution). All inhibitory effects of cetrorelix and/or TB were reversible after cessation of treatment. These findings demonstrate that cetrorelix reversibly inhibits spermatogenesis in a non-human primate model. Although TB maintained the GnRH antagonist-induced suppression of spermatogenesis, azoospermia was not achieved. This latter effect may reflect either a direct spermatogenesis-supporting effect of the high dose of TB or the partial recovery of inhibin secretion (indirectly reflecting FSH secretion) or a combination of both.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional and clinical consequences of mutations in the FSH receptor.

The follicle-stimulating hormone (FSH) is essential for normal gametogenesis. In females FSH is required for ovarian development and follicle maturation whereas in males FSH determines Sertoli cell number and quantitatively and qualitatively normal spermatogenesis. FSH action is mediated by a G-protein coupled receptor expressed solely in granulosa and Sertoli cells. The FSH-receptor (FSHR) gene is localized on chromosome 2 p21 and spans a region of 54 kb. It consists of ten exons; exon one to nine encode the large extracellular domain and the transmembrane domain is comprised of exon ten. Mutations in the FSHR gene could severely affect gametogenesis and result in infertility. Therefore screening programs have been initiated, in which patients with disturbed fertility were searched for mutations in the FSHR gene. Several Finnish families were identified displaying an inherited pattern of ovarian dysgenesis, a disease leading to streaky underdeveloped ovaries and primary amenorrhea. By genetic linkage the locus of the genetic defect was confined to chromosome 2 p21. Analysis of the FSHR gene resulted in the identification of a mutation (Ala189Val) homozygous in all affected females. Functional studies revealed that the mutation affects the proper protein folding and thereby inactivates the receptor. In a male patient hypophysectomized because of a pituitary tumor, who despite undetectable serum gonadotropins had normal semen parameters, we hypothesized an activating mutation of the FSHR. Screening of exon ten of the FSHR gene resulted in the identification of a Asp567Gly transition in the third intracytoplasmatic loop. Functional studies resulted in a 1.5-fold increase in basal cAMP production compared to wild type FSHR, indicating that the heterozygous mutation leads to a ligand-independent constitutive activation of the FSHR. This patient provides an exceptional model of nature defining the role of FSH in human spermatogenesis. Mutations of the FSHR might have differential effects in each gender. For example activating mutations have not been described in women, therefore it is not clear whether the constitutive activity of the receptor could disturb normal follicular development resulting in certain infertility.