G. Grande

Male fertility and reduction in semen parameters: A single tertiary-care center experience

Background. Infertility is both a clinical and a public problem, affecting the life of the couple, the healthcare services, and social environment. Standard semen analysis is the surrogate measure of male fertility in clinical practice. Objective. To provide information about the relationship between semen parameters and spontaneous conception. Methods. We evaluated retrospectively 453 pregnancies that occurred among 2935 infertile couples evaluated at an infertility clinic of a tertiary-care university hospital, between 2004 and 2009. Results. Normal semen analysis was present only in 158 patients; 295 subfertile patients showed alterations in at least one seminal parameter. A reduction in all seminal parameters was observed in 41 patients. Etiological causes of male infertility were identified in 314 patients. Conclusion. Our data highlights the possibility of a spontaneous conception with semen parameters below WHO reference values. Therefore, we support the importance of defining reference values on a population of fertile men. Finally, we analyzed the related ethical issues.

Soluble urokinase-type plasminogen activator receptor as a putative marker of male accessory gland inflammation.

The association between male accessory gland infection/inflammation (MAGI) and infertility is well‐known in clinical practice. Standard semen analysis, leukocytospermia, and microbiological tests are often not enough accurate for a diagnosis. A large amount of biochemical parameters in seminal plasma have been suggested as inflammation markers, however, there is not yet a sensitive and specific biomarker that accurately identifies MAGI. We investigated the presence of soluble urokinase‐type plasminogen activator receptor (suPAR), known marker of systemic inflammation, in the seminal plasma to evaluate its possible involvement in urogenital tract inflammation. On the basis of andrological evaluation, including spermiogram and ultrasound findings, we selected 76 patients with MAGI and 30 healthy men as control group. Patients were classified according to the results of the semen culture in group A (n = 28) presenting a bacterial MAGI and group B (n = 48) with abacterial MAGI. C‐reactive protein (CRP), total protein (TP), procalcitonin (PCT), leukocytes peroxidase (LP), and suPAR concentrations were assayed on seminal plasma. Spermiogram parameters were significantly lower in the patients with MAGI than in controls. CRP, TP, PCT, and LP did not differ in MAGI vs. controls. suPAR was detectable in all semen samples; it was significantly increased in A and B groups (86.6 ± 30.7 ng/mL vs. 39.7 ± 17.2 ng/mL) with an inverse correlation with sperm parameters. We selected by receiver operating characteristic curve a suPAR cut‐off value of 55.3 ng/mL as a diagnostic threshold for the diagnosis of MAGI. We report in this study the first evidence of suPAR presence in seminal plasma, focusing on its interesting role as reliable and sensitive marker of inflammation for the differential diagnosis of MAGI.

Pituitary-tumour-transforming-gene 1 expression in testicular cancer

Genomic instability is a feature of germ cell tumours. The pituitary‐tumour‐transforming‐gene 1 (PTTG1) is the major effector of chromosome segregation during mitosis, protecting the cell from aneuploidy. The protein expression of this gene has been evaluated in testicular tumours by immunohistochemistry. Formalin‐fixed and paraffin‐embedded specimens of testicular tissues from 83 patients undergoing therapeutic orchidectomy for seminomas (n = 53), embryonal carcinoma (n = 10), yolk sac tumour (n = 10) and teratoma (n = 10) were examined. Seminoma was associated with in situ carcinoma (CIS) in 23 samples. PTTG1 immunostaining was performed using rabbit anti‐PTTG1 as a primary antibody. In CIS, only isolated cells showed nuclear staining for PTTG1. In the peripheral area of seminoma, PTTG1 was mostly detected as localised in the nucleus; in the central area of seminoma, PTTG1 staining was more intense in cytoplasm. PTTG1‐positive cells were also present in the areas of seminoma infiltration. On the other hand, in embryonal carcinoma, cells had a diffuse positive immunostaining, mainly cytoplasmatic, while we did not observe an expression of PTTG1 in yolk sac tumour and mature teratoma. We firstly identified the PTTG1 expression pattern in normal testis, CIS and testicular cancer. Further investigation is needed to clarify the functional activity of PTTG1 in testicular oncogenesis.

Prednisone treatment in infertile patients with oligozoospermia and accessory gland inflammatory alterations

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.

Seminal suPAR Levels as Marker of Abacterial Male Accessory Gland Inflammation in Hypogonadism

BACKGROUNDnRecent evidences suggest that hypogonadism is an important risk factor for lower urinary tract symptoms and benign prostatic hyperplasia. Several papers have discussed the role of chronic inflammation in the development of BPH, which may be modulated by the hypogonadal state. Soluble Urokinase-type Plasminogen Activator Receptor (suPAR), known protein marker of systemic inflammation, can be assayed in the seminal plasma and represents a reliable and sensitive marker of inflammation for the Male Accessory Gland Inflammation (MAGI).nnnOBJECTIVEnThe aim of this study has been to investigate if seminal suPAR is elevated in MAGI with hypogonadism and if suPAR represent a useful marker of abacterial inflammation in hypogonadism.nnnMETHODSnWe included in the study twenty male patients aged between 25 and 55 year-old with secondary postsurgical hypogonadism. The same patients were also evaluated after a 3-month of Testosterone Replacement Therapy (TRT), to evaluate the effect of androgen replacement therapy on suPAR. Ten fertile men have been enrolled as a control group in the protocol. SuPAR concentrations were assayed on seminal plasma using an Enzyme-Linked Immunosorbent Assay (ELISA) kit.nnnRESULTSnHypogonadic patients presented significantly increased levels of seminal suPAR respect to controls (86.1±36.8 vs 55.2±20.0 ng/mL, p<0.05). TRT in hypogonadic patients has been associated with a significant reduction of suPAR levels as reported in the control group (50.9±22.91 vs 86.1±36.8 ng/ml p<0.05).nnnCONCLUSIONSnThese results confirm the role of suPAR as a protein marker of MAGI and support the hypothesis that hypogonadism induces a state of inflammation in male accessory glands which is involved in male infertility. Moreover demonstrated that testosterone treatment probably exerts a positive effect on MAGI and infertility as documented by reduction of suPAR levels in hypogonadic treated patients.

Semen Proteomics Reveals the Impact of Enterococcus faecalis on male Fertility

BACKGROUNDnInfectious etiologies contribute to 15% of male factor infertility. Enterococcus faecalis (E. faecalis) is commonly identified in semen culture of infertile men and it is associated with significantly poorer semen quality.nnnOBJECTIVEnAim of this study was to identify new seminal biomarkers for the male tract infection by E. faecalis, using proteomic profiling, in order to understand the effect of E. faecalis on the physiopathology of male reproduction.nnnMETHODSnWe included in the study ten patients seeking medical care for primary infertility with prostate-vesicular-epidydimitis and with microbiological analysis on semen and/or prostatic secretions positive for E. faecalis. Ten fertile men have been enrolled as a control group in the protocol. An aliquot of each seminal plasma was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLCnano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer.nnnRESULTSnEight proteins have not been identified in the group of controls and have been observed in a remarkable proportion of patients, mainly involved in immune system activity (CD177, Swiprosin-1 and 2-oxoglutarate dehydrogenase). Arylsulfatase has been identified in the group of controls and was absent in all patients with infection. Three proteins (TIMP-1, WFDC domain protein 2 and Carboxypeptidase E) have been observed significantly different in patients versus controls, mainly related with inflammation.nnnCONCLUSIONSnThis is the first application of MS-based proteomics aimed to reveal an array of proteins in the seminal plasma and reflecting the effect of the infection by E. faecalis on semen composition.

Proteomic Markers in Cervical Mucus

BACKGROUNDnCervical Mucus (CM) is a viscous fluid produced by the secretory cells of the cervical crypts. The CM undergoes modifications throughout the cycle that make it have different biochemical and biophysical characteristics, becoming a crucial element for the identification of ovulation. Since CM is rich in secreted proteins, it may represent moreover a source of biomarkers for female reproductive tract diseases.nnnOBJECTIVEnThis review is an attempt to collect relevant knowledge about the physicochemical properties and functions of the cervical mucus, including its important role as a clinical marker of female fertility, and draws attention to CM as a source of potential proteomic biomarkers.nnnFINDINGSnAll the assessed studies evidenced that the observation of the CM allows the identification of the days with the highest probability of pregnancy. CM proteome changes throughout the menstrual cycle have been revealed. Few proteomic studies on the constitutive protein composition of CM of fertile women have been conducted to date. In the CM of patients affected by endometriosis have been identified some proteins that could represent potential biomarkers of the disease.nnnCONCLUSIONnThere is still limited knowledge about the physicochemical properties and functions of the CM and how these undergo to changes during menstrual cycle. CM is a reliable predictor of fertility. Further characterization of CM proteins would contribute to a better understanding of the key role they have on fertility, reproduction and biological regulation. CM may represent moreover a source of biomarkers for gynecological diseases.

Olfactory Receptors in Semen and in the Male Tract: From Proteome to Proteins

The estimated number of testicular olfactory receptors (ORs) in mammals range between 20 and 66. Previous data reported the role of hOR17-4 and mOR23 in sperm–oocyte chemiotaxis. Proteomic analysis was performed to understand which are the ORs expressed in seminal plasma. Seminal samples by four fertile men were analyzed by an Ultimate 3000Nano/Micro-HPLC apparatus coupled with an LTQ-Orbitrap XL hybrid mass spectrometer. Western blot analysis confirmed the expression of three identified ORs. The expression of ORs in sperm cells, testis, and epididymis was evaluated by confocal microscopy analysis. In seminal plasma eight different ORs were identified by proteomics and three ORs have been confirmed by western blot. Confocal microscopy analysis revealed that OR4S1, OR4C13, and OR1I1 are expressed on the surface of sperm cells. In testicular tissue, OR4S1 and OR1I1 are expressed in spermatocytes and spermatids and OR4C13 is expressed throughout all the tubules. In patients with spermatocyte maturation arrest OR4S1 and OR1I1 expression was reduced and a weak positivity for OR4C13 was detected in the spermatogonia. OR4S1, OR4C13, and OR1I1 had intense and diffuse staining in the epididymis. This study initiated a new methodology for screening OR repertoire in sperms, testis and epididymis. Our results open new insights into OR involvement in sperm maturation and migration.

Nuovi problemi derivanti dall’impiego delle tecniche di fecondazione artificiale

Le tecniche di fecondazione artificiale (PMA) pongono al medico che opera nel campo delle metodiche della riproduzione e al bioeticista notevoli problemi di ordine morale, per la cui risoluzione e necessaria una base scientifica a monte della valutazione etica. Questo studio analizza, dunque, gli aspetti piu propriamente scientifici in merito alla Intra Cytoplasmic Sperm Injection (ICSI). Tale tecnica e oggi quella piu usata nei centri di PMA ed ha rivoluzionato l’approccio alla sterilita di coppia. Pur tuttavia essa pone notevoli problemi, sia in merito all’impiego di seme di soggetti infertili sia connessi alla stessa tecnica. Per quanto riguarda i primi, si distinguono aspetti correlati: 1. alla presenza di anomalie citogenetiche parentali e delle cellule spermatiche, onde e stato dimostrato che pazienti infertili ammessi a programmi ICSI hanno un alto tasso di aneuploidie negli spermatozoi, inversamente correlate con i parametri seminali e il numero di forme morfologicamente normali dopo selezione; 2. all’esistenza di microdelezioni del cromosoma Y. Su tale aspetto ci si sofferma, evidenziando il ruolo dei segmenti genici coinvolti, nonche il rischio di trasmissione della microdelezione ai figli concepiti via ICSI. Sono quindi descritte le principali problematiche connesse sia alla tecnica sia all’abilita dell’operatore, evidenziando il carattere di sperimentalita della tecnica in esame. Considerando, poi, i risultati delle procedure ICSI, occorre considerare sia il tasso di fecondazione e l’outcome ostetrico sia il seppur lieve aumento di incidenza di malformazioni ed anomalie citogenetiche. Inoltre, il tentativo di ottenere gravidanze da situazioni maschili sempre piu severe, pone il problema di valutare i risultati in rapporto al quadro eziologico di partenza, ma vi sono poche osservazioni in tal senso. In conclusione vengono valutati i nuovi campi di indagine, con particolare riferimento alla round spermatid injection (ROSI), alla round spermatid nuclear injection (ROSNI) ed all’impiego di spermatogoni, fino alle forme di “semi-clonazione”, e gli ulteriori problemi che permangono ancora aperti, quali gli aspetti immunologici o la trasmissibilita dell’infezione da Human Immunodeficency Virus (HIV). ---------- The reproductive technologies (RTs) puts to the physician, involved in the reproductive medicine, and the bioethicist remarkable moral problems; therefore a solid scientific ground is necessary for an ethical evaluation. This study analyses the more properly scientific aspects, particularly related to Intracytoplasmic Sperm Injection (ICSI). Such technique, that is today the more used in the RTs centres, upsets the approach to couple sterility. It places, however, remarkable problems, both about the use of infertile man sperm and about the same technique. As to the first point, we can distinguish aspects connected to the presence of parental citogenetic anomalies and in sperm cells, so it was demonstrated that infertile patients admitted to ICSI programs have a high rate of aneuploidies in the spermatozoa, inversely correlated with the sperm parameters and the number of morphologically normal shapes after selection, and to the existence of micro-deletion of the Y chromosome. About this we meant to stop to us, evidencing the role of the deleted genic segments and the risk of transmission of the microdeletion to sons conceived via ICSI. Then the authors describe the major problems connected to the technique and to the ability of the operator, evidencing the experimentation of this technology. Considering, then, the outcomes of ICSI procedures, it is necessary to consider both the fecondation rate and the obstetrical outcome, and the slight increase of the incidence of malformations and citogenetical anomalies. Moreover, the attempt to obtain pregnancies from more and more severe male situations places the problem to estimate the outcome referred with the etiology, but there are only few observations about it. In conclusion we considered the new perspectives, specially about round spermatid injection (ROSI), round spermatid nuclear injection (ROSNI) and the use of spermatogonia, until “the semi-cloning reproduction” and the other problems still opened, as the immunological aspects or the transmission of the Human Immunodeficiency Virus (HIV).