Mazzilli R et al., 2023·Journal of endocrinological investigation·Free full text on PubMed Central
Infertility, which is defined as the inability to conceive after at least 12 months of regular unprotected sexual intercourses, affects about 15-20% of couples worldwide and a male factor is involved in about half of the cases. The development of assisted reproductive technology (ART) made it possible to conceive also to individuals affected from severe oligospermia or azoospermia. However, the impact of the male factor on embryo development, implantation, prevalence of chromosomal abnormalities, genetic and epigenetic alterations, and clinical and obstetric outcomes is still controversial.
This narrative review examines the indications, minimum access criteria, and outcomes by individual ART technique in relation to the male factor.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Rocca MS et al., 2023·The Journal of clinical endocrinology and metabolism
Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity.
The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males.
A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses.
We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic.
This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.
Pappalardo C et al., 2023·Frontiers in cell and developmental biology·Free full text on PubMed Central
Environmental pollutants are claimed to be major factors involved in the progressive decline of the fertility rate worldwide. Exposure to the heavy metal Cadmium (Cd) has been associated with reproductive toxicity due to its ionic mimicry. However, the possible direct accumulation of Cd in human sperm cells has been poorly investigated. In this study, we aimed to clarify the possible direct effect of Cd exposure on sperm function through the analysis of its cell accumulation. Semen samples from 30 male subjects residing in high environmental impact areas and adhering to the "Exposoma e Plurifocalità nella Prevenzione Oncologica" campaign for testis cancer prevention were compared with semen samples from 15 males residing in low exposure areas. Semen levels and cell Cd content were quantified by inductively coupled plasma (ICP) spectroscopy. Cell Cd distribution was assessed by scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). The impact of Cd on sperm function was evaluated by the in vitro exposure to the heavy metal, whilst possible scavenging approaches/agents were assessed. In addition to higher values of semen Cd, exposed subjects showed a reduction in total motile sperm fraction compared to not-exposed controls (59.6% ± 13.6% vs. 66.3% ± 7.3%, p = 0.037). Semen Cd levels were also significantly correlated with SEM-EDS signals of Cd detected on the head and neck of sperm (respectively p = 0.738, p < 0.001 and ρ = 0.465, p < 0.001). A total of 2 h of in vitro exposure to 0.5 μM Cd was associated with a significant reduction of sperm progressive motility. Scavenging approaches with either hypo-osmotic swelling or 10 μM reduced glutathione were ineffective in blunting cell Cd and restoring motility. The reduction of exposure levels appears to be the main approach to reducing the reproductive issues associated with Cd.
Guidelines by Clinical Area · Bone and Endocrine Guidelines
Corona G et al., 2023·Journal of endocrinological investigation·Free full text on PubMed Central
Erectile dysfunction (ED) is one of the most prevalent male sexual dysfunctions. ED has been in the past mistakenly considered a purely psycho-sexological symptom by patients and doctors. However, an ever-growing body of evidence supporting the role of several organic factors in the pathophysiological mechanisms underlying ED has been recognized.
The Italian Society of Andrology and Sexual Medicine (SIAMS) commissioned an expert task force involving several other National Societies to provide an updated guideline on the diagnosis and management of ED. Derived recommendations were based on the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system.
Several evidence-based statements were released providing the necessary up-to-date guidance in the context of ED with organic and psychosexual comorbidities. Many of them were related to incorrect lifestyle habits suggesting how to associate pharmacotherapies and counseling, in a couple-centered approach. Having the oral therapy with phosphodiesterase type 5 inhibitors as the gold standard along with several other medical and surgical therapies, new therapeutic or controversial options were also discussed.
These are the first guidelines based on a multidisciplinary approach that involves the most important Societies related to the field of sexual medicine. This fruitful discussion allowed for a general agreement on several recommendations and suggestions to be reached, which can support all stakeholders in improving couple sexual satisfaction and overall general health.
Pasquali D et al., 2022·Journal of endocrinological investigation·Free full text on PubMed Central
Klinefelter syndrome (KS) is frustratingly under-diagnosed. KS have a broad spectrum of clinical features, making it difficult to identify. We describe KS clinical presentation in a large Italian cohort.
This is the first observational cohort study within a national network, the Klinefelter ItaliaN Group (KING). Primary outcomes were to describe the basic clinical features and the actual phenotype of KS in Italy. Secondary outcomes were to determine age at diagnosis and geographical distribution.
We performed a basic phenotyping and evaluation of the hormonal values of 609 adult KS patients.
Mean age at diagnosis was 37.4 ± 13.4 years. The overall mean testicular size was 3 ml, and 2.5 ml in both testes in untreated KS group. BMI was 26.6 ± 5.8 kg/m2, and 25.5% of KS had metabolic syndrome (MetS). LH and FSH were increased, and mean total testosterone were 350 ± 9.1 ng/dl. A descriptive analysis showed that 329 KS patients were evaluated in Northern Italy, 76 in Central and 204 in Southern Italy. Analysis of variance demonstrated significant statistical differences (p < 0001) between the age at diagnosis of the three geographical groups. Compared with the expected number among male patients matched for age in Italy, only 16% of KS patients received a diagnosis.
These data are the results of the only national database available that collects the clinical and hormonal data of the KS patients, currently referred at the KING centers. In Italy the typical KS patient is overweight, with small testes, and elevated LH and FSH. Only 25.5% of them are diagnosed with MetS. Early detection and timely treatment are mandatory.
Grande G et al., 2022·Springer book chapter (Practical Clinical Andrology)·Free to read
Abstract Male infertility may depend by pre-testicular (for example, hypothalamic or pituitary diseases), testicular, and post-testicular (for example, obstructive pathologies of seminal ducts) causes. However, a large proportion (30–60%) of infertile males does not receive a clear diagnosis. In these cases, generally reported as idiopathic infertility, there is a strong suspicion of genetic factors yet to be discovered. Furthermore, male fertility may be influenced by a host of lifestyle risk factors such as environment, nutrition, exposure to infections, and smoking. Therefore, lifestyle and environment risk factors may have a role in many cases of idiopathic male infertility. In this chapter, we focus our attention on these risk factors, discussing three paradigmatic situations of interference between environment/lifestyle and male fertility, thus providing the pathophysiological basis of their detrimental impact on male fertility: exposure to environmental endocrine disruptors, such as perfluoro-alkyl substances (PFAS); exposure to viruses, such as HPV; effect of nutritional status and obesity.
Ferlin A et al., 2022·Journal of endocrinological investigation
Infertility affects 15-20% of couples and male factors are present in about half of the cases. For many aspects related to the diagnostic and therapeutic approach of male factor infertility, there is no general consensus, and the clinical approach is not uniform.
In the present document by the Italian Society of Andrology and Sexual Medicine (SIAMS), endorsed by the Italian Society of Embryology, Reproduction, and Research (SIERR), we propose evidence-based recommendations for the diagnosis, treatment, and management of male factor infertility to improve patient and couple care.
Components of the initial evaluation should include at minimum medical history, physical examination, and semen analysis. Semen microbiological examination, endocrine assessment, and imaging are suggested in most men and recommended when specific risk factors for infertility exist or first-step analyses showed abnormalities. Full examination including genetic tests, testicular cytology/histology, or additional tests on sperm is clinically oriented and based on the results of previous investigations. For treatment purposes, the identification of the specific cause and the pathogenetic mechanism is advisable. At least, distinguishing pre-testicular, testicular, and post-testicular forms is essential. Treatment should be couple-oriented, including lifestyle modifications, etiologic therapies, empirical treatments, and ART on the basis of best evidence and with a gradual approach.
These Guidelines are based on two principal aspects: they are couple-oriented and place high value in assessing, preventing, and treating risk factors for infertility. These Guidelines also highlighted that male infertility and in particular testicular function might be a mirror of general health of a man.
Garolla A et al., 2021·Andrology·Free full text on PubMed Central
Scrotal color Doppler ultrasonography and transrectal ultrasonography provide crucial information about the clinical status of testes and male accessory glands.
To analyze the impact of ultrasound in the evaluation of infertile males.
A total of 1120 records from infertile men were retrospectively evaluated (from January 2016 up to June 2020). Data on physical examination, semen analysis, sperm culture, scrotal color Doppler ultrasonography and transrectal ultrasonography, as well as sex hormones were analyzed. Among them, 238 reports from oligozoospermic/azoospermic infertile patients (P) fulfilling the inclusion criteria were considered for data analysis. Patients were subdivided into two groups according to follicle-stimulating hormone (FSH) values (Pa with FSH < 8 U/L and Pb with FSH ≥ 8 U/L). Sixty-three fertile volunteers (mean ± SD years) were enrolled as controls (C).
A higher prevalence of ultrasound abnormalities was recorded in P compared to C. Pb group had significantly lower bitesticular volume compared to Pa and C. Pa had a higher prevalence of transrectal ultrasonography abnormalities than Pb (69.9% vs. 38.4%), whereas Pb had a higher prevalence of abnormalities at scrotal color Doppler ultrasonography (60.0% vs. 28.3%, both p < 0.01). Bitesticular volume was inversely proportional to the number of altered seminal parameters and able to predict gonadotropin levels. A bitesticular volume <17 cc was associated with a higher risk of azoospermia (odds ratio = 1.799). Intratesticular vascularization was inversely correlated with gonadotropin levels and directly correlated with sperm count. A higher prevalence of prostate and seminal vesicle alterations was detected in patients and in Pa group, when compared with Pb group.
Ultrasound abnormalities are correlated with seminal parameters and may guide the clinician in the diagnostic workflow of male infertility, suggesting spermatogenesis impairment or genital tract obstructions.
Vitagliano A et al., 2021·Nutrients·Free full text on PubMed Central
Infertility is the condition of about 15% of couples that cannot get a conception after one year of unprotected sexual intercourse. In females, the reduced reproductive capacity underlies the most varied causes. Dietary supplements (DS) might be used to improve the pregnancy rate and a wide range of DS are proposed today to support female fertility. Although many authors demonstrated the positive effect of some of these products, the real efficacy of this approach is still debated. In order to evaluate the potential efficacy of DS for female infertility, we analysed the products marketed in Italy, using an original approach. A review of literature was performed to evaluate the effect of nutraceuticals on various female reproductive outcomes and to detect the minimal effective daily dose (mED) able to improve at least one of these. Thereafter, we conceived a formula to classify the expected efficacy of each DS. Each DS was scored and included into three classes of expected efficacy: higher, lower, and none. Ten out of 24 supplements (41.7%) resulted in the higher and 8 (34.3%) in the lower efficacy group, the remaining 6 DS (25.0%) were expected to have no efficacy. DS marketed in Italy are usually blends of many substances that are frequently employed at a negligible dose or without any evidence of efficacy. These findings raise serious doubt about the potential effectiveness of most commercial DS for female infertility.
Barbonetti A et al., 2021·The journal of sexual medicine
Only few studies have assessed sexual dysfunction in men with Klinefelter syndrome (KS).
To define pooled prevalence estimates and correlates of erectile dysfunction (ED) and decreased libido (DL) in KS.
A thorough search of Medline, Embase and Web of Science was performed to identify suitable studies. Quality of the articles was scored using the Assessment Tool for Prevalence Studies. Data were combined using random effect models and the between-studies heterogeneity was assessed by the Cochrane's Q and I2. The sources of heterogeneity were investigated by meta-regression and sub-group analyses. Funnel plot, Begg's rank correlation and trim-and-fill test were used to assess publication bias.
The pooled prevalence of ED and DL in KS as well as 95% confidence intervals (CIs) were estimated from the proportion of cases of sexual dysfunction and the sample size. Variables that could affect the estimates were identified by linear meta-regression models.
Sixteen studies included collectively gave information about ED and DL in 482 and 368 KS men, respectively, resulting in a pooled prevalence of 28% (95% CI: 19%-36%) for ED and 51% (95% CI: 36%-66%) for DL, with a large heterogeneity. The trim-and-fill adjustment for publication bias produced a negligible effect on the pooled estimates. At the meta-regression analyses, a higher prevalence of ED was significantly associated with an older age but not with lower testosterone levels. In series with a mean age >35 years, the ED prevalence estimate increased up to 38% (95% CI: 31%-44%) with no heterogeneity (I2=0.0%, P=0.6). On the contrary, the prevalence of DL increased significantly as testosterone levels decreased, without a significant relationship with age.
While DL would largely reflect an androgen deficiency, in older men with KS, erectile function should be assessed irrespective of testosterone levels.
This is the first meta-analysis defining pooled prevalence estimates and correlates of ED and DL in KS. Nevertheless, caution is required when interpreting results, due to the high risk of bias in many studies, as well as the dearth of data about psychosocial and/or psychosexological variables and age at the diagnosis.
ED and DL represent common clinical complaints in KS. While the prevalence of ED would increase with age, DL gets more common as serum testosterone decreases. Further studies are warranted to elucidate the pathogenetic mechanism(s) underlying the age-dependent increase in the prevalence of ED, apparently unrelated to the androgenic status. A Barbonetti, S D'Andrea, W Vena, et al. Erectile Dysfunction and Decreased Libido in Klinefelter Syndrome: A Prevalence Meta-Analysis and Meta-Regression Study. J Sex Med 2021;18:1054-1064.
Male Endocrine and Genetic Factors · Male Hormonal Axis
Some evidence suggests that infertile men, who are at increased risk for hypogonadism, metabolic derangements, and osteoporosis, have higher long-term morbidity and mortality than controls, but data are scarce and not conclusive.
We tested whether semen quality and reproductive function could represent a marker of general male health.
DESIGN, SETTING, A retrospective study of 5177 individuals from a prospectively collected database of 11516 males of infertile couples who had semen analysis in a tertiary university center.
OUTCOME MEASUREMENTS Of them, 5177 had all data for reproductive hormones, testis ultrasound, and biochemical determinations for glucose and lipid metabolism. Hypogonadism was defined as testosterone <10.5nmol/l and/or luteinizing hormone >9.4 IU/l. Individuals with a total sperm count of <10 million had genetic testing (karyotype, Y chromosome microdeletions, and CFTR gene mutations) and those with hypogonadism underwent dual-energy x-ray absorptiometry for bone mineral density. Descriptive statistics and odds ratio (OR) calculation were used.
Men with a low sperm count (<39 million/ejaculate) are at a high risk of hypogonadism (OR 12.2, 95% confidence interval [CI] 10.2-14.6) and have higher body mass index, waist circumference, systolic pressure, low-density lipoprotein cholesterol, triglycerides, and homeostatic model assessment (HOMA) index; lower high-density lipoprotein cholesterol; and a higher prevalence of metabolic syndrome (OR 1.246, 95 CI 1.005-1.545). All data are worse in men with hypogonadism, but a low sperm count per se is associated with a poor metabolic parameter. Men with hypogonadism have lower bone mineral density and 51% prevalence of osteoporosis/osteopenia. Longitudinal studies are necessary to support these data.
This is the largest study with comprehensive evaluation of semen quality and reproductive function, etiology and risk factor determination, and metabolic, cardiovascular, and osteoporosis risk assessment, performed in men referred for fertility evaluation. A low sperm count is associated with poorer metabolic, cardiovascular, and bone health. Hypogonadism is mainly involved in this association, but a low sperm count in itself is a marker of general health.
This large study evaluated semen quality, reproductive function, and metabolic risk in men referred for fertility evaluation, and showed that a man's semen count is a marker of his general health. Men with low sperm counts are more likely than those with normal sperm counts to have greater body fat, higher blood pressure, higher "bad" (low-density lipoprotein) cholesterol and triglycerides, and lower "good" (high-density lipoprotein) cholesterol. They also have a higher frequency of metabolic syndrome and insulin resistance, a condition that can lead to diabetes. Men with low sperm counts had a 12-fold increased risk of hypogonadism or low testosterone levels, and half of them had osteoporosis or low bone mass. Fertility evaluation gives men the unique opportunity for health assessment and disease prevention.
Garolla A et al., 2021·Frontiers in endocrinology·Free full text on PubMed Central
Dietary supplements (DS) for male infertility marketed in Italy were evaluated for composition, concentration of ingredients, and recommended daily dose. A systematic review of literature identified ingredients potentially effective on sperm parameters and their minimal effective daily dose (mED).
This study was conducted in order to critically evaluate the composition and efficacy of DS marketed in Italy.
DESIGN This was a systematic review of randomized controlled trials.
A formula allowed us to classify the expected efficacy of each DS, based on composition. Each DS was scored and included into three classes of expected efficacy: high, low, and none.
Among 24 supplements, 3 (12.5%) fall in high, 9 (37.5%) in lower, and 12 (50.0%) in no expected efficacy class. DS composition showed 36 substances, 18 with no literature on male fertility and 18 showing positive effect on sperm parameters, thus considered potentially active ingredients (PAI). All DS were mixtures of ingredients, containing from 2 to 17 different substances. Fifteen supplements (65.2%) contained at least 1 ingredient without evidence of efficacy and 21 formulations had PAI dosed below mED. Some PAI were associated to the improvement of specific sperm parameters.
DS were usually blends of many substances that are frequently employed at negligible dose or without any evidence of efficacy on male reproduction. Some ingredients have been demonstrated to be effective on specific sperm parameters by RCTs. We report a list of ingredients with potential efficacy on specific sperm parameters, aimed to allow a tailored use of DS.
The market of DS for male infertility offers products with potential efficacy in the improvement of sperm parameters but also many with uncertain effects. Based on current scientific literature, our study can help in the choice of DS that are more likely to be effective on specific sperm alterations, so providing the best supplementation for each patient.
Rocca MS et al., 2021·Genes·Free full text on PubMed Central
Telomeres are considered to be an internal biological clock, and their progressive shortening has been associated with the risk of age-related diseases and reproductive alterations. Over recent years, an increasing number of studies have focused on the association between telomere length and fertility, identifying sperm telomere length (STL) as a novel biomarker of male fertility. Although typically considered to be repeated DNA sequences, telomeres have recently been shown to also include a long non-coding RNA (lncRNA) known as TERRA (telomeric repeat-containing RNAs). Interestingly, males with idiopathic infertility show reduced testicular TERRA expression, suggesting a link between TERRA and male fertility. The aim of this study was to investigate the role of seminal TERRA expression in embryo quality. To this end, STL and TERRA expression were quantified by Real Time qPCR in the semen of 35 men who underwent assisted reproductive technologies (ART) and 30 fertile men. We found that TERRA expression in semen and STL was reduced in patients that underwent ART (both p < 0.001). Interestingly, TERRA and STL expressions were positively correlated (p = 0.010), and TERRA expression was positively associated with embryo quality (p < 0.001). These preliminary findings suggest a role for TERRA in the maintenance of sperm telomere integrity during gametogenesis, and for the first time, TERRA expression was found as a predictive factor for embryo quality in the setting of assisted reproduction.
Rocca MS et al., 2020·Molecular genetics & genomic medicine·Free full text on PubMed Central
The androgen receptor (AR) is a nuclear receptor, encoded by the AR gene on the X chromosome. Within the first exon of the AR gene, two short tandem repeats (STR), CAG and GGC, are a source of polymorphism in the population. Therefore, high-throughput methods for screening AR, such as next-generation sequencing (NGS), are sought after; however, data generated by NGS are limited by the availability of bioinformatics tools. Here, we evaluated the accuracy of the bioinformatics tool HipSTR in detecting and quantify CAG repeats within the AR gene.
The AR gene of 228 infertile men was sequenced using NGSgene panel. Data generated were analyzed with HipSTR to detect CAG repeats. The accuracy was compared with the results obtained with Sanger.
We found that HipSTR was more accurate than Sanger in genotyping normal karyotype men (46,XY), however, it was more likely to misidentify homozygote genotypes in men with Klinefelter syndrome (47,XXY).
Our findings show that the bioinformatics tool HipSTR is 100% accurate in detecting and assessing AR CAG repeats in infertile men (46,XY) as well as in men with low-level mosaicism.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Rocca MS et al., 2020·Journal of assisted reproduction and genetics·Free full text on PubMed Central
To develop and assess a novel custom next-generation sequencing (NGS) panel for male infertility genetic diagnosis.
A total of 241 subjects with diagnosis of idiopathic infertility ranging from azoospermia to normozoospermia were sequenced by a custom NGS panel including AR, FSHB, FSHR, KLHL10, NR5A1, NANOS1, SEPT12, SYCP3, TEX11 genes. Variants with minor allele frequency < 1% were confirmed by Sanger sequencing.
Nineteen missense variants were detected in 23 subjects with abnormal sperm count, whilst no variants were identified in normozoospermic men. Of identified variants, we prioritized variants classified as pathogenic and of uncertain significance (VUS) (63.1%, 12/19). No missense variants were found in males with normal seminal parameters (0/67). Therefore, the prevalence of variants was significantly higher in patients with spermatogenic impairment (16/174 vs 0/67, p = 0.007).
This study confirms the utility to apply NGS panel for infertility diagnosis in order to find new genetic variants potentially linked to male infertility with much higher accuracy than standard tests suggested by guidelines. Indeed, based on biological significance, prevalence in the general population and clinical data of patients, it is plausible that identified variants in this study might be linked to quantitative spermatogenic impairment, although further studies are needed.
Flesia L et al., 2020·Sexual medicine·Free full text on PubMed Central
Young adult males are more likely to demonstrate health-risk behaviors than other individuals. The use of specific data about health-risk behaviors within this population might be important to promote effective preventive psychosocial and educational programs and interventions.
To provide a detailed description of health-related lifestyles, substance-related behaviors, and sexual habits that can negatively affect fertility, sexual sphere, and health in a large sample of Italian young adult males.
A sample comprising 2,170 males aged 18-21 years, attending the last year of high school, was administered an online questionnaire made up of 39 multiple-choice questions. The questionnaire explored health-related lifestyles, substance-related behaviors, and sexuality and sexual habits. Descriptive analyses were conducted.
The outcome measures included data about health-related lifestyles, substance-related behaviors, and sexuality and sexual habits reported by Italian young adult males.
Health-related lifestyles: 92.9% of the sample reported practicing some physical activity during the week. 90.3% declared a Mediterranean diet and 8.1% a hyperproteic diet. Substance-related behaviors: 33.8% of the sample reported having smoked tobacco at least once in their lives; among them, 71% reported current daily smoking. 40.2% declared drinking alcohol from 5 to 7 days in a week. 32.9% of the sample reported currently using a substance. Sexuality and sexual habits: 97.1% of the sample self-defined themselves as heterosexual. 73.3% of participants rated their knowledge about sexuality as "excellent/good," 58.7% about sexually transmitted infections. Only 4.8% reported having had a seminal liquid examination. Half of the sample (52.2%) declared having had sexual intercourses, in the largest proportion protected sex. 14.7% of the sample reported having at least one sexual dysfunction. 88.6% of participants reported having used pornography, 18.7% every day.
The present study highlighted the need to empower the number and efficacy of preventive interventions to promote health-related behaviors among Italian young male population. Flesia L, Cavalieri F, Angelini S, et al. Health-Related Lifestyles, Substance-Related Behaviors, and Sexual Habits Among Italian Young Adult Males: An Epidemiologic Study. Sex Med 2020;8:361-369.
Ferlin A et al., 2020·Journal of clinical medicine·Free full text on PubMed Central
About one-fifth of couples has fertility problems in Western countries. Male factors are present in about half of them, either alone or in combination with female causes. Therefore, both partners should be evaluated simultaneously. The fertility status and/or specific conditions of each partner influence the clinical and treatment approach. This article summarizes in a practical way when, how, and why the male partner of an infertile couple should be investigated. The available evidence and international guidelines were used, interpreting, discussing, and expanding them from personal decades-long experience in this field. The aim is to delineate the most appropriate clinical approach for the male partner of infertile couples, considering traditional and emerging technologies and laboratory analyses in the context of their clinical significance. Components of the initial evaluation in men without known risk factors for infertility should include at minimum medical history, physical examination, and semen analysis. Semen microbiological examination, endocrine assessment, scrotal ultrasound, and transrectal ultrasound are suggested in most men and are mandatory when specific risk factors for male infertility are known to be present or when the initial screening demonstrated abnormalities. Full examination, including genetic tests, testicular histology, or additional tests on sperm, is clinically oriented and/or suggested after the results of initial investigations.
Metabolic and Endocrine Agents · GLP-1 Receptor Agonists
Corona G et al., 2020·Reviews in endocrine & metabolic disorders
The association between diabetes mellitus (and its micro- and macro-vascular complications) and erectile dysfunction is widely known and the presence of hypogonadism may further complicate sexual dysfunction and quality of life, given the association between hypogonadism and reduced libido, ejaculatory disorders, and depressive symptoms. However, the recent introduction of novel antidiabetic agents with a wide range of mechanism of action may have a significant impact both on male and female sexuality directly (by inducing side effects as urinary tract infections) and indirectly (improving metabolic status and reducing diabetes complications behind sexual dysfunctions). To date only few papers are reporting the sexual effects of these treatments and, often, these are not comparable in their results. Conversely, female sexual dysfunctions are somehow under-investigated. Data on prevalence is heterogeneous and specific pathogenic mechanisms, as well as the burden of psychological factors, are still heatedly debated. The aim of this narrative review is to summarize current knowledge and stressing out the need to diagnose male and female sexual dysfunctions also in light of the impact of treatments with novel antidiabetic agents. This would highlight the still unmet needs for sexual care in a diabetes care setting and could represent an incentive for future discussions, as well as a required theoretical starting point for studies on this subject.
Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of β-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.
Garolla A et al., 2020·Front Endocrinol (Lausanne)·Free full text on PubMed Central
This expert opinion summarizes current knowledge on risk factors for infertility and identifies a practical clinical and diagnostic approach for the male and female partners of an infertile couple aimed to improve the investigation and management of fertility problems. Infertility represents an important and growing health problem affecting up to 16% of couples worldwide. In most cases, male, female, or combined factor can be identified, and different causes or risk factors have been related to this condition. However, there are no standardized guidelines on the clinical-diagnostic approach of infertile couples and the recommendations concerning infertility are sometimes lacking, incomplete, or problematic to apply. The aim of this work is to provide an appropriate clinical and diagnostic pathway for infertile couples designed by a multidisciplinary-team of experts. The rationale is based on the history and physical examination and then oriented on the basis of initial investigations. This approach could be applied in order to reduce variation in practice and to improve the investigation and management of fertility problems. Prominent Italian experts of the main specialties committed in the ART procedures, including gynecologists, andrologists, embryologists, biologists, geneticists, oncologists, and microbiologists, called "InfertilItaly group", used available evidence to develop this expert position. Starting from the individuation of the principal risk factors that may influence the fertility of females and males and both genders, the work group identified most appropriate procedures using a gradual approach to both partners aimed to obtain a precise diagnosis and the most effective therapeutic option, reducing invasive and occasionally redundant procedures. This expert position provides current knowledge on risk factors and suggests a diagnostic workflow of infertile couples. By using this step-by-step approach, health care workers involved in ART, may individuate a practical clinical management of infertile couples shared by experts.
Guidelines by Issuing Body · European and United Kingdom Bodies
Corona G et al., 2020·Journal of endocrinological investigation·Free full text on PubMed Central
The recent pandemic of severe acute respiratory syndrome (SARS) due to coronavirus (CoV) 2 (SARS-CoV-2) has raised several concerns in reproductive medicine. The aim of this review is to summarize available evidence providing an official position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) A comprehensive Pubmed, Web of Science, Embase, Medline and Cochrane library search was performed. Due to the limited evidence and the lack of studies, it was not possible to formulate recommendations according to the Oxford 2011 Levels of Evidence criteria.
Several molecular characteristics of the SARS-CoV-2 can justify the presence of virus within the testis and possible alterations of spermatogenesis and endocrine function. Orchitis has been reported as a possible complication of SARS-CoV infection, but similar findings have not been reported for SARS-CoV-2. Alternatively, the orchitis could be the result of a vasculitis as COVID-19 has been associated with abnormalities in coagulation and the segmental vascularization of the testis could account for an orchitis-like syndrome. Finally, available data do not support the presence of SARS-CoV-2 in plasma seminal fluid of infected subjects.
Data derived from other SARS-CoV infections suggest that in patients recovered from COVID-19, especially for those in reproductive age, andrological consultation and evaluation of gonadal function including semen analysis should be suggested. Studies in larger cohorts of currently infected subjects are warranted to confirm (or exclude) the presence of risks for male gametes that are destined either for cryopreservation in liquid nitrogen or for assisted reproduction techniques.
Pizzocaro A et al., 2020·Journal of endocrinological investigation
Low testosterone (T) in Klinefelter's syndrome (KS) can contribute to typical features of the syndrome such as reduced bone mineral density, obesity, metabolic disturbances and increased cardiovascular risk. The aim of the present study is to review and meta-analyze all available information regarding possible differences in metabolic and bone homeostasis profile between T treated (TRT) or untreated KS and age-matched controls.
We conducted a random effect meta-analysis considering all the available data from observational or randomized controlled studies comparing TRT-treated and untreated KS and age-matched controls. Data were derived from an extensive MEDLINE, Embase, and Cochrane search.
Out of 799 retrieved articles, 21 observational and 22 interventional studies were included in the study. Retrieved trials included 1144 KS subjects and 1284 healthy controls. Not-treated KS patients showed worse metabolic profiles (including higher fasting glycemia and HOMA index as well as reduced HDL-cholesterol and higher LDL-cholesterol) and body composition (higher body mass index and waist circumference) and reduced bone mineral density (BMD) when compared to age-matched controls. TRT in hypogonadal KS subjects was able to improve body composition and BMD at spinal levels but it was ineffective in ameliorating lipid and glycemic profile. Accordingly, TRT-treated KS subjects still present worse metabolic parameters when compared to age-matched controls.
TRT outcomes observed in KS regarding BMD, body composition and glyco-metabolic control, are similar to those observed in male with hypogonadism not related to KS. Moreover, body composition and BMD are better in treated than untreated hypogonadal KS. Larger and longer randomized placebo-controlled trials are advisable to better confirm the present data, mainly derived from observational studies.
Risk factors established during adolescence affect health outcomes in adulthood, although little is known about how adolescent health risk behaviours (HRBs) affect testicular development and reproductive health.
To assess prevalence of HRBs among last year high school students; to describe the most prevalent andrological disorders in this cohort; to explore HRBs associated with andrological disorders and investigate factors possibly associated with impaired testicular development in puberty.
The Amico-Andrologo Survey is a permanent nationwide surveillance programme conducted by the Italian Society of Andrology and Sexual Medicine and supported by the Ministry of Health. A nationally representative survey of final-year male high school students was conducted using a validated structured interview (n = 10124) and medical examination (n = 3816).
Smoking (32.6%), drinking (80.6%) and use of illegal drugs (46.5%) are common in adolescence. 16.6% of subjects were overweight, 3.1% were underweight and 2.3% were obese. Among sexually active students (60.3%), unprotected sex was very common (48.3%). Only 11.6% had been treated for andrological disorders, despite an abnormal clinical examination in 34.6%. Bilateral testicular hypotrophy (14.0%), varicocoele (27.1%) and phimosis (7.1%) were the most prevalent disorders; 5.1% complained of premature ejaculation and 4.7% had an STI. Underweight and heavy alcohol or drug use were associated with testicular hypotrophy. HRBs emerged as significant predictors of testicular hypotrophy, explaining up to 9.6% of its variance. Limitations include risk of selection bias for voluntary physical examination and recall bias for the self-compiled questionnaire.
There is an emerging global adverse trend of HRBs in male high school students. A significant proportion of adolescent males with unsuspected andrological disorders engage in behaviours that could impair testicular development.
Greater attention to the prevention of andrological health in adolescence is needed.
Klinefelter syndrome is a frequent cause of hypogonadism, but despite hundreds of publications on different aspects of Klinefelter syndrome, only a few studies dealt with sexual dysfunction. In particular, testosterone is critical for various aspects of sexual response, but its role on sexuality in Klinefelter syndrome patients is debatable and no studies have evaluated the efficacy of testosterone treatment on sexual dysfunction in these subjects. Furthermore, the impact of psychological and relational aspects on sexual function of Klinefelter syndrome subjects is poorly defined. In this study, we aimed to determine the presence and type of sexual dysfunctions in Klinefelter syndrome subjects; to correlate them with testosterone levels and psychosexological and relational domains; and to evaluate the effects of testosterone therapy. We studied 62 non-mosaic naïve Klinefelter syndrome patients and 60 age-matched controls by means of medical history, psychosexological history, 15-item International Index of Erectile Function questionnaire, endocrine assessment, and dynamic penile color Doppler ultrasound. Twenty-five hypogonadal Klinefelter syndrome patients were studied after 6 months of testosterone replacement therapy. Klinefelter syndrome subjects have reduced 15-item International Index of Erectile Function scores regarding sexual desire, intercourse satisfaction, and overall satisfaction with respect to controls, and these aspects were significantly associated with testosterone levels. Klinefelter syndrome subjects had also higher prevalence of erectile dysfunction, but no relation with testosterone levels was evident. A high prevalence of a range of psychological disturbances was present in Klinefelter syndrome subjects with erectile dysfunction with respect to those without erectile dysfunction. No statistical difference in the prevalence of premature and delayed ejaculation was observed between Klinefelter syndrome and control subjects. Testosterone replacement therapy improved sexual desire, intercourse satisfaction, and overall satisfaction scores, but had no effect on erectile function. Penile color Doppler ultrasound was normal in all subjects. This study shows that sexual dysfunction in Klinefelter syndrome is multifactorial and related only in part to hypogonadism and largely to psychological disturbances. Evaluation and therapy of sexual dysfunction should include a combined andrological and psychosexological approach.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
To investigate whether sperm recovery is related to clinical features, hormone parameters and testosterone replacement therapy (TRT) in patients with Klinefelter syndrome (KS).
This study provides three interesting insights: (i) the probability to retrieve sperm is not related to testicular volume; (ii) TRT does not affect sperm retrieval rate (SRR); and (iii) reduced levels of LH and FSH represent a negative predictor of sperm retrieval in patients with TRT.
Classical KS shows a karyotype with one extra X chromosome in all of somatic cells and clinical manifestations characterized by hypergonadotropic hypogonadism and infertility.
STUDY DESIGN, We performed a retrospective cohort study. Data from 111 consecutive KS azoospermic patients undergoing testicular sperm extraction (TESE) were collected from 2005 to 2016.
PARTICIPANTS/MATERIALS, Data on anthropometric parameters, reproductive hormones and testicular volumes were collected. SRR was related to clinical characteristics and compared between TRT and untreated patients.
MAIN A total of 38 patients (34.2%) had successful sperm recovery. The comparison of clinical characteristics did not differ between patients with and without sperm recovery. Sperm retrieval was successful also in subjects with smaller testes. The comparison of SRR in patients with or without TRT was not different (33.3% vs 34.6%). In TRT group, LH and FSH levels were significantly lower in subjects with no sperm retrieval (P values, respectively, <.05 and <.001).
LIMITATIONS Well-designed controlled studies are necessary to confirm these data aimed to set the best therapeutic approach for fertility management in hypogonadal patients with nonmosaic KS.
Age at TESE, anthropometric measures, testis volume, sex hormones levels and semen parameters are not predictive parameters of SRR. Among TRT patients, reduced gonadotropin is related to failure in sperm retrieval.
The association between inflammation of the male reproductive system and oligozoospermia has been frequently reported in the clinical work-up of male infertility. To improve sperm parameters in infertile patients with genital inflammation, many phytochemical and nutraceutical drugs are currently being used. However, their use is still empirical and no conclusive data have been provided about their efficacy. The treatment with steroid anti-inflammatory drugs might be useful in reducing inflammation and improving sperm parameters, thus increasing the fertility outcome. The aim of this study was to evaluate if glucocorticoid treatment improves seminal parameters in infertile oligozoospermic patients presenting signs of accessory gland inflammation at genital ultrasound. A total of 90 infertile patients were enrolled in the study. They presented normal testicular volume, normal FSH plasma levels, the presence of various degrees of oligozoospermia, associated with scrotal and trans-rectal ultrasound signs indicative of accessory gland inflammation, but negative microbiological analysis on semen and/or prostatic secretions. Patients were randomly allocated into three groups of treatment, receiving, respectively, 5, 12.5, and 25 mg daily oral Prednisone for one month. Seminal parameters were evaluated at admission and after treatment. In patients undergoing Prednisone treatment at a daily dose of 5 mg we observed a significant increase in total sperm count. At a daily dose of 12.5 mg, Prednisone treatment improved sperm concentration, total sperm count, and the percentage of sperm motility. Twenty-five mg of Prednisone led to significant improvement in all the sperm parameters, except for semen volume. These results clearly demonstrate that Prednisone treatment can significantly improve sperm parameters in a selected population of oligozoospermic patients. These findings suggest that Prednisone treatment should be considered in idiopathic oligozoospermic patients with supposed normal spermatogenesis and accessory gland inflammatory alterations, in order to improve sperm parameters and fertility outcome.
Male Reproductive Surgery · Sperm Retrieval Procedures
Specific factors underlying successful surgical sperm retrieval rates (SRR) or pregnancy rates (PR) after testicular sperm extraction (TESE) in adult patients with Klinefelter syndrome (KS) have not been completely clarified.
The aim of this review was to meta-analyse the currently available data from subjects with KS regarding SRRs as the primary outcome. In addition, when available, PRs and live birth rates (LBRs) after the ICSI technique were also investigated as secondary outcomes.
An extensive Medline, Embase and Cochrane search was performed. All trials reporting SRR for conventional-TESE (cTESE) or micro-TESE (mTESE) and its specific determinants without any arbitrary restriction were included.
Out of 139 studies, 37 trials were included in the study, enrolling a total of 1248 patients with a mean age of 30.9 ± 5.6 years. The majority of the studies (n = 18) applied mTESE, 13 applied cTESE and in one case testicular sperm aspiration (TESA) was used. Additionally, four studies used a mixed approach and in one study, the method applied for sperm retrieval was not specified. Overall, a SRR per TESE cycle of 44[39;48]% was detected. Similar results were observed when mTESE was compared to cTESE (SRR 43[35;50]% vs 45[38;52]% for cTESE vs micro-TESE, respectively; Q = 0.20, P = 0.65). Meta-regression analysis showed that none of the parameters tested, including age, testis volume and FSH, LH and testosterone (T) levels at enrollment, affected the final SRR. Similarly, no difference was observed when a bilateral procedure was compared to a unilateral approach. No sufficient data were available to evaluate the effect of previous T treatment on SRR. Information on fertility outcome after ICSI was available for 29 studies. Overall a total of 218 biochemical pregnancies after 410 ICSI cycles were observed (PR = 43[36;50]%). Similar results were observed when LBR was analyzed (LBR = 43[34;53]%). Similar to what was observed for SRR, no influence of KS age, mean testis volume, LH, FSH or total T levels on either PR and LBR was observed. No sufficient data were available to test the effect of the women's age or other female fertility problems on PR and LBR. Finally, no difference in PR or LBR was observed when the use of fresh sperm was compared to the utilization of cryopreserved sperm.
The present data suggest that performing TESE/micro-TESE in subjects with KS results in SRRs of close to 50%, and then PRs and LBRs of close to 50%, with the results being independent of any clinical or biochemical parameters tested.
Andrologists, cardiologists and diabetologists (and general practitioners) have the great opportunity to collaborate and find shared clinical workup for the benefit of a large number of men. Several evidence established a link between erectile dysfunction (ED), cardiovascular disease (CVD), diabetes, and metabolic syndrome. Not only these conditions share many risk factors and pathophysiological mechanisms but also an emerging paradigm indicates that ED is, in fact, an independent marker of cardiovascular disease risk, CV events and CV mortality. However, there is no consensus on the best cardiologic investigation in men with ED with no known CVD and, on the contrary, on what is the clinical and prognostic role of detecting ED during cardiovascular investigation and CVD risk assessment. Only vasculogenic ED, which represents the most common type of organic ED, indeed represents a harbinger of CVD, especially for younger patients, and might be diagnosed by dynamic penile color doppler ultrasonography, which represents a real cardiovascular imaging technique that give evidence on the presence of systemic endothelial dysfunction and atherosclerosis. Furthermore, assessment of glucose and lipid metabolism is warranted as first step workup in all ED patients, and diabetologists should ask their patients for erectile function, address ED patients to andrologists, and consider vasculogenic ED in the context of the cardiovascular and metabolic workup and in the context of diabetic complications. Sexual symptoms (and testosterone levels) should sound as harbinger for cardiovascular and metabolic investigation and cardiologists and diabetologists have the opportunity to have a symptom (erectile dysfunction) and a vascular test (penile color doppler) that help them in better management of patients, their comorbidities and complications.
Nutrition and Metabolic Health · Blood Sugar and Insulin
Diabetes is an established risk factor for erectile dysfunction (ED). The pathophysiology of ED in diabetic men is multifactorial, but it mainly involves a vascular disorder related to a reduction of endothelial function. Recently, several studies have correlated ED risk factors with vitamin D deficiency. In this study, we evaluate the relationship between 25-hydroxyvitamin D [25(OH)D] levels, erectile dysfunction, and vascular disease, in type 2 diabetes mellitus men (T2DM). In this observational study, 92 T2DM males (58.83 ± 9.73 years) underwent medical history collection, International Index of Erectile Function (IIEF-5) questionnaire, that allows the identification and grading of DE, physical examination, biochemical/hormonal blood tests, and penile echo-color Doppler ultrasonography. T2DM patients with lower 25(OH)D levels (<25 nmol/l) showed higher penile IMT (p < 0.05), waist circonference (p < 0.05), glucose concentrations (p < 0.05), and lower IIEF-5 score (p < 0.005), testosterone concentrations (p < 0.05), and cavernous peak systolic velocity (PSV) (p < 0.05), compared to patients with 25(OH)D >50 nmol/l. 25(OH)D levels were directly correlated with IIEF-5 (R = 0.39; p = 0.0001), testosterone (R = 0.24; p = 0.02), and PSV (R = 0.24; p = 0.04) and inversely with waist (R = -0.33; p = 0.002), HbA1c (R = -0.22; p = 0.03), triglyceride (R = -0.21; p = 0.06), and penile IMT (R = -0.30; p = 0.009). At multivariate analysis, 25(OH)D deficiency remained an independent predictor of DE. We demonstrate a significant association between 25(OH)D deficiency and erectile dysfunction in T2DM men. This association may be due to the influence of 25(OH)D deficiency on cardiovascular risk factor (glycaemia, HDL cholesterol, and triglycerides), testosterone plasma levels and endothelial dysfunction.
Querin G et al., 2016·Journal of neurology, neurosurgery, and psychiatry·Free full text on PubMed Central
To carry out a deep characterisation of the main androgen-responsive tissues involved in spinal and bulbar muscular atrophy (SBMA).
73 consecutive Italian patients underwent a full clinical protocol including biochemical and hormonal analyses, genitourinary examination, bone metabolism and densitometry, cardiological evaluation and muscle pathology.
Creatine kinase levels were slightly to markedly elevated in almost all cases (68 of the 73; 94%). 30 (41%) patients had fasting glucose above the reference limit, and many patients had total cholesterol (40; 54.7%), low-density lipoproteins cholesterol (29; 39.7%) and triglyceride (35; 48%) levels above the recommended values. Although testosterone, luteinising hormone and follicle-stimulating hormone values were generally normal, in one-third of cases we calculated an increased Androgen Sensitivity Index reflecting the presence of androgen resistance in these patients. According to the International Prostate Symptom Score (IPSS), 7/70 (10%) patients reported severe lower urinal tract symptoms (IPSS score >19), and 21/73 (30%) patients were moderately symptomatic (IPSS score from 8 to 19). In addition, 3 patients were carriers of an indwelling bladder catheter. Videourodynamic evaluation indicated that 4 of the 7 patients reporting severe urinary symptoms had an overt prostate-unrelated bladder outlet obstruction. Dual-energy X-ray absorptiometry scan data were consistent with low bone mass in 25/61 (41%) patients. Low bone mass was more frequent at the femoral than at the lumbar level. Skeletal muscle biopsy was carried out in 20 patients and myogenic changes in addition to the neurogenic atrophy were mostly observed.
Our study provides evidence of a wide non-neural clinical phenotype in SBMA, suggesting the need for comprehensive multidisciplinary protocols for these patients.
De Toni L et al., 2016·The Journal of clinical endocrinology and metabolism
The G protein-coupled receptor GPRC6A is an emerging effector with multiple endocrine roles, including stimulation of T production from the testis. Recently, two men with an inactivating mutation (F464Y) of GPRC6A have been identified, and they showed primary testicular failure and deranged spermatogenesis. Furthermore, one of them also reported cryptorchidism at birth. In addition, a polymorphism (rs2274911, Pro91Ser) in GPRC6A is associated with prostate cancer, a typical androgen-sensitive cancer.
To study the possible association between rs2274911 polymorphism and male fertility and/or cryptorchidism. Design, Patients, Settings: A total of 611 subjects, including 343 infertile patients, 197 normozoospermic controls, and 71 cryptorchid newborns, were retrospectively selected.
Sequencing analysis for rs2274911 polymorphism and F464Y mutation, and serum levels of FSH, LH, and T were assessed. In vitro functional studies for rs2274911 and F464Y were also performed.
Homozygous subjects for the risk allele A of rs2274911 had a 4.60-fold increased risk of oligozoospermia and 3.52-fold increased risk of cryptorchidism. A significant trend for increased levels of LH in the GA and AA genotypes, compared with GG homozygotes, was detected in men with azoospermia/cryptozoospermia (P for trend = .027), further supporting an association with primary testicular failure. The mutation F464Y was found in one cryptorchid child (one in 71; 1.41%). Functional studies showed that the A allele of rs2274911 and the F464Y substitution were associated with lower exposition of the receptor on the cell membrane and a reduced downstream phosphorylation of ERK1/2 with respect to wild type.
Our results suggest that GPRC6A inactivation or sub-function contributes to reduced exposure to androgens, leading to cryptorchidism during fetal life and/or low sperm production in adulthood.
Rocca MS et al., 2016·Human reproduction (Oxford, England)
Could sperm telomere length (STL) represent a novel parameter and biomarker of sperm quality?
STL is associated with standard semen quality parameters and, more importantly, it is significantly associated with levels of DNA fragmentation and sperm protamination.
Telomeres are fundamental for genome integrity. Recent studies have demonstrated that STL increases with age and men with oligozoospermia have shorter sperm telomeres than normozoospermic men.
STUDY DESIGN, SIZE, Cohort study conducted from September 2014 to June 2015 on 100 subjects with normal standard semen parameters.
PARTICIPANTS/MATERIALS, SETTING, STL was measured indirectly by quantitative polymerase chain reaction using telomere/single-copy gene ratio, sperm DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and protamination by aniline blue staining. Data were analyzed for determining the relationships between STL, standard semen parameters and DNA fragmentation and protamination.
MAIN Among standard semen parameters, STL was positively associated with progressive motility (P = 0.004) and vitality (P = 0.007). STL was significantly and negatively associated with sperm DNA fragmentation (P = 0.001) and significantly and positively associated with protamination (P = 0.002). The role of chance was limited and the findings have biological relevance and a pathophysiological explanation.
LIMITATIONS, For the present study, we deliberately selected only men with normozoospermia to better analyze whether STL might represent a biomarker of sperm quality beyond traditional sperm parameters. Additional studies in proven fertile men with normal sperm parameters are needed.
The measurement of STL is a simple and rapid method that offers further information about the quality of sperm. The results of this study demonstrate that STL could be considered as an additional sperm parameter and opens new perspectives in the evaluation of the infertile male. Additional studies will clarify the significance of this parameter also as a prognostic biomarker in assisted reproduction.
No external funding was either sought or obtained for this study. There are no conflicts of interest to be declared.
Hormones and Aging Tissues · Musculoskeletal Aging
Foresta C et al., 2015·Asian journal of andrology·Free full text on PubMed Central
Late-onset hypogonadism is defined as a combination of low testosterone (T) levels and typical symptoms and signs. A major area of uncertainty is whether T concentrations are always really sufficient to fully reflect Leydig cell (dys)function. Mild testicular alteration could be diagnosed only by additional biochemical markers, such as luteinizing hormone (LH) and 25-hydroxyvitamin D levels. These markers help in identifying the so-called "subclinical" hypogonadism (normal T, high LH levels). Patients with hypogonadism have frequently low levels of 25-hydroxyvitamin D due to impairment of the hydroxylating enzyme CYP2R1 in the testis. However, no data have been published dealing with the best treatment option (cholecalciferol - the Vitamin D precursor, or calcidiol - 25-hydroxylated form of Vitamin D) in these patients. We studied 66 patients with classic hypogonadism (total T [TT] <12 nmol l-1 , LH ≥ 8 IU l-1 ) (n = 26) and subclinical hypogonadism (TT ≥ 12 nmol l-1 , LH ≥ 8 IU l-1 ) (n = 40) and low 25-hydroxyvitamin D (<50 nmol l-1 ). Subjects received cholecalciferol (5000 IU per week) (n = 20) or calcidiol (4000 IU per week) (n = 46), and 25-hydroxyvitamin D and parathyroid hormone (PTH) were evaluated after 3 months of therapy. Supplementation with calcidiol significantly increased 25-hydroxyvitamin D and significantly decreased PTH levels in both groups of men with hypogonadism (primary, n = 16 and subclinical, n = 30), whereas supplementation with cholecalciferol did not modify their levels. This study shows for the first time that the administration of the 25-hydroxylated form of Vitamin D (calcidiol), and not the administration of the precursor cholecalciferol, restores 25-hydroxyvitamin D levels in subjects with hypogonadism.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Garolla A et al., 2015·Human reproduction (Oxford, England)
Is there a difference between molecular karyotype of single sperm selected by high-magnification microscopy from infertile patients with testicular damage and from proven fertile controls?
The molecular karyotype of single sperm from patients with testiculopathy had a significantly higher percentage of chromosomal alterations than fertile controls.
Infertile patients with testicular impairment have many sperm with aneuploidies and/or increased structural chromosome alterations. In these patients, sperm use by ICSI has poor outcome and raises concerns about the possible impact on pregnancy loss and transmission of genes abnormalities in offspring. High-magnification microscopy has been recently introduced to select morphologically better sperm aimed at improving ICSI outcome. However, there are no studies evaluating the molecular karyotype of sperm selected by this method.
STUDY DESIGN, SIZE, Three consecutive infertile patients with oligozoospermia due to testicular damage and three age-matched proven fertile men attending a tertiary care center, were enrolled in the study from September to November 2014. Inclusion criteria of patients were age ≥30 ≤35 years, at least 2 years of infertility, oligozoospermia (sperm count below 10 million), reduced testicular volumes high FSH plasma levels and absence of altered karyotype, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator gene mutations, sperm infections, cigarette smoking, varicocele, obesity.
PARTICIPANTS/MATERIALS, SETTING, Participants were evaluated for sperm parameters, sex hormones and testicular color-doppler ultrasound. From each semen sample, 20 sperm with large vacuoles (LVs), 20 with small vacuoles (SVs) and 20 with no vacuoles (NVs) were retrieved individually by a micromanipulator system. Each cell was further analyzed by whole genome amplification and array comparative genomic hybridization (aCGH).
MAIN The aCGH allowed us to detect chromosomal aneuploidies, unbalanced translocations and complex abnormalities. Sperm selected from infertile patients showed a higher percentage of abnormal molecular karyotypes than controls (19.4 versus 7.7%, respectively, P < 0.001). In particular, sperm with LV and SV showed 38.3 and 20.0% abnormal karyotype in infertile men versus 18.3 and 5.0% in controls, respectively (both P < 0.01). Complex abnormalities were found only in the LV category. An abnormal karyotype was never found in NV sperm from both patients and controls.
LIMITATIONS The main limitation of this study is the low number of included subjects. Moreover, a time of writing we have no data regarding the ICSI outcome using LV, SV or NV sperm. This is the first study evaluating the molecular karyotype of single sperm selected by high-magnification microscopy and further confirmation of the data is needed.
Our data showed that sperm from infertile patients with testicular impairment have a higher percentage of abnormal molecular karyotypes than sperm from fertile controls. Therefore, if confirmed, our data suggest that the use of individually retrieved NV sperm may improve ICSI outcome in infertile men with testicular damage.
To study the role of NR5A1 in cryptorchidism and male factor infertility. Mutations in NR5A1 have been initially associated with primary adrenal insufficiency and 46,XY gonadal dysgenesis and more recently with less severe phenotypes, including preliminary descriptions in severe forms of male factor infertility. Far less clear is the possible involvement of NR5A1 mutations in cryptorchidism.
Retrospective cross-sectional cohort study and functional analysis of mutant proteins.
University department.
PATIENT(S): Nine hundred fifty-nine subjects, including children with cryptorchidism and adults with different semen phenotypes associated or not associated with a history of cryptorchidism.
INTERVENTION(S): None.
MAIN OUTCOME MEASURE(S): Mutation screening of NR5A1 by sequencing all exons. Functional analysis of mutant proteins by transactivation assays of CYP11A1 and CYP17A1 promoters.
RESULT(S): We identified seven undescribed and one previously described missense mutation in subjects with severe spermatogenic impairment, without (4/236, 1.7%) and with (3/85, 3.5%) a history of cryptorchidism. Newborns with cryptorchidism carry NR5A1 mutations at low frequency (0.7%), whereas no mutations were found in milder forms of infertility and normozoospermia, irrespective of the presence of cryptorchidism. The mutant proteins showed impaired transactivation of gonadal promoters. A single nucleotide polymorphism (rs1110061; c.437 G→C; p.Gly146Ala) was also associated with more severe forms of spermatogenic impairment with cryptorchidism.
CONCLUSION(S): This study, combined with what is already known about NR5A1-associated phenotypes, suggests considering mutations in this gene as a novel genetic cause of more severe forms of male factor infertility, especially when associated with a history of cryptorchidism.
Ferlin A et al., 2015·Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA
This manuscript describes the role of low vitamin D in bone metabolism of Klinefelter subjects. Low vitamin D is frequent in this condition and seems to be more important than testosterone in inducing low bone mineral density (BMD) and osteoporosis. Supplementation with vitamin D restores BMD after 2 years of treatment, whereas testosterone alone seems to be ineffective.
Decreased bone mineral density (BMD) in Klinefelter syndrome (KS) is frequent, and it has been traditionally related to low testosterone (T) levels. However, low BMD can be observed also in patients with normal T levels and T replacement therapy does not necessarily increase bone mass in these patients. Nothing is known about vitamin D levels and supplementation in KS. In this study, we determine vitamin D status and bone mass in KS subjects and compare the efficacy of T therapy and vitamin D supplementation on BMD.
A total of 127 non-mosaic KS patients and 60 age-matched male controls were evaluated with reproductive hormones, 25-hydroxyvitamin D, PTH, and bone densitometry by dual-energy X-ray absorptiometry (DEXA). Patients with hypogonadism and/or 25-hydroxyvitamin D deficiency were treated with T-gel 2% and/or calcifediol and re-evaluated after 24 months of treatment.
25-hydroxyvitamin D levels were significantly lower in KS patients with respect to controls, and they had significantly lower lumbar and femoral BMD. The percentage of osteopenia/osteoporosis in subjects with 25-hydroxyvitamin D deficiency was higher with respect to subjects with normal 25-hydroxyvitamin D and was not related to the presence/absence of low T levels. Subjects treated with calcifediol or T + calcifediol had a significant increase in lumbar BMD after treatment. No difference was found in T-treated group.
These data highlight that low 25-hydroxyvitamin D levels seem to have a more critical role than low T levels in inducing low BMD in KS subjects. Furthermore, vitamin D supplementation seems to be more effective than T replacement therapy alone in increasing BMD.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Ferlin A et al., 2014·Current opinion in obstetrics & gynecology
The purpose of this review is to highlight the most important advances in the field of genetics of male infertility, with particular attention to primary articles dealing with the identification of new genetic and epigenetic markers that could be translated into clinical practice in the near future.
Copy number variations (CNVs) of the Y chromosome (gr/gr) deletions could already be included in the diagnostic workup of infertile men, although confirming studies are needed for CNVs on the X chromosome, as well for polymorphisms in some autosomal genes and telomere length in sperm. Methods need to be further standardized before sperm DNA analysis could be included in clinical practice, although they can help in defining some forms of idiopathic infertility. Epigenetic biomarkers are potentially important in elucidating the cause of idiopathic male infertility. Polymorphisms in FSHB/FSHR could be used in clinical practice to diagnose some forms of male infertility and as a pharmacogenetic marker for FSH treatment.
New genetic causes and genetic risk factors have been identified in recent years and new technologies for genomic and postgenomic analyses (arrays, next-generation sequencing, proteomics, metabolomics, global methylome analysis and so on) are promising research fields. It is presumed that some of these genetic and epigenetic tests will be introduced in clinical practice in the near future.
Selice R et al., 2013·European journal of endocrinology
Klinefelter syndrome (KS) is a chromosomal alteration characterized by increased risk of metabolic syndrome, mainly caused by visceral obesity. In the last years, obesity has been studied as a potential risk factor for prostate disease and recently a link has been demonstrated between visceral adiposity with prostate volume. The aim of this study was to analyze the relationship between obesity and prostate volume and growth during testosterone therapy in KS subjects.
We evaluated reproductive hormones, metabolic parameters, anthropometric measures, PSA, and prostate volume in 121 naïve non-mosaic KS patients and 60 age-matched healthy male controls. Fifty-six KS hypogonadic subjects were treated with testosterone-gel 2% and reevaluated after 18 months of treatment.
Prostate volume in KS was positively related to waist circumference (WC). The KS group with WC ≥94 cm had significantly higher prostate volume, BMI, insulin plasma levels, homeostasis model assessment index, total cholesterol, triglycerides, and glycemia with respect to the KS group with WC <94 cm. After testosterone replacement therapy, only hypogonadic KS men with WC ≥94 cm had a statistically significant increase in prostate volume. Furthermore, in untreated KS subjects, prostate volume showed a statistically significant increase after 18 months of follow-up only in subjects with WC ≥94 cm.
This study showed that visceral obesity, insulin resistance, and lipid and glucose metabolism alterations are associated with prostate volume and growth during testosterone replacement therapy in KS, independently from androgen or estrogen levels. These latter findings might provide the basis for a better management and follow-up of KS subjects.
Garolla A et al., 2012·Frontiers in endocrinology·Free full text on PubMed Central
Testicular cancer represents the more frequent solid tumor affecting males aged 15-35 years. In the last decades, its incidence showed a progressive increased probably due to genetic and environmental factors. Despite exposure to some viruses such as HIV, HCV, EBV, and HPV is frequently related to cancer development, there are no studies aimed to evaluate the possible implication of viral infections in the pathogenesis of testicular cancer. In this study, we analyzed sperm parameters and prevalence of HPV on sperm in 155 testicular cancer patients at diagnosis (T-1), after orchiectomy (T0) and after 12 months from surgery or from the end of adjuvant treatments (T12). All patients showed a significantly higher prevalence of semen infection than controls (9.5% and 2.4% respectively,) and altered sperm parameters both at T-1 and T0. Considering sperm parameters, at T-1 we observed a reduction of progressive motility, and after orchiectomy patients showed a reduction of sperm concentration and count and a further worsening of motility. Thereafter, patients were assigned to three groups on the basis of medical option after surgery: S = surveillance, R = radiotherapy, and C = chemotherapy +/- radiotherapy. At T12, untreated patients had an improvement of sperm parameters while R group and even more C group had a strong decrease of sperm number (p < 0.01 both vs. T0 and S group). Moreover, patients who received radio and/or chemotherapy had a very high prevalence of HPV semen infection (S = 7.7%, R = 30.8%, and C = 61.5%). In conclusion, patients with testicular cancer had frequently altered sperm parameters and higher prevalence of HPV semen infection that were worsened after radio and chemotherapy. Because HPV infection is a risk factor for cancer development and it may further reduce fertility, we suggest screening for HPV in testicular cancer patients at diagnosis and particularly after adjuvant treatments.
Ferlin A et al., 2011·The Journal of clinical endocrinology and metabolism
Klinefelter syndrome (KS) is a chromosomal alteration characterized by supernumerary X-chromosome(s), primary hypogonadism, decreased pubertal peak bone mineral density (BMD), and accelerated bone loss during adulthood. Decreased bone mass has been traditionally related to low testosterone levels. However, testosterone replacement therapy does not necessarily increase bone mass in these patients, and low BMD can be observed also in patients with normal testosterone levels. The androgen receptor (AR) gene CAG polymorphism seems to modulate the sensitivity to testosterone and previous studies have related it to some clinical aspects of KS, to include BMD, gynecomastia, testes and prostate volume, and hemoglobin concentration.
To analyze the relation between bone mass, testosterone, and AR CAG polymorphism in men with KS.
Cross-sectional cohort study.
University department.
One hundred twelve consecutive treatment-naïve 47,XXY Klinefelter patients (mean age 33.5 ± 4.7 yr) and 51 age-matched normal male controls.
Dual-energy x-ray absorptiometry, CAG repeat length polymorphism, X-chromosome inactivation, and testosterone levels.
Forty-nine of 112 KS subjects (42.5%) had low bone mass (osteopenia or osteoporosis). Lumbar and/or femoral T-scores were lower in KS patients compared with controls. No significant relationship was observed between testosterone levels and bone parameters, and the prevalence of osteopenia/osteoporosis was similar in subjects with normal and low testosterone levels (43.7% and 40.5%, respectively). The mean CAG repeat length calculated after X-chromosome inactivation analysis showed no differences between patients with normal and low bone mass.
Testosterone levels and AR CAG polymorphism are not associated with bone mass phenotype in KS.
Foresta C et al., 2011·PloS one·Free full text on PubMed Central
Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases in females and males. Precise data about the presence, mechanism of infection and clinical significance of HPV in the male reproductive tract and especially in sperm are not available. Here we show that HPV can infect human sperm, it localizes at the equatorial region of sperm head through interaction between the HPV capsid protein L1 and syndecan-1. Sperm transfected with HPV E6/E7 genes and sperm exposed to HPV L1 capsid protein are capable to penetrate the oocyte and transfer the virus into oocytes, in which viral genes are then activated and transcribed. These data show that sperm might function as vectors for HPV transfer into the oocytes, and open new perspectives on the role of HPV infection in males and are particularly intriguing in relation to assisted reproduction techniques.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Selice R et al., 2011·International journal of andrology
In the last years, follice-stimulating hormone (FSH) receptor (FSHR) gene polymorphisms have been studied as potential risk factors for spermatogenetic failure. In this study, we have evaluated the response of FSH treatment in terms of sperm production on the basis of Ala307Thr-Asn680Ser polymorphisms in the FSHR gene in a group of oligozoospermic subjects with hypospermatogenesis and normal FSH levels. Patients were randomized into two groups: 70 treated with recombinant FSH (150 IU thrice per week for 3months) and 35 without treatment. After 3months of treatment, we observed significant increase in total sperm count, sperm concentration, forward motility, percentage of normal morphology forms and total motile sperm. When 70 treated subjects were subdivided based on FSHR genotype, only subjects with at least one serine in position 680 showed a statistically significant increase in these sperm parameters, whereas subjects with homozygote Thr307-Asn680 showed no difference in any seminal parameters evaluated. Non-treated subjects showed no differences in any parameter evaluated. This study suggests that the analysis of this gene represents a valid pharmacogenetic approach to the treatment of male infertility, confirming also the importance of strict criteria for the selection of patients to be treated with FSH.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
To verify in another population (Italians) whether a single-nucleotide polymorphism in the FSHB gene promoter previously associated with serum FSH levels in Estonians is indeed associated with sperm count and FSH plasma levels, and especially to verify whether it could be a pharmacogenetic tool for the treatment of male infertility with FSH.
Cross-sectional and prospective study.
Infertility center at a university hospital.
PATIENT(S): Five hundred fourteen subjects with nonobstructive azoospermia and oligozoospermia and 248 subjects with normozoospermia.
INTERVENTION(S): None.
MAIN OUTCOME MEASURE(S): Semen parameters, reproductive hormone levels, and FSHB -211 G/T polymorphism (rs10835638).
RESULT(S): FSHB -211 TT genotype was associated with significantly lower FSH levels (mean ± SD: 3.3 ± 2.5 IU/L vs. 9.1 ± 8.9 IU/L in GG homozygotes). TT homozygotes were seen in 25% of subjects with azoo-oligozoospermia and low FSH levels (≤1.5 IU/L). We did not observe this genotype in men with high FSH levels (>8 IU/L) or in men with normozoospermia. Treatment with FSH induced a significantly higher improvement in sperm count and quality in TT homozygotes regarding carriers of the G allele.
CONCLUSION(S): FSHB -211 TT genotype might represent a novel treatable form of male infertility characterized by severe spermatogenic impairment and low or inappropriately normal FSH plasma levels. This genetic marker could represent a valid pharmacogenetic approach for identification of potential responders to FSH treatment.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Selice R et al., 2010·Journal of endocrinological investigation
Klinefelter syndrome (KS) (47,XXY) is the most common sex chromosomal disorder, and it is a frequent form of male hypogonadism and infertility. Although the majority of these patients are azoospermic, they might have severe oligozoospermia or residual single-residual foci with spermatogenesis in the testis.
We report our experience on sperm retrieval in the ejaculate and testis, and evaluate the frequency of chromosome abnormalities in sperm of KS.
Eighty-four 47,XXY KS were evaluated with seminal analysis, body hair distribution, reproductive hormones, ultrasonographic scanning of the testis and prostate, bilateral testicular sperm extraction (TESE), sperm or testicular cells sex chromosomes aneuploidies.
Out of 84 patients, 7 (7/84; 8.3%) had sperm in the ejaculate. Out of the 77 azoospermic patients, 24 underwent TESE and 9 (9/24; 37.5%) had successful sperm recovery. The comparison of reproductive hormones, age and testicular volume did not show significant differences between patients with and without successful sperm recovery in semen or TESE . Patients without successful sperm recovery in semen analysis or TESE had signs of hypoandrogenism more evident than patients with successful sperm recovery. Patients with KS produced a higher number of sperm aneuploidy with respect to normozoospermic fertile controls and non-genetic severely oligozoospermic men.
Men with KS are not always sterile. In some of these patients sperm can be found in semen or in the testis, but the proportion of sperm aneuploidy is high. Signs of hypoandrogenism seem to be associated with low sperm recovery rate.
To determine whether changes in intratesticular microcirculation perfusion affect spermatogenesis in patients with left varicocele we performed testicular contrast harmonic imaging.
A total of 90 patients with left varicocele (oligospermia in 50 and normozoospermia in 40) and 36 controls without varicocele (oligospermia in 16 and normozoospermia in 20) were enrolled in the study. Before contrast harmonic imaging all participants were evaluated by clinical examination, hormonal analysis, semen sample and scrotal ultrasound. We calculated contrast material arrival time in the arteriolar circulation (wash-in), time to peak in arterial circulation, arrival time in the venular circulation (washout) and mean transit time in each testis on contrast harmonic imaging.
We found no difference in the distribution rate of varicocele grade in patients with vs without oligospermia. All contrast harmonic imaging parameters were significantly higher in patients with varicocele plus normozoospermia or oligospermia and controls. We found no significant differences in contrast harmonic imaging parameters in patients with lower varicocele grading with respect to the higher grades. In patients with varicocele we found a negative linear correlation between total sperm count and left mean transit time (r = -0.29). In a multivariate model left mean transit time was the only independent predicting parameter of oligospermia (p <0.05). Mean transit time greater than 36 seconds predicted oligospermia in patients with left varicocele with 78% sensitivity and 58% specificity.
To our knowledge we report for the first time that testicular contrast harmonic imaging may be a new diagnostic tool able to improve our knowledge about the influence of varicocele on intratesticular microcirculation.
Endothelial progenitor cells (EPCs) are bone marrow-derived cells required for endothelial repair. Circulating EPC concentration is low in conditions characterized by endothelial dysfunction but their number can be increased by treatment with phosphodiesterase-5 (PDE5) inhibitors. EPCs are also reduced in hypogonadal men and testosterone (T) treatment restores their concentration.
To evaluate the relationship between the effect of PDE5 inhibitors and T on EPCs, we analysed the acute effect of vardenafil on the number of EPCs in hypogonadotrophic hypogonadal (HH) patients, before and after T treatment.
A case-control study at a university andrology centre.
Fifteen HH subjects and 25 aged-matched controls.
The number of circulating EPCs and progenitor cells (PCs) in HH patients was evaluated after acute vardenafil administration at baseline and after 6 months of T supplementation.
At baseline, HH men had significantly lower numbers of PCs and EPCs with respect to controls and vardenafil administration had no effect on the number of these cells. After 6 months of T treatment, all HH patients were eugonadal. With respect to baseline, PCs and EPCs were significantly higher and reached the levels observed in controls. Vardenafil administration in HH men at the end of T treatment induced a significant increase in PCs and EPCs in a manner similar to that in controls.
This study showed that normal T levels are necessary to restore the responsiveness of EPCs to PDE5 inhibitors, suggesting that T positively modulates PDE5 in bone marrow.
Foresta C et al., 2009·Expert opinion on biological therapy
FSH plays a crucial role in human reproduction. Treatment with gonadotrophins has been shown to be effective in males affected by hypogonadotrophic hypogonadism. The success of this treatment has brought about the utilization of the same therapy in infertile oligozoospermic subjects, aimed at obtaining increased sperm count. This physiological role in spermatogenesis has induced various attempts to treat idiopathic oligozoospermic men with FSH, often inducing the restoration of normal spermatogenesis and spontaneous pregnancy.
To evaluate clinical efficacy of recombinant FSH in male infertility.
Evaluation of pharmacokinetic, pharmacodynamic properties, efficacy and safety of this hormone preparation, on the basis of the data published in literature.
Recombinant FSH is effective, safe and well tolerated. Treatment with this hormone may represent a valid tool for infertile men. However it should be performed on selected patients utilizing some predictive parameters able to identify a priori responder subjects with high probability.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Mutations in the androgen receptor (AR) gene can cause the androgen insensitivity syndrome (AIS). For complete and severe partial AIS, well-characterized in vitro functional assays can be used for genotype-phenotype correlation; however, for mild forms of AIS, as associated with male infertility, experimental evidence is scarce or lacking. In particular, optimal in vitro functional tests informative about the genotype-phenotype relation have not been described.
The objective of this study was to investigate the association among genotype and phenotype for AR mutations found in infertile males by conventional functional assays and additional in-depth studies performed with several gene reporters.
To this aim, we selected four AR missense mutations associated with isolated male infertility (L547F and two novel mutations A474V and S650G) or partial AIS (Y571H). After introduction of the specific mutations in AR expression plasmid, we performed classical in vitro studies (Western immunoblotting, electrophoretic mobility shift assay, hormone-response curves) and transactivation assays with different reporter constructs (MMTV, Sc-ARU-TK, TAT-GRE- 2X, Slp-ARU-TK and PEM).
Our results showed that standard functional tests provide sufficient information only for severe AR mutations, whereas for AR mutations found in mild AIS patients with male infertility, only an extensive analysis with different in vitro systems, and in particular with PEM promoter, can give information on the functionality of the AR and therefore on the pathogenicity of the mutations and on genotype-phenotype correlation.
Treatment with gonadotrophins is very effective in patients affected by hypogonadotrophic hypogonadism. The success of follicle-stimulating hormone (FSH) treatment in these men has brought the utilization of the same therapy in infertile oligozoospermic patients, aimed at obtaining a quantitative increase in sperm count.
FSH plays a crucial role in human reproduction. This physiological role in spermatogenesis has induced various attempts to treat idiopathic oligozoospermic men with FSH, often inducing the restoration of normal spermatogenesis and spontaneous pregnancy. However, the results obtained so far are still controversial. In this research, attention is focused on the possible criteria able to predict a seminal response to the specific hormonal treatment. Moreover, we have correlated different polymorphisms of FSH receptor gene with the outcome of FSH treatment. In this article, the literature is reviewed, and the authors' experience on using FSH treatment in oligozoospermic patients is discussed.
FSH treatment may represent a valid tool for infertile men. However, it should be performed on selected patients utilizing some predictive parameters able to identify a priori responder patients with high probability.
Cryptorchidism is the most frequent congenital birth defect in male children and represents an important risk factor for infertility and testicular cancer. Major regulators of testicular descent are the hormones insulin-like factor 3 (INSL3) and testosterone, and disruption of these pathways might cause cryptorchidism.
To determine the frequency of genetic alterations in cryptorchidism.
Case-control study in 2 departments of pediatric surgery in Italy between January 2003 and March 2005.
Six hundred male infants with cryptorchidism. Boys were followed up for 2 to 3 years (through January 2008) and orchidopexy was performed in those who were persistently cryptorchid. We analyzed 300 noncryptorchid male children aged 1 to 4 years as controls.
Karyotype anomalies and INSL3, INSL3 receptor, and androgen receptor gene mutations.
The frequency of genetic alterations in boys with cryptorchidism was low (17/600 [2.8%; 95% confidence interval {CI}, 1.7%-4.5%]) and was significantly higher in participants with persistent cryptorchidism (16/303 [5.3%; 95% CI, 3.0%-8.4%]; P = .001) and those with bilateral cryptorchidism (10/120 [8.3%; 95% CI, 4.1%-14.8%]; P = .001) than in controls (1/300 [0.3%; 95% CI, 0.1%-0.8%]). Boys with persistent cryptorchidism had a 17-fold greater odds of having a genetic alteration (odds ratio, 16.7; 95% CI, 2.2-126.5). The most common genetic findings in those with cryptorchidism were 8 cases of Klinefelter syndrome and 5 cases of mutations in the INSL3 receptor gene. Genetic alterations were not found in boys with low birth weight or low gestational age, who had frequent spontaneous descent of the testes.
In a small percentage of the study population, there was a statistically significant association between bilateral and persistent cryptorchidism and genetic alterations, including Klinefelter syndrome and INSL3 receptor gene mutations.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Zuccarello D et al., 2008·Human reproduction (Oxford, England)
Asthenozoospermia (AZS) is a common cause of male infertility characterized by reduced forward motility (WHO grade A+B sperm motility <50% or A < 25%) or absent sperm motility in fresh ejaculate. AZS may exist as an isolated disorder, in combination with other sperm anomalies or as part of a syndromic association. Up to date, only a few genes, constituting the cilia/flagella structure, have been associated with isolated AZS in humans, whereas several other genes are known to be involved in syndromic form of AZS, including primary ciliary dyskinesia (PCD) and Kartagener syndrome (KS). Axonemal ultrastructural defects, including absent or shortened arms of dyneins, can be found in >50% of PCD/KS patients. Approximately 90% of KS male patients are affected by AZS. The majority of KS patients can be ascribed to dynein genes mutations.
Mutation screening of DNAI1, DNAH5 and DNAH11 genes was performed in 90 patients with isolated non-syndromic AZS and 200 controls.
We found three mutations (one in each gene) specifically associated with AZS in seven patients (7.8%). Mutations are inherited from the mothers and may be found in familial clusters. No ultrastructural axonemal anomaly was detected in sperm.
We report for the first time a possible association between mutations in dynein genes and isolated AZS. Male carriers of the mutations always exhibit AZS, whereas female carriers manifest no alterations in either fertility or pulmonary clearance.
Ferlin A et al., 2008·Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research·Free full text on PubMed Central
Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein-coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency.
Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27-41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Real-time cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and muCT, respectively.
Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose- and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor-ligand complex. Consistent with the human phenotype, bone histomorphometric and muCT analyses of Rxfp2(-/-) mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates.
This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Ferlin A et al., 2007·The Journal of clinical endocrinology and metabolism
An explosive growth in Y chromosome long arm (Yq) microdeletion testing demand for male infertility occurred in the past few years. However, despite the progresses in the biology of this chromosome, a number of molecular and clinical concerns are not supported by definitive data.
The objective was to provide information on the type and prevalence of microdeletions in infertile males, indication for testing, genotype-phenotype correlation, sperm aneuploidies, and genetic counseling.
We performed a prospective study from January 1996 to December 2005 in an academic clinic.
We studied 3073 consecutive infertile men, of which 625 were affected by nonobstructive azoospermia and 1372 were affected by severe oligozoospermia. Ninety-nine patients with microdeletions are described here.
Yq microdeletions, seminal analysis, reproductive hormones, testicular cytology/histology, and sperm sex chromosomes aneuploidies were used as outcome measures.
The prevalence of microdeletions was 3.2% in unselected infertile men, 8.3% in men with nonobstructive azoospermia, and 5.5% in men with severe oligozoospermia. Only 2 of 99 deletions were found in men with more than 2 million sperm/ml. No clinical data are useful to identify a priori patients with higher risk of Yq microdeletions. Most deletions are of the AZFc-b2/b4 subtype and are associated with variable spermatogenic phenotype, with sperm present in 72% of the cases. Complete AZFa and AZFb (P5/Proximal P1) deletions are associated with Sertoli cell-only syndrome and alterations in spermatocyte maturation, respectively, whereas partial deletions in these regions are associated with milder phenotype and frequent presence of sperm. Men with AZFc-b2/b4 deletions produce a higher percentage of sperm with nullisomy for the sex chromosomes and XY-disomy.
This extensive clinical research expands the knowledge on genotype-phenotype relationships and confirms that the identification of Yq microdeletions has significant diagnostic and prognostic value, adding useful information for genetic counseling in these patients.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Pengo M et al., 2006·Reproductive biomedicine online
In women, single nucleotide polymorphisms (SNP) of the FSH receptor (FSHR) gene influence FSH concentrations and the sensitivity of the FSHR to FSH in vivo. In contrast, the significance of FSHR R gene SNP in the male is poorly understood. To this aim, the possible role of three FSHR SNP was evaluated in male infertility. SNP in exon 10 (codon 307 and 680) and in the core promoter region (at position -29) of the FSHR gene were analysed by polymerase chain reaction-restriction fragment length polymorphism technique in 150 men representative of the general population, 107 proven fathers, 92 normozoospermic controls, and 215 infertile patients classified according to sperm parameters (38 azoospermia, 53 severe oligozoospermia, 48 moderate oligozoospermia, and 76 slight oligozoospermia). Reproductive hormones were measured in infertile males and normozoospermic controls. No significant difference was found in allelic variants frequency and genotype distribution between each category of subjects when analysing the FSHR exon 10 SNP alone and in combination with the SNP at position -29. Serum FSH concentrations and other andrological parameters did not differ between subjects with different genotype within each group. The data showed that in the Italian population, FSHR genotypes have no influence on FSH concentrations both in normal and infertile males and do not associate with spermatogenetic impairment.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Androgens and a functioning androgen receptor (AR) are essential for development and maintenance of the male phenotype and spermatogenesis. Consistent with this, mutations in the AR gene cause a variety of defects related to androgen insensitivity, ranging from complete feminization to phenotypic males with infertility. The aim of his study was to analyse the prevalence of AR gene mutations in male infertility and to clarify the genotype-phenotype relation.
Males with infertility were recruited consecutively at the Centre for Male Gamete Cryopreservation at the University of Padova from January 1996 to January 2005.
One thousand five hundred and seventeen men with < 10 million sperm/ml and 310 age-matched normozoospermic controls.
Screening for AR gene mutation was done by DHPLC and sequencing, and reproductive hormone concentrations were measured.
We found 20 mutations in 26 of 1517 patients (1.7%) and no mutations in controls. A high number of mutations localized in exon 1 of the AR gene coding for the transactivation domain of the protein. Of 20 mutations, 7 represent novel mutations. With respect to men without AR mutations, subjects with AR mutations have lower ejaculate volume, higher testosterone levels, higher oestradiol levels, and higher androgen sensitivity index. However, the ranges for these variables were highly overlapping between men with and without AR gene mutations. Also clinical manifestations of AR mutations are not unique and 22 men had only spermatogenic impairment.
AR gene mutations are quite frequent in unselected infertile men but no specific hormonal or clinical data could be used to preselect patients at risk of mutations.
Ferlin A et al., 2006·Journal of endocrinological investigation
Insulin-like factor 3 (INSL3) is expressed in Leydig cells of the testis and theca cells of the ovary. This peptide affects testicular descent by acting on gubernaculum via its specific receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8). From initial animal data showing the cryptorchid phenotype of Insl3/Lgr8 mutants, an extensive search for mutations in INSL3 and LGR8 genes was undertaken in human patients with cryptorchidism, and a frequency of mutation of 4-5% has been detected. However, definitive proofs of a causative role for some of these mutations are still lacking. More recent data suggest additional paracrine (in the testis and ovary) and endocrine actions of INSL3 in adults. INSL3 circulates at high concentrations in serum of adult males and its production is dependent on the differentiation effect of LH. Therefore, INSL3 is increasingly used as a specific marker of Leydig cell differentiation and function.
Foresta C et al., 2006·The Journal of clinical endocrinology and metabolism
Endothelial dysfunction seems to be the first step of the atherosclerotic process. In the past few years, it has been demonstrated that injured endothelial monolayer is restored by a premature pool of circulating progenitor cells (PCs) and a more mature one of circulating endothelial PCs (EPCs). Even though there is increasing evidence that estrogens play a beneficial role on EPCs and, even if debated, on the cardiovascular system, less is known about androgens.
Our objective was to evaluate the levels of circulating PCs and EPCs in men with hypogonadotropic hypogonadism (HH) and the effect of prolonged testosterone (T) replacement therapy on these cells.
We conducted a prospective study on males with HH at a university andrological center.
The study included 10 young HH patients (28.6 +/- 3.1 yr) and 25 age-matched controls.
Idiopathic HH patients were treated with T gel therapy, 50 mg/d for 6 months.
We assessed circulating PC and EPC concentrations and immunocytochemistry for androgen receptor expression on cultured EPCs.
At baseline, HH patients showed a significant reduction of both PCs and EPCs with respect to controls. T replacement therapy induced a significant increase of these cells with respect to baseline. Immunocytochemistry on cultured EPCs showed strong expression of the androgen receptor.
Hypotestosteronemia is associated with a low number of circulating PCs and EPCs in young HH subjects. T treatment is able to induce an increase in these cells through a possible direct effect on the bone marrow.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Ferlin A et al., 2005·Journal of medical genetics·Free full text on PubMed Central
Complete deletions of the AZFc region in distal Yq are the most frequent molecular genetic cause of severe male infertility. They are caused by intrachromosomal homologous recombination between amplicons--large, nearly identical repeats--and are found in 5-10% of cases of azoospermia and severe oligozoospermia. Homologous recombination may also generate different partial deletions of AZFc, but their contribution to spermatogenic impairment has not been confirmed.
In this study we analysed the prevalence and characteristics of different partial AZFc deletions and their association with spermatogenic failure. We studied 337 infertile men with different spermatogenic impairment and 263 normozoospermic fertile men using AZFc specific sequence tagged site markers and DAZ specific single nucleotide variants.
We identified 18 cases of partial AZFc deletions in the infertile group (5.3%) and one case in the control group (0.4%). Seventeen deletions had the "gr/gr" pattern, one the "b2/b3" pattern, and one represented a novel deletion with breakpoints in b3 and b4 amplicons. Partial AZFc deletions were associated with different spermatogenic phenotypes ranging from complete azoospermia to only moderate oligozoospermia.
Together with published data, our analysis of DAZ gene copy suggested that the contribution of the different deletions to male infertility varies: only partial AZFc deletions removing DAZ1/DAZ2 seem to be associated with spermatogenic impairment, whereas those removing DAZ3/DAZ4 may have no or little effect on fertility. These data show that, beside complete AZFc deletions, specific partial deletions represent a risk factor for male infertility, even if with different effect on spermatogenesis.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Ferlin A et al., 2005·Cytogenetic and genome research
The most common type of karyotype abnormality detected in infertile subjects is represented by Klinefelter's syndrome, and the most frequent non-chromosomal alteration is represented by Y chromosome long arm microdeletions. Here we report our experience and a review of the literature on sperm sex chromosome aneuploidies in these two conditions. Non mosaic 47,XXY Klinefelter patients (12 subjects) show a significantly lower percentage of normal Y-bearing sperm and slightly higher percentage of normal X-bearing sperm. Consistent with the hypothesis that 47,XXY germ cells may undergo and complete meiosis, aneuploidy rate for XX- and XY-disomies is also increased with respect to controls, whereas the percentage of YY-disomies is normal. Aneuploidy rates in men with mosaic 47,XXY/46,XY (11 subjects) are lower than those observed in men with non-mosaic Klinefelter's syndrome, and only the frequency of XY-disomic sperm is significantly higher with respect to controls. Although the great majority of children born by intracytoplasmic sperm injection from Klinefelter subjects are chromosomally normal, the risk of producing offspring with chromosome aneuploidies is significant. Men with Y chromosome microdeletions (14 subjects) showed a reduction of normal Y-bearing sperm, and an increase in nullisomic and XY-disomic sperm, suggesting an instability of the deleted Y chromosome causing its loss in germ cells, and meiotic alterations leading to XY non-disjunction. Intracytoplasmic injection of sperm from Y-deleted men will therefore transmit the deletion to male children, and therefore the spermatogenic impairment, but raises also concerns of generating 45,X and 47,XXY embryos.
Caretta N et al., 2005·Journal of endocrinological investigation
Aging in men is accompanied by a decrease in libido and sexual activity. Recently, it has been demonstrated that a significant proportion of men >60 yr of age has biochemical hypogonadism. Hypoandrogenism, which is associated with other conditions that negatively influence erectile activity, may be an important cofactor in the induction of erectile dysfunction and in the response to phosphodiesterase type 5 (PDE5) inhibitors in aging. In these patients, administration of 50 mg sildenafil or 3 mg apomorphine has no influence on erectile function. Recently we showed that, in hypogonadal subjects, testosterone treatment might stimulate endothelial and neuronal production of vasoactive substances like nitric oxide (NO). Furthermore it can improve endothelial repair mechanisms by increasing bone marrow-derived endothelial progenitor cell (EPC) number in the peripheral circulation. Finally, the normal response to pharmacological stimulation with apomorphine or sildenafil, observed in hypogonadal men treated with testosterone, indicates a positive role of this hormone in the central and peripheral control of erection, confirming the possible positive influence on endothelial function and PDE5 expression. Therefore, we suggest measuring plasma testosterone levels in aged men with erectile dysfunction and recommend treating them barring clinical contraindications. Testosterone plus PDE5 inhibitors may be beneficial in improving erectile function in hypogonadal men with erectile dysfunction who were unresponsive to PDE5 inhibitors alone.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Foresta C et al., 2005·The Journal of clinical endocrinology and metabolism
Recent reports suggest that children born after intracytoplasmic sperm injection performed for male factor infertility are at increased risk of congenital malformations and chromosome aberrations. To explain these observations, we hypothesized that infertile men may be more likely than fertile men to have genetic abnormalities. We studied 750 severely oligozoospermic men (sperm count <5 million/ml) who were candidates for intracytoplasmic sperm injection, and 303 fertile men. We analyzed the peripheral blood karyotype, the Y chromosome long arm for detection of microdeletions in the azoospermia factors, and mutations in the cystic fibrosis gene and the androgen receptor gene. We also analyzed sperm for chromosome aneuploidies among the 421 men who subsequently entered the in vitro fertilization program. A total of 104 genetic abnormalities were diagnosed, corresponding to a frequency of 13.9% (104 of 750). Chromosomal aberrations were present in 5.6% (42 of 750) of infertile men and 0.3% of controls (one of 295), and they were in most cases alterations of the sex chromosomes. Y chromosome long-arm microdeletions were detected in 6.0% (45 of 750) of infertile men and most frequently included the azoospermia factor c, whereas no cases were found in controls (zero of 210). Mutations in the cystic fibrosis gene were diagnosed in 1.2% (nine of 750) of infertile men and 1.0% of controls (three of 303), and mutations in the androgen receptor gene were found in 1.1% (eight of 750) of infertile men and none of the 188 controls. Sperm sex chromosome aneuploidies were increased in men with karyotype anomalies and Y chromosome microdeletions as well as in subjects without constitutional genetic abnormalities. This study shows that the frequency of genetic alterations is increased among men with severe spermatogenic impairment. Genetic tests and genetic counseling should therefore be considered in oligozoospermic men who are candidates for intracytoplasmic sperm injection.
Ferlin A et al., 2005·Annals of the New York Academy of Sciences
Insulin-like factor 3 (INSL3) affects testicular descent. Mutations in the INSL3 gene or its receptor, LGR8/GREAT, can cause cryptorchidism. Expression of LGR8/GREAT in different tissues and production of INSL3 by adult-type Leydig cells suggest additional roles for this hormonal system in adults. We used a novel radioimmunoassay kit to measure INSL3 concentrations in the serum of normal men and those with different testicular pathologies. We demonstrate that INSL3 circulates in adult men and is almost exclusively of testicular origin. Subjects with severe testicular damage (infertility) produce small amounts of INSL3, and concentrations of this hormone seem to reflect the functional status of the Leydig cells. Analysis of men treated with different combinations of hormones of the hypothalamus-pituitary-testis axis suggests that the production of INSL3 is related to the luteinizing hormone.
Male Reproductive Surgery · Sperm Retrieval Procedures
Bettella A et al., 2005·Asian journal of andrology·Free to read
To report the fine needle aspiration cytology (FNAC) of the testes used as a diagnostic tool in non-obstructive azoospermic patients.
One hundred and twenty-five non-obstructive azoospermic male candidates to intracytoplasmic sperm injetion (ICSI) were analysed for follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone and inhibin B plasma levels. They were classified into three groups on the basis of 1) Sertoli cell-only syndrome (SCOS) (70); 2) severe hypospermatogenesis (42); and 3) maturation arrest (13). Then, all men underwent testicular sperm extraction (TESE) for sperm recovery for ICSI.
Mature spermatozoa were detected by FNAC in 24 of 42 men with severe hypospermatogenesis and nine of 13 men with maturation arrest; while they were retrieved by TESE in 29 of 70 men with SCOS, 35 of 42 men with severe hypospermatogenesis (including the 24 by FNAC) and 10 of 13 men with maturation arrest (including the nine by FNAC). The sensitivity and specificity of FNAC were 44.6 % and 100 %, respectively. There was no difference on testicular volume and hormonal parameters in men with and without sperm retrieved.
These findings suggest that FNAC may be a simple and valid diagnostic parameter in non-obstructive azoospermic men and it may represent a valid positive prognostic parameter for sperm recovery at TESE.
To evaluate the effects of treatment with FSH on seminal parameters and spontaneous pregnancy in male infertility.
Prospective, controlled, randomized clinical study.
Infertility center at a university hospital.
PATIENT(S): One hundred twelve men affected by idiopathic oligozoospermia.
INTERVENTION(S): Patients were randomized into two groups: 62 subjects were treated with 100 IU of recombinant human FSH on alternate days for 3 months, and 50 patients did not receive any treatment. Semen analysis was performed in all subjects at the end of this period of treatment and after the following 3 months. Subjects who had not reached spontaneous pregnancy underwent assisted reproductive techniques.
MAIN OUTCOME MEASURE(S): Seminal parameters, testicular cytologic analysis, FSH, LH, T, and inhibin B concentrations.
RESULT(S): The treatment group considered as a whole did not show modifications in sperm parameters. However, a subgroup of these (30, 48.4%) had a significant increase of sperm count (responder group). In the period including 3 months after the withdrawal of FSH therapy, we observed a significantly higher spontaneous pregnancy rate in the responder group (5 of 30 [16.7%]) with respect to nonresponder and nontreated groups (1 of 32 [3.1%] and 2 of 50 [4.0%], respectively). Furthermore, the improvement of seminal parameters in the responder group allowed these patients to undergo less frequent IVF-ET/intracytoplasmic sperm injection.
CONCLUSION(S): Results from this controlled, randomized clinical trial show that FSH therapy does not improve sperm concentration or pregnancy rate when infertile male patients are chosen solely by the clinical criteria of idiopathic oligospermia and normal FSH concentration. Subgroup analysis, however, does indicate that patients without maturation arrest in addition to the clinical scenario do benefit from medical therapy.
Foresta C et al., 2004·The Journal of clinical endocrinology and metabolism
Insulin-like factor 3 (INSL3) is a member of the relaxin-insulin family, and it is expressed in pre- and postnatal Leydig cells of the testis. This peptide affects testicular descent during embryonic development, and mutations in INSL3 gene or its receptor LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8)/GREAT (G protein-coupled receptor affecting testicular descent) cause cryptorchidism in humans. The expression of LGR8/GREAT in different tissues and the production of INSL3 also by adult-type Leydig cells suggest additional roles of this hormonal system in adulthood. In this preliminary report we performed the first analysis in humans of INSL3 using a novel RIA kit to measure INSL3 concentrations in serum of normal men and with different testicular pathologies. The results show that INSL3 is circulating in adult men, and it is almost exclusively of testicular origin. Subjects with severe testicular damage, such as men with severe infertility, produce low amount of INSL3, and the concentrations of this hormone seem to reflect the functional status of the Leydig cells. In particular, INSL3 concentrations may be an even more sensitive marker of Leydig cell function than testosterone itself. Analysis of men treated with different combinations of hormones of the hypothalamus-pituitary-testis axis suggests that the production of INSL3 is related to LH in a manner similar to that of the LH-testosterone axis.
To investigate whether partial deletions of the DAZ gene family on the Y chromosome are associated with cryptorchidism, similar to that found for complete AZF deletions.
Prospective study.
University hospital.
PATIENT(S): A total of 193 azoospermic and severely oligozoospermic men: 95 with a history of cryptorchidism and 98 classified as idiopathic.
INTERVENTION(S): A two-part study for Y chromosome microdeletions was performed: a polymerase chain reaction (PCR)-based analysis for complete AZF deletions and partial DAZ gene analysis by PCR-restriction digestion assay for single-family variants.
MAIN OUTCOME MEASURE(S): Presence and type of AZF deletions and number of DAZ genes present.
RESULT(S): The frequency of complete AZF deletions was similar in idiopathic (13.3%) and cryptorchid men (11.6%), but partial DAZ deletions were found only in infertile subjects without cryptorchidism (7.1%). The testicular phenotype was similar in men with complete AZF deletions and partial DAZ deletions, therefore the contribution of the other AZF genes in determining the spermatogenic impairment is still unclear.
CONCLUSION(S): Our findings suggest that the loss of only some copies of DAZ is sufficient to lead to severe male infertility, but it is not a frequent finding in cryptorchid men.
Erectile response in mammals is centrally and peripherally regulated by androgens. Severe hypogonadism in men usually results in loss of libido and potency. The present studies were designed to evaluate the possible influence of circulating androgens in the regulation of sexual function.
A total of 15 men with severe hypogonadism (testosterone less than 2.0 ng/ml) were recruited. The control group consisted of 20 patients with psychogenic erectile dysfunction. All subjects underwent nocturnal penile tumescence (NPT) and rigidity monitoring during 2 consecutive nights, evaluation of cavernous artery flow using penile color duplex ultrasound (P-CDU), and real-time visually stimulated erection evaluation at baseline and after administration of 50 mg sildenafil or 3 mg apomorphine. NPT, P-CDU and visually stimulated erection were evaluated after 6 months of therapy with a 5 mg daily testosterone patch.
NPT analysis in subjects with severe hypogonadism showed a significant decrease in sleep related erections. P-CDU showed a partial erectile response with an alteration of the parameters analyzed, and the visually stimulated erections test was pathological. Administration of 3 mg apomorphine and 50 mg sildenafil has no influence on erectile function. Testosterone treatment for 6 months induced normalization of NPT and P-CDU parameters, and of visually stimulated erection effects with the restoration of a normal response to pharmacological stimulation with apomorphine or sildenafil.
Our results show that testosterone has a key role in the central and peripheral modulation of erectile function even if the accurate testosterone plasma level threshold that may influence these processes remains to be established.
Male Endocrine and Genetic Factors · Testicular Function
Foresta C et al., 2004·Reproductive biomedicine online
Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in human males. INSL3 and LGR8/GREAT proteins seem to act as ligand and receptor respectively, and to have a role in gubernaculum development involved in testicular descent. Mutations in the INSL3 gene or LGR8/GREAT were found to be associated with cryptorchidism in humans. In a cohort of 135 ex-cryptorchid patients and 100 controls, mutations were sought in INSL3 and LGR8/GREAT genes by sequencing. Six patients were found with mutations in the INSL3 gene and four patients with LGR8/GREAT mutation (10/135, 7.4%). The 10 patients show different phenotypes, ranging from normozoospermia to complete azoospermia, and from bilateral cryptorchidism to retractile testes. Furthermore, the endocrine function of the testis appeared normal in all subjects. These findings demonstrate that INSL3-LGR8/GREAT mutations are frequently associated with human cryptorchidism, and that the only clinical consequence of alterations of the INSL3-LGR8/GREAT system seems to be failure of the testis to descend normally in the scrotum during embryonic development, without affecting the spermatogenic and endocrine components of the testis itself. The first analysis in humans of INSL3 was then performed using a novel radioimmunoassay kit to measure INSL3 concentrations in serum of adults. The results show that INSL3 circulates in adult men, it is a male-specific hormone, and it is of almost exclusively testicular origin. The role of this hormonal system in adulthood is, however, to date unknown.
Male Endocrine and Genetic Factors · Male Hormonal Axis
Foresta C et al., 2004·Human reproduction (Oxford, England)
In vitro continuous stimulation of Sertoli cells with FSH leads to a desensitization of these cells to FSH action. To evaluate the presence of a desensitization of FSH receptor on Sertoli cells in vivo, we performed a controlled clinical study in 97 men affected by severe oligozoospermia.
On the basis of FSH and inhibin B plasma concentrations, these subjects were divided into three groups: group A, 33 subjects with high FSH and low inhibin B plasma levels; group B, 32 subjects with high FSH plasma levels and inhibin B concentrations at the lower limit of the normal range; and group C, 32 subjects with normal FSH and inhibin B plasma levels. Patients with high FSH plasma levels (groups A and B) were prospectively randomized into two subgroups, called A1, A2, B1 and B2. Patients of groups A1 and B1 were treated with a GnRH agonist, leuprolide acetate, to induce a hypogonadotrophic state and then were treated with recombinant human FSH (r-hFSH; 100 IU/day) and hCG (2000 IU/twice a week) for 2 months. Subjects of groups A2, B2 and C were treated only with r-hFSH for the same period.
In patients of group A1, inhibin B remained unmodified during the whole period of study, whereas in subjects of group B1, we observed a significant reduction of this hormone during the hypogonadotrophic period and then an increase of inhibin B plasma levels that were higher that those observed before therapy. In patients of groups A2 and B2, FSH treatment did not induce a significant increase in inhibin B concentrations. In patients of group C, FSH induced a significant increase in inhibin B plasma levels.
In infertile men, suppression of the high endogenous levels of plasma FSH associated with much lower exogenous FSH levels is able to evoke higher inhibin B production, which may indicate improved Sertoli cell function and the possibility that this could have a positive effect on spermatogenesis.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Ferlin A et al., 2002·Journal of endocrinological investigation
The deleted-in-azoospermia (DAZ) gene family constitutes the major candidate for the AZFc (azoospermia factor c) phenotype of male infertility, being deleted in about 10% of azoospermic and severely oligozoospermic subjects. Four DAZ genes are arranged in two clusters in AZFc, and standard analysis by PCR cannot distinguish among the different copies. Therefore only deletions of the entire gene cluster can be identified. We developed a PCR amplification-restriction digestion assay able to distinguish from DAZ genes for single nucleotide variants. Then we applied this approach to screen a group of idiopathic infertile men in which the DAZ genes presence was previously assessed by standard PCR analysis. Two patients out of 25 showed deletion of two copies of DAZ (DAZI and 2), suggesting that this mutation was actually the cause of spermatogenic damage. This preliminary screening demonstrates that deletions of copies of DAZ genes may be often found in severely infertile men and it strengthens the role of this gene family in spermatogenesis. Furthermore, this simple method, being able to distinguish among the different DAZ copies, could be used to screen a larger number of patients and to perform a more accurate diagnosis.
Guidelines by Clinical Area · Fertility and Infertility Guidelines
Foresta C et al., 2002·European journal of human genetics : EJHG
Research on genetic causes of male and female infertility rapidly expanded in the last years, following the development of in vitro fertilising techniques. Genetic tests are now available to explore the cause of the infertility and assess the risk of a given couple to transmit its genetic characteristics. This allows at-risk couples to take an informed decision when electing for a medically assisted reproduction. It also allows the professionals to offer a prenatal diagnosis when appropriate. Thus, the genetic work-up of the infertile couple has become good practice for an appropriate diagnosis, treatment and prognostic assessment. The lack of national or international rules for the genetic approach to the infertile couple, prompted the Italian community of professionals in the field of reproductive medicine to join and set up guidelines for the genetic diagnosis of male and female infertility. The group of clinical and research experts is representative of 12 national scientific societies and was supported by external experts from four international societies. We examine the clinically relevant genetic causes of male and female infertility and suggest the category of patients for which each genetic test is recommended or optional, both for an accurate diagnosis and prior to ART.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Foresta C et al., 2002·European journal of endocrinology
The DAZ (deleted in azoospermia) gene family on the Y chromosome long arm is the major candidate for the AZFc (azoospermia factor c) phenotype of male infertility and it is expressed only in germ cells. The aim of the study was to assess Sertoli cell function in subjects with AZFc deletions.
Case-control, prospective study.
We have studied six severely oligozoospermic subjects with AZFc-DAZ deletions, and looked whether they responded in terms of inhibin B production to a 1 month FSH treatment. These patients were compared with three groups of patients affected by different spermatogenic alterations not related to deletions on the Y chromosome.
Although affected by severe testiculopathy, patients with AZFc-DAZ deletions had only slightly elevated FSH, and normal inhibin B plasma concentrations. Inhibin B responded normally during FSH treatment, supporting the hypothesis that Sertoli cells are not altered. On the contrary, other severe testiculopathies not related to Y chromosome deletions showed high FSH and low inhibin B concentrations, with no response to FSH treatment. In these cases the cause of the spermatogenic defect probably damaged both germ and Sertoli cells. Finally, idiopathic patients with a hormonal status similar to Y-deleted patients (slightly elevated FSH and normal inhibin B concentrations) did not respond to FSH treatment, suggesting that Sertoli cells were already at their maximal functional capability.
These data confirm that Sertoli cell function is not damaged in patients with AZFc-DAZ deletions and that the strong reduction of germ cells does not affect the FSH-inhibin B feedback loop.
To evaluate the effects of treatment with recombinant human FSH (r-hFSH) on seminal parameters and seminiferous epithelium in idiopathic patients with oligozoospermia with normal FSH plasma levels.
Randomized single-blind study.
Academic setting.
PATIENT(S): Forty-five subjects with idiopathic oligozoospermia (sperm count <10 x 10(6)/mL) and normal FSH and inhibin B plasma levels.
INTERVENTION(S): Three months of treatment with r-hFSH 50 IU (15 patients) or with r-hFSH 100 IU on alternate days (15 patients) or no treatment (15 patients); bilateral testicular fine-needle aspiration (FNA) performed before and after therapy; FSH and inhibin B plasma levels evaluated during treatment.
MAIN OUTCOME MEASURE(S): Seminal parameters; testicular cytological features evaluated by FNA; plasma levels of FSH, LH, T, and inhibin B.
RESULT(S): Treatment with r-hFSH at a dose of 50 IU induced no increase in sperm concentration, while treatment with r-hFSH at a dose of 100 IU induced a significant increase in sperm concentration. In particular, in 11/15 patients a doubling of the pretreatment sperm concentration was observed. No significant increase in sperm parameters was observed in the control group. In both groups of patients treated with r-hFSH, the cytological analysis before treatment showed hypospermatogenesis. An increase in the percentage of spermatogonia and spermatocytes was observed only after the treatment with r-hFSH at a dose of 100 IU.
CONCLUSION(S): The findings of this study demonstrate that r-hFSH at a dose of 100 IU, as previously seen with highly purified FSH, increases the spermatogonial population and sperm production in idiopathic patients with oligozoospermia with normal FSH and inhibin B plasma levels and a cytological picture of hypospermatogenesis.
Foresta C et al., 2001·Annali italiani di medicina interna : organo ufficiale della Societa italiana di medicina interna
Microdeletions of the Y chromosome represent the most frequent cause of male infertility, being responsible for 10-15% of cases of azoospermia or severe oligozoospermia. Such mutations localize in one or more loci named azoospermia factor (AZF) a, b and c. Mutations more frequently involve the DAZ gene in AZFc, and could determine both azoospermia and severe oligozoospermia. It is therefore difficult to find a clear relationship between genotype and phenotype. DAZ is present in multiple copies in AZFc, and this causes the gene to be difficult to analyze. In fact, polymerase chain reaction, the principal technique utilized for detection of the deletions, cannot distinguish among the different copies of the gene. Furthermore, it is not clear if all the DAZ copies are expressed in the testis, and other genes, such as CDY1, map in AZFc; therefore their alteration may play a role in determining the phenotype. In this review we report the current knowledge on the function of the Y chromosome in human spermatogenesis. In particular we analyze some of our experimental studies on the role of the DAZ gene family. Expression studies allowed us to clarify that an altered expression of DAZ might cause infertility in patients with severe testiculopathies. Furthermore, we describe for the first time a deletion not involving all the DAZ copies in a patient with severe hypospermatogenesis and we clarify that CDY1 is not involved in the testicular damage observed in patients with deletions of DAZ. These studies elucidate the role of DAZ and have important clinical consequences in the diagnostic and therapeutic approach of the infertile patient, above all when he is a candidate for assisted reproduction techniques, due to the possibility of transmitting the genetic alteration to the offspring.
Marin P et al., 2001·Journal of endocrinological investigation
Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male, but its etiology is for the most part unknown. Evidence suggests that a possible genetic cause may be involved. Animal models support this hypothesis, and in particular INSL3 (Leydig insulin-like 3 hormone) has been proposed as putative gene for cryptorchidism, since male mice mutant for Insl3 exhibit bilateral abdominal cryptorchidism due to alteration of gubernaculum development. In this study, we analyzed whether mutations in INSL3 could be associated with human cryptorchidism. Heteroduplex analysis and sequencing of both exons of INSL3 in 65 ex-cryptorchid patients and a group of control subjects allowed us to find four nucleotide changes in the sequence of exon I. These mutations are all single base substitutions from G to A at position 27, 96, 126 and 178. Only the 178G-->A substitution changes codon 60 from alanine to threonine (A60T). All mutations were found in comparable distribution in ex-cryptorchid patients and non-cryptorchid men. Therefore, all mutations represent neutral polymorphisms not associated with phenotype. This study confirms previous observations and demonstrates a novel polymorphism in the INSL3 gene. In contrast to that described for the mutant mouse, these data indicate that mutations of INSL3 do not seem to represent a frequent cause of cryptorchidism.
Foresta C et al., 2001·Human reproduction (Oxford, England)
Y chromosome microdeletions represent the most frequent genetic alteration in azoospermic and severely oligozoospermic men, and screening for microdeletions in AZFa, b and c are routinely performed in the major andrology and infertility centres. Since patients with Y microdeletions often require intracytoplasmic sperm injection (ICSI), the question of whether the type of the microdeletion present could have prognostic value for the presence of spermatozoa in the ejaculate or in the testes [by testicular sperm extraction (TESE)] is an interesting one. The review of the literature on this topic showed that there is still no clear genotype--phenotype relationship, i.e. similar testicular alterations may be caused by different types of microdeletions, and apparently identical microdeletions may be associated with diverse tubular damage. Even in azoospermic men, the localization of the microdeletion cannot be used as a valid prognostic parameter before TESE--ICSI to identify patients with spermatozoa in their testes. The only finding with absolute negative prognostic value is the presence of complete AZFa--c deletions, which are invariably associated with an absence of spermatozoa. Microdeletions in AZFa or AZFb seem to have promising prognostic value, but more data and gene-specific deletions have to be provided to draw clear conclusions. The absence of a clear genotype--phenotype relationship, and therefore of a prognostic value of Y deletion analysis, is probably due to the current methods used for the screening of the microdeletions. In fact, to date most centres do not use gene-specific markers but instead use anonymous primers that contribute little information to the pathogenic role of the microdeletions.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Foresta C et al., 2001·The Journal of clinical endocrinology and metabolism
Deletions of the azoospermia factors on the Y chromosome long arm are an important cause of male infertility, and they may involve germ cell-specific genes or ubiquitously expressed genes. To date, no clinical or hormonal parameters have yet been found to distinguish patients with and without Yq microdeletions. In particular, Sertoli cell function, as evaluated by inhibin B, has not yet been described. Our hypothesis was that microdeletions involving genes specifically expressed in germ cells should not alter Sertoli cell function. To do this, we have evaluated the testicular hormonal function in infertile patients affected by severe testiculopathies with and without Yq microdeletions, with particular emphasis on Sertoli cell function. We studied 102 well-characterized infertile patients; 27 had Yq microdeletions, and 75 were classified as idiopathic infertiles. Patients with Yq microdeletions had lower FSH and higher inhibin B plasma concentrations with respect to patients without microdeletions, suggesting that Sertoli cell function in Yq-deleted men is only partially altered. Furthermore, patients with deletions involving germ cell-specific genes had higher concentrations of inhibin B with respect to patients with deletions of ubiquitously expressed genes. These results suggested that a specific alteration of germ cells only partially influences Sertoli cell function. Hormonal status of patients without deletions suggested that in such cases the cause that has determined the spermatogenic defect may have damaged both Sertoli and germ cells. Inhibin B production in patients with Yq deletions was about 70% higher than the nondeleted patients, and the functional relationship between FSH and inhibin B was normally preserved. This study elucidated the multifactorial mechanisms underlying spermatogenic defects, where Sertoli cells may be normally functioning or damaged depending on the primary cause that has determined the testicular damage.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Three different spermatogenesis loci have been mapped on the Y chromosome and named "azoospermia factors" (AZFa, b, and c). Deletions in these regions remove one or more of the candidate genes (DAZ, RBMY, USP9Y, and DBY) and cause severe testiculopathy leading to male infertility. We have reviewed the literature and the most recent advances in Y chromosome mapping, focusing our attention on the correlation between Y chromosome microdeletions and alterations of spermatogenesis. More than 4,800 infertile patients were screened for Y microdeletions and published. Such deletions determine azoospermia more frequently than severe oligozoospermia and involve especially the AZFc region including the DAZ gene family. Overall, the prevalence of Y chromosome microdeletions is 4% in oligozoospermic patients, 14% in idiopathic severely oligozoospermic men, 11% in azoospermic men, and 18% in idiopathic azoospermic subjects. Patient selection criteria appear to substantially influence the prevalence of microdeletions. No clear correlation exists between the size and localization of the deletions and the testicular phenotype. However, it is clear that larger deletions are associated with the most severe testicular damage. Patients with Y chromosome deletions frequently have sperm either in the ejaculate or within the testis and are therefore suitable candidates for assisted reproduction techniques. This possibility raises a number of medical and ethical concerns, since the use of spermatozoa carrying Y chromosome deletions may produce pregnancies, but in such cases the genetic anomaly will invariably be passed on to male offspring.
Foresta C et al., 2000·Molecular and cellular endocrinology
The aim of this study was to individuate parameters able to distinguish oligozoospermic subjects who will respond to follicle-stimulating hormone (FSH) therapy. A group of 135 oligozoospermic subjects was divided in three groups considering basal FSH and inhibin B concentrations: group A (normal FSH and inhibin B) characterized by moderate hypospermatogenesis sometimes associated to partial spermatidic arrest; group B (high FSH and normal inhibin B) characterized by hypospermatogenesis associated or not to spermatogonial/spermatocytic arrest; group C (high FSH and low inhibin B) characterized by severe hypospermatogenesis. Seventy-eight patients were treated with FSH at the dose of 75 IU on alternate days while 57 were treated with the same dose every day for 3 months. After FSH treatment a significant increase in ejaculated sperm concentration was observed only in oligozoospermic subjects with normal basal FSH and inhibin B plasma levels (group A) showing a testicular cytological picture of moderate hypospermatogenesis. In these subjects no differences in sperm production were observed between the two protocols of therapy. In the remaining patients of group A, characterized by hypospermatogenesis associated with maturation arrest at spermatidic level and in group B and C, no increase in sperm concentration was observed after therapy. These data suggest that FSH treatment may have a role in oligozoospermic subjects only when the spermatogenetic alterations consist in germ cell depopulation without maturative disturbances and with normal FSH concentrations.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Moro E et al., 2000·The Journal of clinical endocrinology and metabolism
Deletions in distal Yq interval 6 represent the cause of 10-15% of idiopathic severe male infertility and map to a region defined AZFc (azoospermia factor c). The testis-specific gene DAZ is considered a major AZFc candidate, and its deletion has been associated with a severe disruption in spermatogenesis. However, DAZ is actually a multicopy gene family consisting of seven clustered copies spanning about 1 megabase. Only deletions removing the entire DAZ gene cluster together with other genes have been reported in infertile males. Because no case of spermatogenic failure has been traced to intragenic deletions, point mutations, or even deletions not involving all the DAZ copies, the definitive proof for a requirement of DAZ for spermatogenesis is still debatable. Here we report the first case of a partial deletion of the DAZ cluster removing all but one of the copies. This deletion is present in a patient affected with severe oligozoospermia who had a testicular phenotype characterized by a great quantitative reduction of germ cells (severe hypospermatogenesis). The absence of this deletion in the fertile brother of the patient suggests that this de novo mutation indeed caused the spermatogenic failure.
Foresta C et al., 1999·Annali italiani di medicina interna : organo ufficiale della Societa italiana di medicina interna
We investigated the possible role of Y chromosome microdeletions in regions previously shown to be important for male germ cell development in unilateral ex-cryptorchid subjects manifesting important bilateral testiculopathy, in order to clarify whether cryptorchidism could be the expression of an intrinsic congenital testicular abnormality. Microdeletion analysis of the Y chromosome long arm was performed by polymerase chain reaction and confirmed by Southern blot in 40 selected unilateral ex-cryptorchid patients with azoospermia or severe oligozoospermia sustained by severe bilateral testiculopathy (Sertoli cell-only syndrome and severe hypospermatogenesis, respectively), 20 unilateral ex-cryptorchid men with moderate oligozoospermia and normal function of the descended testis, 110 patients affected by severe idiopathic primary testiculopathies, 20 patients affected by moderate idiopathic testiculopathy and, as controls, 50 patients affected by known causes of testiculopathy and 50 fertile controls. Eleven out of the 40 (27.5%) unilateral ex-cryptorchid patients affected by bilateral testiculopathy and 28 out of 110 (25.4%) patients affected by severe idiopathic primary testiculopathy showed microdeletions in the Y chromosome long arm, while all other subjects were normal. Male relatives of patients with deletions were also normal. Microdeletions were distributed in different regions of the Y chromosome long arm, including known regions involved in spermatogenesis (DAZ and RBM genes, AZFa, b and c regions) and other still poorly defined loci. No difference in localization of deletions was evident between ex-cryptorchid and idiopathic patients. Microdeletions in the Y chromosome may be responsible for bilateral severe testicular damage: the clinical consequence, other than idiopathic azoospermia and severe oligozoospermia, may also be unilateral cryptorchidism, probably due to altered testicular responses to mechanisms regulating testicular descent.
Ferlin A et al., 1999·Human reproduction (Oxford, England)
Deletions of the DAZ (deleted in azoospermia) gene family are frequently responsible for male infertility and are generally assessed by analyses of genomic DNA extracted from peripheral leukocytes. The multicopy nature of this gene prevents the distinction of intragenic deletions or deletions not involving the whole DAZ gene cluster. Thus it is still unclear whether each DAZ copy is effectively expressed in the testis. We analysed, by reverse transcription-polymerase chain reaction (RT-PCR), the expression of DAZ, RBM and SRY genes, in testicular cells from infertile men affected by idiopathic severe hypospermatogenesis, obstructive azoospermia and Sertoli cell-only syndrome. Normal mRNA for DAZ, RBM and SRY were observed in obstructive azoospermia, whereas only SRY transcripts were detected when only Sertoli cells were present. Nine out of 10 patients affected by idiopathic severe hypospermatogenesis had normal expression of SRY, RBM and DAZ, while in one patient no DAZ transcript was detected, suggesting that his testiculopathy was related to the absence of DAZ expression. The lack of DAZ mRNA in testicular cells with an apparently normal DAZ gene constitution on DNA extracted from leukocytes may be explained by different hypotheses: (i) not all the copies of the DAZ gene cluster are transcribed in the germ cells and the reported patient had a small deletion involving only the active ones; (ii) the patient may be mosaic for the DAZ gene having a normal constitution in leukocytes and be deleted for DAZ gene in the testis; (iii) abnormalities of DAZ transcription may exist. These findings highlight the intrinsic interpretative difficulties of normal PCR analysis for DAZ and RBM on leukocytes and suggest caution in the use of germ cells for assisted reproductive techniques in these cases to avoid transmission of genetic abnormalities to male offspring.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Foresta C et al., 1999·The Journal of clinical endocrinology and metabolism
Azoospermic subjects affected by Klinefelter's syndrome may occasionally show the presence of intratesticular residual foci of spermatogenesis, and the retrieval of mature spermatozoa from the testis may permit fertility and paternity by means of intracytoplasmic sperm injection. Previous studies have demonstrated that these subjects show the presence of an increased incidence of hyperaploid spermatozoa. Here we analyzed, by fluorescence in situ hybridization using specific probes for chromosomes 8, X, and Y, the spermatogenic process and the meiotic progression of 47,XXY germ cells retrieved by fine needle aspiration of the testis in ten azoospermic patients affected by classic Klinefelter's syndrome. All patients had lower testicular volume, higher gonadotropins, and lower testosterone plasma levels compared with control subjects. Cytological analysis of the testicular cells retrieved by fine needle aspiration showed the presence of Sertoli cells only in eight subjects, while germ cells were observed in two patients. In each patient Sertoli cells showed a 47,XXY karyotype, and the same chromosome pattern was observed in spermatogonia and primary spermatocytes of patients presenting a residual spermatogenesis. Secondary spermatocytes, spermatids, and mature spermatozoa showed different sex chromosome patterns, reflecting their origin from 47,XXY spermatogonia. In conclusion, this study demonstrated that, in subjects affected by Klinefelter's syndrome, residual germ cells may be present in the testis and that 47,XXY spermatogonia are able to undergo and complete the spermatogenic process leading to mature spermatozoa. These data further suggest the need to evaluate the sex chromosome status of sperm from patients affected by Klinefelter's syndrome undergoing assisted reproductive techniques.
Ferlin A et al., 1999·Human reproduction (Oxford, England)
Microdeletions in Yq11 overlapping three distinct 'azoospermia factors' (AZFa-c) represent the aetiological factor of 10-15% of idiopathic azoospermia and severe oligozoospermia, with higher prevalence in more severe testiculopathies, such as Sertoli cell-only syndrome. Using a PCR-based screening, we analysed Yq microdeletions in 180 infertile patients affected by idiopathic Sertoli cell-only syndrome and different degrees of hypospermatogenesis, compared with 50 patients with known causes of testicular alteration, 30 with obstructive azoospermia, and 100 normal fertile men. In idiopathic severe testiculopathies (Sertoli cell-only syndrome and severe hypospermatogenesis), a high prevalence of microdeletions (34.5% and 24.7% respectively) was found, while milder forms were not associated with Yq alteration. No deletions were found in testiculopathies of known aetiology, obstructive azoospermia, normal fertile men and male relatives of patients with deletions. Deletions in the AZFc region involving the DAZ gene were the most frequent finding and they were more often observed in severe hypospermatogenesis than in Sertoli cell-only syndrome, suggesting that deletions of this region are not sufficient to cause complete loss of the spermatogenic line. Deletions in AZFb involving the RBM gene were less frequently detected and there was no correlation with testicular phenotype, with an apparent minor role for such gene in spermatogenesis. The DFFRY gene was absent in a fraction of patients, making it a candidate AZFa gene. Our data suggest that larger deletions involving more than one AZF-candidate gene are associated with a more severe testicular phenotype.
Foresta C et al., 1999·The Journal of clinical endocrinology and metabolism
To clarify whether cryptorchidism might be the expression of an intrinsic congenital testicular abnormality, we investigated the frequency of Y chromosome long arm (Yq) microdeletions in unilateral excryptorchid subjects manifesting an important bilateral testiculopathy. Microdeletion analysis of Yq was performed by polymerase chain reaction in the following 40 unilateral excryptorchid patients with azoospermia or severe oligozoospermia due to a bilateral severe testiculopathy (Sertoli cell-only syndrome or severe hypospermatogenesis); 20 unilateral excryptorchid men with moderate oligozoospermia and a normal testicular cytological picture in the contralateral testis; 110 patients affected by idiopathic severe primary testiculopathies; 20 patients affected by idiopathic moderate testiculopathy; and, as controls, 50 patients affected by known causes of testiculopathy and 100 fertile men. Eleven of 40 (27.5%) unilateral excryptorchid patients affected by bilateral testiculopathy and 28 of 110 (25.4%) patients affected by idiopathic severe primary testiculopathy showed Yq microdeletions, whereas no microdeletions were found in all the other subjects, nor in male relatives of patients with deletions. Microdeletions were located in different parts of Yq, including known regions involved in spermatogenesis (DAZ and RBM, AZFa, b, and c) and other loci still poorly defined. No difference in localization of deletions was evident between cryptorchid and idiopathic patients. Microdeletions in Yq may be responsible for severe bilateral testicular damage that could be phenotypically expressed by unilateral cryptorchidism, as well as by idiopathic infertility.
Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility
Rossato M et al., 1998·Molecular human reproduction
A case is reported in which a high fertilization rate was achieved by conventional in-vitro fertilization (IVF), using spermatozoa from an oligozoospermic man carrying a microdeletion of the long arm of the Y chromosome. The patient presented with idiopathic infertility of 10 years duration; the fertility status of his wife was completely normal. After IVF, five out of eight oocytes retrieved showed normal fertilization and four showed normal embryo cleavage. Four embryos were transferred; however, pregnancy did not result. These results demonstrate that spermatozoa from oligozoospermic patients carrying a Yq microdeletion are fully competent in achieving capacitation, acrosome reaction and fertilizing ability during IVF. Therefore, although definitive conclusions cannot be made from a single case report, we suggest that Yq microdeletion analysis should be considered in oligozoospermic patients undergoing conventional IVF.
Foresta C et al., 1998·Human reproduction (Oxford, England)
Azoospermia frequently represents the end-point of different pathological conditions that cause important quantitative and qualitative alterations of both spermatogenesis and testicular structure, including intratesticular blood vessels. In this study we performed colour Doppler ultrasound of the testis in 12 azoospermic subjects affected by primary testicular pathology (four bilateral post-orchitis, four postradiotheraphy for cancer, four post-traumatic) aged 28.2+/-3.3 (mean+/-SD) years, in six subjects affected by obstructive azoospermia aged 29.7+/-2.4 years and in 20 age-matched fertile subjects (aged 28.6+/-2.5 years). The analysis of intratesticular vessels per organ was quantified using a semiquantitative score: category 0, no vessels visible; category 1, between one and three intratesticular vessels visible; and category 2, more than three vessels visible. In obstructive azoospermic patients and in fertile subjects there were always more than three intratesticular vessels. No intratesticular vessels were detected in eight testes (33.3%) and fewer than three vessels in 16 testes (66.6%) in subjects affected by primary testicular pathology. In azoospermic subjects the testicular structure of the testis was evaluated by diagnostic fine needle aspiration cytology (FNAC) performed in the middle portion of the testis. In non-obstructive azoospermic patients this procedure showed the presence of only Sertoli cells in all cases. When detectable vessels were present, a new aspiration was performed in these areas. In 12 out of 16 cases, spermatogenetic cells including mature spermatozoa, were found when the FNAC was performed in testicular regions showing the presence of blood vessels. These results indicate that colour Doppler sonography of the testis may be useful in the differential diagnosis of azoospermia and suggest the evaluation of the intratesticular blood vessel distribution before performing any method to retrieve intratesticular spermatozoa for intracytoplasmic sperm injection.
To evaluate the effects of treatment with FSH on seminal indices and on the seminiferous epithelium of oligozoospermic subjects with normal FSH plasma levels.
Placebo-controlled, double-blind randomized study.
Academic setting.
PATIENT(S): Ninety subjects with idiopathic oligozoospermia (sperm count of < 10 x 10(6)/mL) and normal plasma levels of FSH.
INTERVENTION(S): Three months of treatment with FSH (60 patients) or placebo (30 patients); bilateral testicular fine-needle aspiration.
MAIN OUTCOME MEASURE(S): Seminal indices; testicular cytologic features; plasma levels of FSH, LH, and testosterone; and ultrasonographic testicular examination.
RESULT(S): According to seminal indices, patients treated with FSH and placebo were classified as nonresponders or as responders (as determined by at least a doubling of sperm count). No placebo-treated patients responded to treatment. Among FSH-treated patients, 20 responded to hormonal treatment and 40 did not. The results of pretreatment cytologic examination of testicular specimens from patients who did not respond to FSH treatment were consistent with hypospermatogenesis associated with maturational disturbances at the spermatid level. In contrast, patients who responded to treatment with FSH had isolated hypospermatogenesis without maturational disturbances. After FSH therapy, we detected an increase of spermatogonia and spermatocyte population in both the responder and nonresponder subjects. This increase was associated with an activation of spermatogenic and spermiogenic processes and with a rise in ejaculated sperm concentration only when isolated hypospermatogenesis was present (responder patients).
CONCLUSION(S): The findings of this study demonstrate that FSH treatment increases the spermatogonial population in men. This treatment may be appropriate for oligozoospermic subjects who have normal FSH plasma levels and a testicular evaluation characterized by hypospermatogenesis without maturational disturbances.
Foresta C et al., 1998·Human reproduction (Oxford, England)
Idiopathic Sertoli cell-only syndrome (SCOS) is characterized by azoospermia, small testes, absence of germ cells in the testes, elevated follicle stimulating hormone and normal testosterone concentrations. The Y-chromosome is involved in the regulation of spermatogenesis and in the pathogenesis of a fraction of idiopathic male infertility. An azoospermia factor (AZF) is present on the Y-chromosome long arm euchromatic region (Yq11) and two gene families (DAZ and RBM) have been identified within this region. The aim of this study was to investigate whether a specific pattern of Yq11 microdeletions may be associated with idiopathic SCOS. Eighteen idiopathic subjects showing a testicular cytological picture of bilateral SCOS were selected and tested by polymerase chain reaction for a set of 29 Y-specific sequence-tagged sites (STS). We found Yq microdeletions in 10 out of 18 patients (55.5%) while the fathers or brothers of six out of 10 patients deleted for Yq were shown to carry an intact Y-chromosome. These deletions may therefore be considered as de-novo deletions and the cause of SCOS. The analysis of the microdeletions allowed us to identify two homogeneous regions that have a high incidence of deletion. The smallest deletion, common to all patients, is located in Yq interval 5. We therefore speculate that there is a relationship between specific, well-characterized Yq11 microdeletions and a testicular picture of SCOS, identifying an Y-related region frequently deleted in this syndrome. In conclusion, the findings of this study demonstrate that a large percentage of idiopathic SCOS may be genetically determined and identify an Y-related region that seems to possess one or more still unknown genes essential for spermatogenesis.
Foresta C et al., 1997·The Journal of clinical endocrinology and metabolism
A genetic etiology has been recently proposed for some severe forms of idiopathic male infertility and a region of the Y chromosome long arm (Yq) defined AZF is thought to be critical for the regulation of spermatogenesis. To date, two genes, YRRM and DAZ, have been identified in AZF, but the actual relationship between genotype and phenotype related to AZF deletions is not well characterized. By means of a PCR strategy we typed Yq microdeletions in 16 azoospermic and 22 severely oligozoospermic subjects whose testicular cytological picture (assessed by fine needle aspiration) was that of Sertoli cell-only syndrome and severe hypospermatogenesis, respectively. Microdeletions in AZF were found in 37.5% of azoospermic men and in 22.7% of severely oligozoospermic men, suggesting that very frequently these genetic abnormalities determine a severe quantitative defect in spermatogenesis. Furthermore, DAZ and YRRM do not seem to be the sole genes regulating spermatogenesis, as deletions in these genes were observed in only 6 of the 11 deleted cases. No correlation between the spermatogenic defect and the type of Yq deletion exists. Intracytoplasmic sperm injection performed using spermatozoa of these Y-deleted patients will invariably pass this defect onto their male offspring. Screening for deletion within AZF or at least an informed consent should, therefore, be obtained in all idiopathic infertile male undergoing a program of intracytoplasmic sperm injection of a spermatozoon into the oocyte.
To characterize the testicular cytologic pictures in cryptorchild and contralateral testis related to seminal pattern.
Controlled clinical study.
Andrological and urologic academic setting.
One hundred nine patients orchidopexied because of unilateral cryptorchidism compared with 35 normospermic subjects.
Bilateral testicular fine-needle aspiration cytology.
Seminal parameters; testicular cytologic features; plasma levels of FSH, LH, and T; and ultrasound testicular examination.
The cytologic analysis revealed an important quantitative and qualitative impairment of spermatogenic line in all the excryptorchid testes. These alterations are present in the contralateral testes only when azoospermia or severe oligozoospermia were observed, whereas in moderate oligozoospermic subjects a normal tubular status was evident. A compensatory activity in normally descended testis of normozoospermic patients was present.
In unilateral cryptorchidism, a frequent testicular damage in the orchidopexied and in the contralateral descended testis suggests that this condition is the end point of different pathological conditions, including testicular intrinsic (congenital) and extrinsic (anatomical) causes. Fine-needle aspiration cytology of both testes represents a tool in the assessment of the tubular status in excryptorchid subjects.
The recent advances in assisted fertilization and gamete micromanipulation techniques have enabled fertilization in some forms of azoospermia; for example, epididymal sperm aspiration in obstructive azoospermia. Therefore knowledge of the specific degree of testicular damage is of primary importance, since other clinical parameters, such as FSH plasma levels and testicular volume, do not discriminate between the different testiculopathies. In order to further characterize the specific testicular conditions present in azoospermia, we have examined a large group of azoospermic subjects on the basis of testicular cytological analysis obtained by fine needle aspiration.
One hundred and twenty-two infertile, azoospermic men were studied by physical examination, FSH radioimmunoassay, testicular ultrasound examination and fine needle aspiration of the testes. Thirty-five infertile normozoospermic subjects were studied as controls.
The cytological analysis identified five different sub-types in azoospermic I, Sertoli cell-only syndrome; II, hypospermatogenesis; III, spermatogonial and/or spermatocytic arrest; IV, spermatidic arrest; and V, normal germ line. The testicular volume was reduced in groups I and II, while the FSH plasma levels were increased in groups I, II and III, suggesting a primary role of spermatids in the control of FSH secretion.
In azoospermic subjects, testicular cytological analysis permits the identification of different sub-types and this classification may be very important in determining therapy, particularly the choice between surgical treatment and the hypothetical use of assisted fertilization techniques by retrieval of epididymal or intratesticular spermatozoa or spermatids.