Clinical and seminal parameters associated with testicular microlithiasis and its severity in males from infertile couples.

STUDY QUESTION: Is there an association of testicular microlithiasis (TM) and its severity with testicular dysfunction in men from infertile couples? SUMMARY ANSWER: The presence of ≥5 testis microcalcifications per sonogram at the scrotal ultrasonography (US) of infertile males was associated with a more severe testicular dysfunction as compared to males with limited, or without, TM. WHAT IS KNOWN ALREADY: TM, representing an incidental finding in the scrotal US, is associated with male infertility and a higher risk for testicular cancer as compared to that in infertile males without TM. Still, there are unresolved questions on the relation between TM severity and testicular dysfunction in infertile men, as well as on the identification of risk factors for TM. STUDY DESIGN, SIZE, DURATION: This study was an observational, retrospective, case-control investigation involving males who underwent clinical evaluation, measurement of reproductive hormones, seminal analysis and scrotal US as part of diagnostic work-up for couple infertility at an andrology clinic, between January 2004 and December 2018. One hundred patients, out of the 2112 scored men, were found to have TM during the US evaluation. One hundred male partners from 100 infertile couples without TM, comprising the control group, were selected through a matched analysis by age and date of evaluation to reduce the confounding effect of both age and technique variability all along the long period of observation. PARTICIPANTS/MATERIALS, SETTING, METHODS: TM was defined as limited TM (LTM) or classical TM (CTM), when the maximum number of hyperecogenic spots per sonogram was <5 or ≥5, respectively. CTM, LTM and control groups were compared for clinical variables, serum levels of FSH, LH, and total testosterone, as well for semen parameters and scrotal US features. MAIN RESULTS AND THE ROLE OF CHANCE: After the exclusion of cases with testicular nodules to eliminate the possible confounding effect of testis cancer on testicular dysfunction, cases with CTM showed a lower mean testis volume (P = 0.03) and a lower sperm concentration (P = 0.03) as compared to the other two groups. A higher FSH level was observed in the CTM group compared to the LTM group (P = 0.02) and in controls (P = 0.009). The multiple logistic regression analysis showed that only a smaller testicle volume exhibited an independent significant association with a higher odds of detecting CTM (odds ratio = 0.84, 95% CI: 0.75-0.94; P = 0.02). No significant differences were observed between groups in the prevalence of risk factors for testicular cancer, or in the prevalence of conditions associated with TM. LIMITATIONS, REASONS FOR CAUTION: The retrospective design of the study did not allow conclusions to be drawn about the possible underlying links in the associations of TM with defective spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: Males from infertile couples who exhibit a reduced testicular volume should undergo scrotal US, independent of sperm parameters, to exclude CTM and, eventually, testis cancer, although the association of CTM and current or future testis cancer risk is not yet clear. Evidence is provided here demonstrating that the presence of LTM has no clinical relevance in males from infertile couples. STUDY FUNDING/COMPETING INTEREST(S): Investigation was funded by Ministero dell'Università e della Ricerca, PRIN 2018, Italy. The authors have not declared any competing interests. TRIAL REGISTRATION NUMBER: N/A.

Relationship of Vitamin D status with testosterone levels: a systematic review and meta-analysis.

PURPOSE: Despite a biological plausibility of a direct link between low vitamin D and androgen deficiency, the association remains inconclusive in epidemiological studies. Therefore, this systematic review and meta-analysis of case-control studies aim to assess whether and in what populations such an association can be demonstrated. METHODS: A systematic search was performed in PubMed, EMBASE, Cochrane Library, Web of science, Science Direct, and CINAHL. Standardized mean differences (SMDs) and 95% confidence intervals (CIs) in total testosterone (TT) levels between men with 25-hydroxyvitamin D (25(OH)D) <20 and ≥20 ng/mL were combined using random-effects models. Funnel plot and trim-and-fill analysis were used to assess publication bias. Heterogeneity source was explored by a sub-group analysis according to health-related characteristics of the study populations. RESULTS: Eighteen included studies collectively gave information on 9892 men with vitamin D deficiency and 10,675 controls. The pooled SMD revealed a slight, albeit just significant, positive association between 25(OH)D and TT (pooled SMD: -0.23, 95% CI: -0.45 to -0.01; P = 0.04) with a large between-study heterogeneity (I2 = 98%, Pfor heterogeneity < 0.00001). At the sub-group analysis, a significant positive association, along with noticeable decrease in heterogeneity, could only be demonstrated in studies of patients with frailty states (pooled SMD: -0.19; 95% CI: -0.27, -0.10, P < 0.0001; I2 = 51%, Pfor heterogeneity = 0.06). A sensitivity analysis revealed a high stability of the result and the trim-and-fill adjustment for publication bias did not affect pooled estimate. CONCLUSIONS: Both hypovitaminosis D and androgen deficiency should be regarded as markers of a poor health status, sharing common underlying aetiologies and risk factors.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Scrotal ultrasound reference ranges and associations with clinical, seminal, and biochemical characteristics.

BACKGROUND: Scrotal color Doppler ultrasound (CDUS) still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study to assess the CDUS characteristics of healthy fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report and discuss the scrotal organs CDUS reference ranges and characteristics in HFM and their associations with clinical, seminal, and biochemical parameters. METHODS: A cohort of 248 HFM (35.3 ± 5.9years) was studied, evaluating, on the same day, clinical, biochemical, seminal, and scrotal CDUS following Standard Operating Procedures. RESULTS: The CDUS reference range and characteristics of the scrotal organs of HFM are reported here. CDUS showed a higher accuracy than physical examination in detecting scrotal abnormalities. Prader orchidometer (PO)- and US-measured testicular volume (TV) were closely related. The US-assessed TV with the ellipsoid formula showed the best correlation with the PO-TV. The mean TV of HFM was ~ 17 ml. The lowest reference limit for right and left testis was 12 and 11 ml, thresholds defining testicular hypotrophy. The highest reference limit for epididymal head, tail, and vas deferens was 12, 6, and 4.5 mm, respectively. Mean TV was associated positively with sperm concentration and total count and negatively with gonadotropins levels and pulse pressure. Subjects with testicular inhomogeneity or calcifications showed lower sperm vitality and concentration, respectively, than the rest of the sample. Sperm normal morphology and progressive motility were positively associated with epididymal head size/vascularization and vas deferens size, respectively. Increased epididymis and vas deferens sizes were associated with MAR test positivity. Decreased epididymal tail homogeneity/vascularization were positively associated with waistline, which was negatively associated with intratesticular vascularization. CDUS varicocele was detected in 37.2% of men and was not associated with seminal or hormonal parameters. Scrotal CDUS parameters were not associated with time to pregnancy, number of children, history of miscarriage. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology, improving its management.

Clinical features and prognosis of COVID-19 in people with spinal cord injury: a case-control study.

STUDY DESIGN: Observational case-control study. OBJECTIVE: Individuals with spinal cord injury (SCI) develop systemic physiological changes that could increase the risk of severe evolution of coronavirus disease 2019 (COVID-19) and result in atypical clinical features of COVID-19 with possible delay in both diagnosis and treatment. We evaluated differences in clinical features and evolution of COVID-19 between people with SCI and able-bodied individuals. SETTING: The study was conducted in an Italian inpatient rehabilitation referral center for individuals with SCI during the lockdown for the COVID-19 pandemic. METHODS: We compared clinical information between patients with SCI and able-bodied healthcare workers of the same center who tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the nasopharyngeal swab polymerase chain reaction. RESULTS: Overall, 15 out of the 25 SCI patients admitted to the center and 17 out of the 69 healthcare workers tested positive for SARS-CoV-2. Patients with SCI exhibited a significantly more advanced age and a higher prevalence of comorbidities. Nevertheless, no significant differences in clinical expression of COVID-19 and treatment strategies were observed between the two groups. All hospitalized subjects were treated in nonintensive care units and no deaths occurred in either group. CONCLUSIONS: This study does not support the supposed notion that COVID-19 could exhibit atypical clinical features or a worse evolution in the frail population of people with SCI.

Relationship between natural and intrauterine insemination-assisted live births and the degree of sperm autoimmunisation.

STUDY QUESTION: What is the relationship between the degree of sperm autoimmunisation, as assessed by IgG-mixed antiglobulin reaction (MAR) test, and natural and intrauterine insemination (IUI)-assisted live births? SUMMARY ANSWER: Compared with a lower degree of positivity (50-99%), a 100%-positive MAR test was associated with a much lower occurrence of natural live births in infertile couples, who could be successfully treated with IUI, as first-line treatment. WHAT IS KNOWN ALREADY: The World Health Organization (WHO) has recommended screening for antisperm antibodies, through either the IgG-MAR test or an immunobead-binding test, as an integral part of semen analysis, with 50% antibody-coated motile spermatozoa considered to be the clinically relevant threshold. However, the predictive value of the degree of positivity of the MAR test above such a cut-off on the occurrence of natural pregnancies remains largely undetermined. Furthermore, the effectiveness of IUI in cases of strong sperm autoimmunisation is not yet well-established. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study on 108 men with a ≥50%-positive MAR test, where the couple had attended a university/hospital andrology/infertility clinic for the management of infertility from March 1994 to September 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: The IgG-MAR test was carried out as an integral part of semen analysis. The patients were divided into two groups: 100% and 50%-99%-positive MAR test. The post-coital test (PCT) was performed in all the couples, and IUI was offered as the first-line treatment. Laboratory and other clinical data were retrieved from a computerised database. Data on subsequent pregnancies were obtained by contacting patients over the telephone. MAIN RESULTS AND THE ROLE OF CHANGE: A total of 84 men (77.8%) were successfully contacted by telephone, and they agreed to participate. Of these, 44 men belonged to the group with a 100%-positive MAR test, while 40 showed lower MAR test positivity. The couples with a 100%-positive MAR test showed a natural live birth rate per couple (LBR) that was considerably lower than that observed with a lower degree of positivity (4.5% vs. 30.0%; P = 0.00001). Among the clinical variables, a significant difference between the two groups was observed only for the PCT outcome, which was poor in the 100%-positive MAR test group. Better PCT outcomes (categorised as negative, subnormal and good) were positively associated with the occurrence of natural live births (6.3, 21.7 and 46.2%, respectively; P = 0.0005 for trend), for which the sole independent negative predictor was the degree of sperm autoimmunisation. IUI was performed as the first-line treatment in 38 out of 44 couples with a 100%-positive MAR test, yielding 14 live births (36.8%). In couples with lower MAR test positivity, the LBR after IUI (26.9%) was similar to the natural LBR in this group (30.0%). LIMITATIONS, REASONS FOR CAUTION: Given the retrospective nature of the study, we cannot exclude uncontrolled variables that may have affected natural pregnancies during the follow up or a selection bias from the comparison of natural live births with those after IUI. WIDER IMPLICATIONS OF THE FINDINGS: The routine use of the IgG-MAR test in the basic fertility workup is justified as it influences decision making. A 100%-positive IgG-MAR test can represent the sole cause of a couple's infertility, which could be successfully treated with IUI. On the other hand, a lower degree of positivity may only represent a contributing factor to a couple's infertility, and so the decision to treat or wait also depends on the evaluation of conventional prognostic factors including the PCT outcome. STUDY FUNDING, COMPETING INTEREST(S): This study was supported by PRIN 2017, Ministero dell'Università e della Ricerca Scientifica (MIUR), Italy. On behalf of all authors, the corresponding author states that there is no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

SARS-CoV-2 infection, male fertility and sperm cryopreservation: a position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) (Società Italiana di Andrologia e Medicina della Sessualità).

PURPOSE: 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) METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Testosterone, level of the lesion and age are independently associated with prostate volume in men with chronic spinal cord injury.

PURPOSE: Although men with spinal cord injury (SCI) exhibit a prostate volume significantly smaller compared to age-matched able-bodied men, the independent association of lower prostate volume with its putative determinants has never been analyzed in this population. This study was designed to identify variables independently associated with prostate volume in men with chronic SCI. METHODS: In this cross-sectional study, prostate volume of 138 men with chronic (> 1 years) SCI, aged 54.5 (25th-75th percentile: 36.0-66.0) years, was evaluated with trans-rectal ultrasonography. All patients underwent a complete neurological exam, as well as biochemical and hormonal assessment, including total testosterone (TT) levels. Free testosterone levels were calculated (cFT) by the Vermeulen formula. RESULTS: The median prostate volume was 23.4 mL. At the univariate analysis, a larger prostate volume was associated with higher TT (p = 0.00001) and cFT (p = 0.001), SCI level below T12 (p = 0.007), more advanced age (p = 0.04), lower body mass index (p = 0.04), higher functional independence score (p = 0.06), higher values of prostate-specific antigen (p = 0.12) and shorter duration of the injury (p = 0.21). However, at the multiple regression analyses, an independent and positive association only persisted between the prostate volume with either TT or cFT levels, and, to a lesser extent, with age and a level of spinal lesion below T12. A prostate volume below the median value was observed in 91.4% (32/35) of patients with both androgen deficiency (TT < 264 ng/dL) and spinal lesion level ≥ T12, but only in 16.5% (2/12) of patients with both normal androgen levels and spinal lesion level below T12 (p < 0.001). CONCLUSIONS: Our data indicate that lower testosterone levels and, to a lesser extent, a younger age and a spinal lesion level ≥ T12 represent the only variables exhibiting an independent association with a smaller prostate volume in men with SCI.

Metabolic syndrome is the key determinant of impaired vaginal lubrication in women with chronic spinal cord injury.

PURPOSE: Spinal cord injury (SCI) affects sexual health of both male and female, but little attention has been given to sexuality of SCI women. Similar to penile erection, vaginal lubrication represents a neurovascular event and then both denervation and vascular damage might contribute to its impairment. Nevertheless, the relative weight of lesion location/degree and vascular risk factors in determining hypolubrication in women with SCI has not yet been investigated. The aim of this study was to recognize among putative determinants of poor sexual arousal in women with SCI, neurogenic and vascular/metabolic independent predictors of vaginal hypolubrication. METHODS: Twenty-eight consecutive female patients admitted to a rehabilitation program because of chronic SCI (≥ 1 year) underwent clinical and biochemical evaluations, including assessment of vaginal lubrication by the Female Sexual Function Index (FSFI). As, in people with SCI, waist circumference overestimates visceral fat mass due to abdominal muscle paralysis, metabolic syndrome (MetS) was defined according to specific criteria proposed for SCI population: BMI ≥ 22 kg/m2 and two or more of the following: triglycerides ≥ 150 mg/dL (or actual treatment), HDL < 50 mg/dL, hypertension (or actual treatment), fasting glucose ≥ 100 mg/dL or diabetes mellitus type 2. RESULTS: A FSFI lubrication sub-score < 3.6, suggestive for impaired vaginal lubrication, was exhibited by 53.7% of the study population. When compared to the group with normal lubrication, a significantly higher proportion of these women had paraplegia (93.3% vs 38.5%, p = 0.003) and met the SCI-specific criteria for MetS (73.4% vs 7.6%, p = 0.0006), whereas, no significant differences were found between the two groups in the proportion of women exhibiting the single components of MetS. At the multiple logistic regression analysis, only the presence of MetS exhibited a significant independent association with impaired vaginal lubrication (OR = 3.1, 95% CI 1.2, 5.8, p = 0.01). CONCLUSIONS: In women with SCI, a clustering of modifiable vascular/metabolic risk factors, constituting the MetS, could contribute to sexual dysfunctions by affecting the vaginal lubrication, independently of the level of the spinal cord lesion.

Thyroid autoimmunity and risk of post-partum depression: a systematic review and meta-analysis of longitudinal studies.

PURPOSE: The aim of this study was to systematically investigate whether, and to what extent, the detection of thyroid autoimmunity during pregnancy and in the weeks after childbirth is associated with an increased risk of developing post-partum depression (PPD), a condition associated with possible adverse outcomes for both mother and offspring. We performed a systematic review and meta-analysis of longitudinal studies, assessing the incidence of PPD in women with and without anti-thyroperoxidase antibody (TPOAb) positivity. METHODS: We searched MEDLINE, EMBASE, Web of Science, Cochrane Library, and CINAHL. Methodological quality of the studies was assessed by the Newcastle-Ottawa Scale. In the presence of even modest between-studies heterogeneity, assessed by Cochrane Q and I2 tests, risk ratios (RRs) for PPD were combined using a random effects model. Funnel plot and trim-and-fill analysis were used to assess publication bias. RESULTS: Five included studies provided information on 449 women with TPOAb-positive and 2483 TPOAb-negative women. Pooled RR indicated a significantly increased risk to develop PPD in TPOAb-positive group (RR 1.49, 95% CI 1.11-2.00; P = 0.008; I2 = 47%, Pfor heterogeneity = 0.11). Consistent with a possible publication bias, the trim-and-fill test detected two putative missing studies in the funnel plot. Nevertheless, the adjustment for publication bias produced a negligible effect on the pooled estimate (adjusted RR 1.41, 95% CI 1.18-1.68, P = 0.0002). CONCLUSIONS: Thyroid autoimmunity during pregnancy and in the weeks after childbirth is associated with an increased risk of developing PPD. Further well-designed studies are warranted to confirm this association and elucidate underlying pathophysiological mechanisms. PROSPERO REGISTRATION: CRD42019129643.

Relationship between leukocytospermia, reproductive potential after assisted reproductive technology, and sperm parameters: a systematic review and meta-analysis of case-control studies.

BACKGROUND: The association of leukocytospermia with male fertility is still under debate. OBJECTIVE: To evaluate the impact of leukocytospermia (≥1 × 106  white blood cells/mL of semen, according to the World Health Organization) in men attending a fertility clinic for couple subfertility, on fertility outcomes after assisted reproductive technology (ART) and on semen quality. MATERIALS AND METHODS: A systematic review with meta-analysis of case-control studies reporting mean ± standard deviation for values of different seminal parameters (sperm concentration, progressive motility, sperm morphology, sperm DNA fragmentation, semen volume, and Ph) and fertilization rate (FR), or the odds ratio (OR) for clinical pregnancy rate (PR) per cycle after ART in leukocytospermic and non-leukocytospermic patients was performed. A literature search was carried out in MEDLINE and SCOPUS for English-language studies published till June 2018. RESULTS: Twenty-eight case-controlled retrospective studies met the inclusion criteria, comparing fertility outcomes after ART or semen parameters in men with or without leukocytospermia. FR and PR after ART were not significantly different in the two groups. Leukocytospermic samples showed a lower sperm concentration (pooled SMD = -0.14; 95% CI: -0.28, -0.01, I2  = 71%, pfor heterogeneity  < 0.00001) and a lower progressive motility (pooled SMD = -0.18; 95% CI: -0.29, -0.06; I2  = 59%, pfor heterogeneity  < 0.0001). However, the significant differences disappeared, along with the large inter-study heterogeneity, when analyses were restricted to studies clearly reporting the inclusion of men without clinical evidence of seminal tract infection. DISCUSSION AND CONCLUSION: Leukocytospermia in men seeking consultation for couple subfertility is not associated with a reduced fertility after ART and with altered semen quality in populations asymptomatic for genital tract infection. Therefore, the current clinical criteria for definition of leukocytospermia should be re-assessed in subfertile couples attending a fertility clinic.

Left spermatic vein reflux after varicocele repair predicts pregnancies and live births in subfertile couples.

PURPOSE: Varicocele repair (VR) is proposed to improve fertility in subfertile couples with a poor semen quality. We explored whether the disappearance of a left continuous spermatic venous reflux (SVR) at scrotal colour-Doppler ultrasound (CDUS), an objective evidence for a VR, is predictive for improved fertility outcome in subfertile couples. METHODS: VR was performed by left internal spermatic vein sclero-embolisation in 88 males seeking a consultation for subfertility. Semen analysis and scrotal CDUS were evaluated before and 6 months after VR. Serum levels of FSH and total testosterone (TT) were obtained at baseline. Telephone interview was used to obtain information about fertility outcome. Significant predictors of pregnancy and live births, selected by univariate analyses, were included into multiple logistic regression models to assess independent associations. RESULTS: At the adjusted model, independent significant predictors of pregnancy after VR were a disappeared SVR at CDUS (OR = 5.5, 97.5% CI 2.01-15.4; p = 0.0009) and an improved sperm total motile count (TMC) (OR = 5.5, 97.5% CI 1.4-27.9; p = 0.02). Even live births were independently associated with both disappeared left SVR at CDUS (OR = 4.3, 97.5% CI = 1.6-11.8; p = 0.003) and improved TMC after VR (OR = 4.8, 97.5% CI 1.3-24.2; p = 0.02). CONCLUSION: The still controversial effect of varicocele repair on fertility in subfertile couples may reside on the undefined objective methods to document a successful VR and its effect on couple fertility. The disappearance of a continuous left SVR at CDUS after VR was the objective best predictor for subsequent improved fertility in subfertile couples.

Prevalence of anti-sperm antibodies and relationship of degree of sperm auto-immunization to semen parameters and post-coital test outcome: a retrospective analysis of over 10 000 men.

STUDY QUESTION: What is the prevalence and the relationship of anti-sperm antibodies (ASA), screened by means of IgG-mixed anti-globulin reaction (MAR) test, to semen quality and post-coital test (PCT) outcome? SUMMARY ANSWER: A 100% positive IgG-MAR test, detected in 2% of the study population, was associated with lower sperm output and progressive motility, and was the sole determinant of higher prevalence of a negative PCT outcome. WHAT IS KNOWN ALREADY: Although ASA may affect sperm fertilizing ability and the IgG-MAR test is recommended by the World Health Organization (WHO) as an integral part of semen analysis for screening the occurrence of ASA, the prevalence and clinical relevance of positive MAR test results remain controversial. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of 12 296 consecutive men who attended a university/hospital andrology clinic for the evaluation of fertility potential was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunological screening with the IgG-MAR test was performed on all ejaculates as an integral part of semen analysis. Positive samples (≥10%) were further tested for IgA-ASA. The prevalence of positive IgG-MAR tests results, along with the relationship of the degree of sperm auto-immunization to semen parameters and PCT outcome, were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: After excluding semen samples showing azoospermia or severe oligo-asthenozoospermia, the prevalence of a positive IgG-MAR test in the remaining 10 025 men was 4%, 3.4% and 2%, with 10%, 50% and 100% thresholds, respectively. The 100%-positive MAR tests exhibited significantly higher consistency over time, and were significantly associated with higher prevalence of a mixed pattern (i.e. when the majority of sperm exhibited beads attached on both the head and along the tail) of positivity as well as with the concomitant occurrence of IgA-ASA. Additionally, the 100%-positive MAR tests were significantly associated with a lower median value of the total number of spermatozoa and progressive motility, compared to samples with a lower degree of positivity or negative samples. In the PCT performed in 120 couples, where ASA were detected in the male partner, the 100%-positive MAR tests were significantly associated with a higher prevalence of negative PCT outcome, in comparison to the lower degree of positivity, independent of, and without any significant contribution from, other determinants (semen and cervical mucus quality). LIMITATIONS, REASONS FOR CAUTION: Only surrogate infertility-related end-points were analyzed in the present study. However, since the impairment of sperm penetration through the cervical mucus represents the primary mechanism of ASA-interference with fertility, PCT outcome may represent a suitable clinical end-point. WIDER IMPLICATIONS OF THE FINDINGS: The present study, being the largest reported to date, provides a reliable estimate of ASA prevalence. Moreover, it indicates that a 50%-positive MAR test, which is suggested by WHO as the clinically-relevant threshold, also includes patients with a degree of sperm auto-immunization that contributes to couple infertility only in the presence of other causal factors; conversely, the 100%-positive MAR test can represent the sole determinant of couple infertility, as it was the sole significant predictor of the highly prevalent negative PCT outcome. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the University of L'Aquila, Italy. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Can the positive association of osteocalcin with testosterone be unmasked when the preeminent hypothalamic-pituitary regulation of testosterone production is impaired? The model of spinal cord injury.

PURPOSE: Osteocalcin (OCN), released from the bone matrix during the resorption phase, in its undercarboxylated form, stimulates testosterone (T) biosynthesis in mouse and a loss-of-function mutation of its receptor was associated with hypergonadotropic hypogonadism in humans. Nevertheless, when population-based studies have explored the OCN-T association, conflicting results have been reported. Hypothesizing that the evidence of a positive association between OCN and T could have been hindered by the preeminent role of a well-functioning hypothalamus-pituitary axis in promoting T biosynthesis, we explored this association in men with chronic spinal cord injury (SCI), exhibiting high prevalence of non-hypergonadotropic androgen deficiency. METHODS: Fifty-five consecutive men with chronic SCI underwent clinical/biochemical evaluations, including measurements of total T (TT), OCN and 25(OH)D levels. Free T (FT) levels were calculated by the Vermeulen formula. Comorbidity was scored by Charlson comorbidity index (CCI). RESULTS: A biochemical androgen deficiency (TT < 300 ng/dL) was observed in 15 patients (27.3%). TT was positively correlated with OCN, 25(OH)D and leisure time physical activity and negatively correlated with age, BMI and CCI. OCN was also positively correlated with calculated FT and negatively correlated with BMI and HOMA-IR. At the multiple linear regression analyses, a positive association of OCN with TT and calculated FT persisted after adjustment for confounders. CONCLUSIONS: The positive association here found between OCN and T levels in men with chronic SCI reinforces the notion that a bone-testis axis is also functioning in humans and suggests that it can be unmasked when the preeminent hypothalamic-pituitary regulation of T production is impaired.

Determination of spermatic vein reflux after varicocele repair helps to define the efficacy of treatment in improving sperm parameters of subfertile men.

PURPOSE: A continuous spermatic venous reflux (SVR) at colour Doppler ultrasound (CDU) is an evidence for varicocele, a frequent correlate for male subfertility. We explored whether SVR after left varicocele repair is predictive for changes in semen quality in subfertile men. METHODS: Blood hormones (FSH, LH, and total testosterone) and scrotal CDU were obtained in subfertile patients with left grade II or grade III varicocele on physical evaluation and a poor sperm quality. Semen analysis and CDU were re-evaluated 6 months after a retrograde internal spermatic vein scleroembolisation. RESULTS: The retrospective study included 100 men with a baseline SVR >3 cm/s; 60 men showed a disappearance (group 1) and 40 men (group 2) showed a reduced SVR after varicocele repair. Total motile sperm count (TMC) was markedly increased after treatment (p < 0.0001; F = 35.79) and the increase was more relevant in group 1 compared to group 2 (p = 0.04; F = 4.20). TMC and left SVR values after varicocele repair were negatively correlated (R = -0.218; p = 0.035). Multivariate analysis showed that adjusted SVR after repair negatively predicted TMC change (TMC after repair minus baseline TMC) (ß = -2.56; p = 0.022). Disappearance of a continuous left SVR at CDU after varicocele repair was associated to a better improvement of semen parameters in subfertile men. CONCLUSION: Recording of a continuous left spermatic vein reflux is an objective method to assess a successful varicocele repair aimed to improve sperm parameters in subfertile men.

2-arachidonoylglycerol levels are increased in leukocytospermia and correlate with seminal macrophages.

Evidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. As eCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of the eCBs, N-arachidonoylethanolamine (AEA), and 2-arachidonoylglycerol (2-AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content of AEA and 2-AG was measured by high-performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106 leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow-cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+, HLA-DR+). The levels of 2-AG, but not of AEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2-AG with macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2-AG are higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.

Semen leukocytes and oxidative-dependent DNA damage of spermatozoa in male partners of subfertile couples with no symptoms of genital tract infection.

The influence of seminal leukocytes on generation of oxidative damage to sperm DNA was here investigated on male partners of subfertile couples asymptomatic for a genital tract infection. The study included 111 ejaculates from men attending the Andrology Centre at University of L'Aquila. Semen leukocytes subset included round cells expressing pan-leukocyte CD45 antigen, monocyte/macrophage lineage antigen CD14, and activated macrophages HLA-DR antigen. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression identified spermatozoa with DNA oxidative adducts while terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labeling (TUNEL) assay detected spermatozoa with DNA fragmentation. Flow cytometry and immunocytochemistry was used for determinations. Main outcome measure was the association of semen leukocyte subpopulations with spermatozoa showing oxidative-related DNA damage and with routine semen parameters. Leukocyte subpopulations were strictly correlated (p < 0.0001), but no association was found between the concentration of leukocytes, semen parameters, the percentage of TUNEL-positive and of 8-OHdG-positive spermatozoa. The percentage of 8-OHdG-positive spermatozoa was positively correlated with the percentage of TUNEL-positive spermatozoa (r = 0.48; p < 0.0001) and negatively correlated with sperm concentration (r = -0.44; p < 0.0001). Sperm concentration and the percentage of TUNEL-positive spermatozoa independently contributed (ß = -0.25, p = 0.008; ß = 0.23, p = 0.05, respectively) to the variation in percentage of 8-OHdG-positive spermatozoa after adjusting for age, abstinence time, and smoking. In conclusion, oxidative-dependent DNA damage in spermatozoa was associated to poor semen quality but not to different leukocyte subpopulations in ejaculates of men asymptomatic for a genital tract infection.

Embolization of left spermatic vein in non-obstructive azoospermic men with varicocele: role of FSH to predict the appearance of ejaculated spermatozoa after treatment.

PURPOSE: Varicocele repair in non-obstructive azoospermia (NOA) was occasionally associated to ejaculated spermatozoa independently from clinical and laboratory measures. We performed a prospective study in infertile men affected by NOA and left side varicocele to find whether or not the appearance of ejaculated spermatozoa after varicocele repair is predicted by baseline measures. METHODS: Patients with NOA and grade II, or grade III left side varicocele were submitted to hormone analysis and to scrotal color Doppler ultrasound (CDU). Azoospermia was confirmed in 23 patients aged 25-47 years who were than submitted to varicocele repair through a retrograde internal spermatic vein embolization. Patients were re-evaluated after 6 months. RESULTS: Six months after varicocele repair 12 patients (52.2 %) were still azoospermic (Group 1) while 11 patients (47.8 %) reported ejaculated spermatozoa (Group 2) [sperm count: 1.3 × 10(6)/mL; 0.5 × 10(6)/mL-1.6 × 10(6)/mL (median 25th-75th centiles)]. Serum baseline FSH was lower in Group 2 compared to Group 1 (p = 0.012), while no differences between groups were revealed for all other clinical and laboratory parameters. ROC analysis indicated that baseline FSH level predicted the appearance of ejaculated spermatozoa after treatment [AUC = 0.811; 95 % Confidence Interval (CI) 0.6-0.9; p = 0.0029]. A cut-off level of FSH <10.06 mIU/mL identified 82.0 % of cases with ejaculated spermatozoa with a specificity of 81.8 % and a sensitivity of 83.3 %. CONCLUSION: Selected patients with NOA may show ejaculated spermatozoa after a non-invasive repair of a left side varicocele, therefore avoiding testicular sperm extraction. Baseline serum FSH was a valuable predictor for ejaculated spermatozoa after treatment.

Correlates of low testosterone in men with chronic spinal cord injury.

Although high rates of serum testosterone deficiency have been reported in men with spinal cord injury (SCI), its determinants and attributes are not yet established. The aim of this study was to recognize, among putative determinants and attributes of androgen deficiency, those significantly associated to low testosterone after adjustment for confounders recognizable in men with chronic SCI. A biochemical androgen deficiency (total testosterone <300 ng/dL) was exhibited by 18 of 51 patients (35.3%). Significant correlates of testosterone levels were as follows: weekly leisure time physical activity (LTPA) explored by the LTPA Questionnaire for people with SCI, body mass index (BMI), homeostatic model assessment of insulin resistance (HOMA-IR), triglycerides and sexual symptoms, explored by the aging males' symptom (AMS) questionnaire. At the multiple linear regression analysis, among putative determinants of low testosterone, only weekly LTPA and BMI exhibited a significant association with testosterone levels, explaining 54.2 and 9.0% of testosterone variability respectively. At the linear regression models, among various putative attributes of androgen deficiency, only lower sexual desire and, at a lesser extent, higher HOMA-IR, exhibited significant associations with lower testosterone levels, after adjustment for BMI, age, comorbidities and weekly LTPA. In conclusion, poor LTPA, high BMI and low sexual desire are independent predictors of low testosterone in men with chronic SCI. This is relevant to clinical practice, as all these features are routinely assessed in rehabilitation settings for SCI. As poor LTPA and high BMI are modifiable life-style related risk factors, prospective studies could clarify whether life-style modification could increase the level of testosterone and improve the low sexual desire, relevant clinical attribute of low testosterone in men with SCI.

Ultrasonographic caput epididymis diameter is reduced in non-obstructive azoospermia compared with normozoospermia but is not predictive for successful sperm retrieval after TESE.

STUDY QUESTION: Is the ultrasonographic determination of the caput epididymis diameter predictive for sperm retrieval after testicular sperm extraction (TESE) in non-obstructive azoospermia (NOA)? SUMMARY ANSWER: Ultrasonographic determination of the caput epididymis diameter did not give any relevant clinical information in NOA and was not predictive for positive sperm retrieval after TESE. WHAT IS KNOWN ALREADY: The diameter of the caput epididymis in ultrasonography (US) has a diagnostic relevance in azoospermic men to correctly identify obstructive azoospermia; however, its clinical value in NOA is not yet determined. STUDY DESIGN, SIZE, DURATION: We performed a retrospective study of 100 azoospermic and 160 normozoospermic men attending a university infertility clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were submitted to scrotal US to determine the mean value of bilateral testicular volumes (ml), the bilateral longitudinal caput diameter (mm) and the antero-posterior diameter of the corpus (mm) epididymis. The number of spermatozoa retrieved after TESE and the testicular histology of azoospermic men was obtained and the percentage of seminiferous tubules with elongated spermatids (%T) was used to classify cases with normal spermatogenesis (obstructive azoospermia) (OA) (n = 20; %T ≥ 80) or with NOA (n = 80; %T < 70). MAIN RESULTS AND THE ROLE OF CHANCE: The US testes volumes and caput diameters were reduced (P < 0.05) in NOA compared with OA and with normozoospermia, but the corpus values were not different. The caput diameter in the side submitted to biopsy was significantly reduced when germinal epithelium was absent (Sertoli cell only) (P < 0.05) and the lowest value of caput diameter was observed when the seminiferous epithelium and tubule lumen were absent (testicular hyalinosis). On the contrary, a total arrest of spermatogenesis at the first meiosis level, or a defect of spermiogenesis resulting in scattered elongated spermatids in each tubule, did not show a reduced diameter of caput epididymis compared with normozoospermia. The caput diameter did not show any difference between NOA patients with or without successful sperm retrieval at TESE. On the contrary testicular volume was significantly reduced in NOA patients with no sperm retrieval (P = 0.0037). The caput diameter was not correlated with the number of retrieved sperm, the serum level of follicle stimulating hormone, or with the percentage of tubules with elongated spermatids at histological analysis. LIMITATIONS, REASONS FOR CAUTION: The aetiology of NOA was not included in the statistical analysis due to the low rate of cases with a specific aetiology for a testicular failure. Larger studies should exclude the possibility that besides testicular histology, aetiology of NOA might influence the diameter of caput epididymis. Moreover, whether a reduced diameter of caput epididymis is only a result of a testicular pathologic phenotype or whether it may underscore a primitive dysfunction influencing the number of ejaculated spermatozoa is not yet determined. WIDER IMPLICATIONS OF THE FINDINGS: We reported that US diameter of the caput epididymis is reduced in cases of NOA but, in contrast with the testicular volume, it is independent of the completion of spermatogenesis and subsequent presence of spermatozoa in the epididymis. Therefore ultrasonographic determination of caput epididymis diameter is not predictive for positive sperm retrieval after TESE in cases of a primitive testicular failure. Our novel findings may help to define which reproducible parameters of scrotal US should be assessed in the work-up of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Ministero dell'Università e Ricerca (I) PRIN 2009. The authors declare no competing interest.

Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa.

Gram-negative bacteria frequently involved in urogenital tract infections release the endotoxin lipopolysaccharide (LPS); its receptor, toll-like receptor-4 (TLR4), has been recently identified in human spermatozoa, and its direct activation has been suggested in mediating adverse effects of LPS on human spermatozoa. However, the underlying signal transduction remains to be clarified. In other cell types, LPS induces the generation of endocannabinoids, which are involved in mediating endotoxin effects. In human spermatozoa, which exhibit a completely functional endocannabinoid system, the activation of cannabinoid receptor-1 (CB1) inhibited sperm mitochondrial membrane potential (ΔΨm). In this study, we tested the hypothesis of a contribution of CB1 activation by sperm-generated endocannabinoids in the adverse effects exerted by LPS on human spermatozoa. The exposure of motile sperm suspensions to E. coli LPS produced a significant decrease in sperm ΔΨm, assessed at flow cytometry with JC-1, similar to that induced by Metanandamide (Met-AEA), a non-hydrolyzable analogue of the endocannabinoid AEA. The LPS-induced inhibition of ΔΨm was prevented by the selective CB1 cannabinoid receptor antagonist, SR141716. However, the inhibition of ΔΨm induced by either LPS or Met-AEA did not affect sperm motility. Consistent with this finding, the CB1-mediated inhibition of ΔΨm was neither associated to mitochondrial generation of reactive oxygen species as evaluated by flow cytometry with MytoSox Red nor to apoptosis pathway activation as evaluated with cytoflorimetric assay for activated caspase-9 and caspase-3. Any oxidative genomic damage was also ruled out with the cytoflorimetric quantification of the oxidized base adduct 8-hydroxy-2'-deoxyguanosine. In conclusion, E. coli LPS inhibited sperm ΔΨm through the activation of CB1, but this effect was not accompanied to the activation of mitochondrial dysfunction-related apoptotic/oxidative mechanisms, which could affect sperm motility and genomic integrity.