Safety issues in semen banks during the COVID-19 pandemic: data from a European survey.

PURPOSE: We performed a survey among European semen banks enquiring safety protocols during the COVID-19 pandemic. We report the experience from a center searching SARS-CoV-2 mRNA in semen of patients undergoing cryopreservation from May 2020 to January 2021. METHODS: A questionnaire was submitted to accredited semen banks of the European Academy of Andrology (EAA) and the Italian Society of Andrology and Sexual Medicine (SIAMS). A total of 22 centers answered to the survey. SARS-CoV-2 mRNA in semen was evaluated by RT-PCR in 111 subjects banking in the Semen Bank of Careggi University Hospital (Florence, Italy). RESULTS: No particularly drastic safety measures were adopted by the majority of the centers to prevent the risk of contamination or transmission of the virus. The most common strategy (77.3%) was the administration of an anamnestic questionnaire. About half of the centers request a negative nasopharyngeal swab (NPS) before cryopreservation. Few centers use a quarantine tank, in case of late response of NPS, and only 4 store in a dedicated tank in case of infection. SARS-CoV-2 mRNA was not found in 111 semen samples cryopreserved in the Florentine bank. CONCLUSIONS: European semen banks use different measures to handle semen samples for cryopreservation during COVID-19 pandemic. The request of NPS is advised to better manage couples undergoing ART and to protect the personnel operating in the bank/ART center. Finally, due to the areas of uncertainties of an almost unknown virus, it is absolutely recommended the use of safe devices for sample handling and storage.

Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

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.

The prevalence of midline prostatic cysts and the relationship between cyst size and semen parameters among infertile and fertile men.

STUDY QUESTION: When is the investigation and treatment of midline prostatic cysts (MPC) of clinical value in the work-up of males of infertile couples? SUMMARY ANSWER: With a prevalence of 10.2% in infertile men, MPC should be investigated according to a seminal algorithm detecting a MPC volume >0.117 ml, which may impair semen parameters, and could be treated to improve sperm count and achieve natural pregnancy. WHAT IS KNOWN ALREADY: MPC are frequent and are considered a correctable cause of male infertility. However, they have been poorly investigated in an infertility setting. In addition, no study has investigated clinical and ultrasound (US) characteristics of men with MPC. STUDY DESIGN, SIZE, DURATION: A cross-sectional analysis was carried out of 693 consecutive subjects consulting for couple infertility from September 2012 to March 2017. As a control group, 103 age-matched healthy, fertile men were studied. Furthermore, a longitudinal evaluation of 11 infertile men undergoing trans-rectal ultrasonically-guided cyst aspiration (TRUCA), semen analyses 1 and 3 months after TRUCA and a follow-up 1 year after TRUCA to assess natural pregnancy were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All subjects underwent, in our outpatient clinic, clinical, hormonal, scrotal and transrectal US evaluation and semen analysis within the same day. Of 693 males of infertile couples, 648 (37.1 ± 7.9 years, mean+SD) without genetic abnormalities were studied, along with 103 fertile men (36.6 ± 5.0 years). Eleven infertile men underwent TRUCA and were followed-up as reported above. MAIN RESULTS AND THE ROLE OF CHANCE: A MPC was present in 66/648 (10.2%) males of infertile couples and in 6/103 (5.8%) fertile men. MPC occurrence and volume were higher in patients with severe oligo- or azoospermia than in fertile men (all P < 0.05). Infertile men with a MPC showed a lower seminal volume and sperm count and a higher prevalence of azoospermia than the rest of the infertile sample or fertile men, and a higher frequency of US signs suggestive of ejaculatory duct obstruction. MPC volume was negatively associated with total sperm count (r = -0.452, P < 0.0001). In fertile men, the highest MPC volume was 0.117 ml, suggesting it as a biological threshold not compromising semen quality. In infertile men, using receiver operating characteristic curve analyses, a MPC volume >0.117 ml identified subjects with severe oligo- or azoospermia with an overall accuracy of ~75% (both P < 0.005). Eleven men with infertility, semen abnormalities and large MPC (>0.250 ml) underwent TRUCA, which led to sperm count improvement in all patients 1 month after surgery. Three months after TRUCA a lower sperm count and a higher MPC volume than 2 months before were observed (P < 0.005 and P < 0.05, respectively), although improved when compared to baseline. After TRUCA a natural pregnancy occurred in four couples. Finally, we propose an algorithm, based on semen parameters, useful in identifying a MPC in males of infertile couples. LIMITATIONS, REASONS FOR CAUTION: Although in line with the sample size of previous studies (n = 7-20), the number of infertile men with MPC evaluated longitudinally after treatment is limited (n = 11). In addition, although a MPC volume >0.117 ml can negatively affect the sperm count, only MPC > 0.250 ml have been treated in this study. WIDER IMPLICATIONS OF THE FINDINGS: First, the algorithm proposed is easy to use and useful for selecting patients who can benefit from a prostate US in the infertility work-up. Second, a MPC volume ≤0.117 ml may not impair semen quality, while a larger volume can lead to severe oligo- or azoospermia and could be treated. Third, TRUCA is effective, and simpler and less invasive than other surgical techniques for MPC treatment. Finally, since the MPC can increase in size and sperm count decrease over time after TRUCA, semen cryopreservation should be considered 1 month after TRUCA. STUDY FUNDING/COMPETING INTEREST(S): Grants from the Ministry of University and Scientific Research (SIR project to F.L., protocol number: RBSI14LFMQ). No conflicts of interest.

Epididymal more than testicular abnormalities are associated with the occurrence of antisperm antibodies as evaluated by the MAR test.

STUDY QUESTION: Is there any association between mixed antiglobulin reaction (MAR) test positivity and clinical features or genital tract ultrasound (US) parameter values in males of infertile and fertile couples? STUDY ANSWER: In males of infertile and fertile couples MAR test positivity was associated with clinical and US features suggestive of chronic epididymal inflammation. WHAT IS KNOWN ALREADY: MAR test positivity has been more often reported in males of infertile couples than in fertile men. A positive MAR test has been detected in men with a history of testicular or post-testicular damage. No previous study has reported US alterations related to MAR test positivity. This is the first study that has systematically evaluated associations between a positive MAR test and clinical, seminal and US characteristics of the entire male genital tract. STUDY DESIGN, SIZE, DURATION: This cross-sectional analysis included 109 fertile men and 699 consecutive subjects seeking medical care for couple infertility from September 2012 to September 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All subjects underwent, in our outpatient clinic, a complete physical, endocrine, scrotal and transrectal US evaluation and semen analysis (including sIL-8) on the same day. Of the 699 males of infertile couples, 181 (age 38.6 ± 6.6 years) had an assessable MAR test, whereas the test was assessable in all 109 fertile men (age 36.6 ± 5.2 years). The associations among MAR test positivity and the other studied parameters were investigated on a caseload of 290 men (patients + fertile men) and in the two cohorts of males of infertile and fertile couples. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 181 men of infertile couples studied, 20 (11%) had a positive MAR test, including 12 (6.6%) who had a MAR test ≥ 50%, which is considered as a pathological threshold according to the WHO. Of the 109 fertile men, four (3.7%) had a positive MAR test, of which one (0.9%) had a MAR test ≥ 50%. MAR test positivity was therefore found more often in men of infertile couples (P < 0.05). In the entire caseload (n = 290) of males of both infertile and fertile couples, no correlations between MAR test positivity and seminal characteristics were observed. A positive MAR test was associated with epididymal US abnormalities, particularly with the mean size of the epididymal body and tail (both P < 0.0001), and in infertile men, a positive MAR test was also associated with an abnormal epididymal echotexture. In addition, subjects with a positive MAR test more frequently showed a history of epididymitis and high sIL-8 levels. Considering endocrine parameters, only a positive correlation between MAR test positivity and LH levels was observed, even after adjusting for age and life-style factors (adj. r = 0.232, P < 0.0001), while no associations with testosterone and FSH levels were found. LIMITATIONS, REASONS FOR CAUTION: Antisperm antibodies (ASA) were detected in this study by using the SpermMAR test IgG, but other tests are available. In addition, for technical reasons, the MAR test is not assessable in subjects with severe oligo-astheno-zoospermia and, therefore, this test may lead to an intrinsic selection bias. Finally, owing to the cross-sectional nature of the study, neither a causality hypothesis nor mechanistic models can be inferred. WIDER IMPLICATIONS OF THE FINDINGS: First, our results indicate that MAR test positivity is associated with clinical and US signs suggestive of chronic epididymal inflammation and not testicular damage. Hence, when investigating a subject with a positive MAR test, the epididymis and not just the testis should be evaluated. Furthermore, MAR test positivity was more often detected in males of infertile couples than in fertile men, but it was not associated with conventional semen parameter values. Our data support a role of ASA in couple infertility, regardless of the conventional sperm analysis. How ASA affects couple fertility needs to be addressed by further studies. STUDY FUNDING/COMPETING INTEREST(S): Grants were received from the Ministry of University and Scientific Research (SIR project to F.L., protocol number: RBSI14LFMQ). There are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Is thyroid hormones evaluation of clinical value in the work-up of males of infertile couples?

STUDY QUESTION: Is thyroid hormones (TH) evaluation of clinical value in the work-up of males of infertile couples? STUDY ANSWER: Our results suggest that TH evaluation is not mandatory in the work-up of male infertility. WHAT IS KNOWN ALREADY: A few previous studies performed on a limited series of subjects reported a negative impact of hyper- and hypo-thyroidism on semen volume, sperm concentration, progressive motility and normal morphology. No previous study has systematically evaluated associations between TH variation, semen parameters and ultrasound characteristics of the male genital tract. STUDY DESIGN, SIZE AND DURATION: Cross-sectional analysis of a consecutive series of 172 subjects seeking medical care for couple infertility from September 2010 to November 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the entire cohort, 163 men (age 38.9 ± 8.0 years) free of genetic abnormalities were studied. All subjects underwent a complete andrological and physical examination, biochemical and hormonal assessment, scrotal and transrectal colour-Doppler ultrasound (CDUS) and semen analysis (including seminal interleukin 8 levels, sIL-8) evaluation within the same day. MAIN RESULTS AND THE ROLE OF CHANCE: Among the patients studied, 145 (88.9%) showed euthyroidism, 6 (3.7%) subclinical hyper- and 12 (7.4%) subclinical hypo-thyroidism. No subjects showed overt hyper- or hypo-thyroidism. At univariate analysis, no associations among thyroid-stimulating hormone (TSH) or TH levels and sperm parameters were observed. Conversely, we observed positive associations among free triiodothyronine (fT3) and free thyroxine (fT4) levels, ejaculate volume and seminal fructose levels. In a multivariate model, after adjusting for confounders such as age, body mass index, smoking habit, sexual abstinence, calculated free testosterone, prolactin and sIL-8 levels, only the associations found for fT3 levels were confirmed. When CDUS features were investigated, using the same multivariate model, we found positive associations between fT3 levels and seminal vesicles (SV) volume, both before and after ejaculation (adj. r = 0.354 and adj. r = 0.318, both P < 0.0001), as well as with SV emptying (ΔSV volume; adj. r = 0.346, P < 0.0001) and echo-texture inhomogeneity. In addition, after adjusting for confounders, negative associations between fT4 levels and epididymal body and tail diameters were found. No significant associations between TSH or TH levels and CDUS features of other organs of the male genital tract, including testis and prostate, were found. Finally, when the features of subjects with euthyroidism, subclinical hypo- and hyper-thyroidism were compared, no significant differences in seminal or hormonal parameters were found. Conversely, evaluating CDUS parameters, subjects with subclinical hyperthyroidism showed a higher difference between the SV longitudinal diameters measured before and after ejaculation when compared with that of subclinical hypothyroid men, even after adjusting for confounders (P < 0.007). All the other male genital tract CDUS characteristics did not differ among groups. LIMITATIONS, REASONS FOR CAUTION: First, the number of patients investigated is relatively small and those with (subclinical) thyroid dysfunctions are an even smaller number; hence, it is therefore difficult to draw firm conclusions. Moreover, the present results are derived from patients consulting an Italian Andrology Clinic for couple infertility, and could have different characteristics from the male general population or from those males consulting general practitioners for reasons other than couple infertility. Finally, due to the cross-sectional nature of the study, neither a causality hypothesis nor mechanistic models can be inferred. WIDER IMPLICATIONS OF THE FINDINGS: Although no associations between TH and sperm parameters were observed, present data support a positive effect of TH on SV size and a permissive role on the ejaculatory machinery, likely through an action on SV and epididymal contractility. This is the first study reporting such evidence. However, in contrast with the view that TH assessment is important for female fertility, our results do not support a systematic evaluation of thyroid function in males of infertile couples. How TH abnormalities impact male fertility needs to be addressed by further studies. STUDY FUNDING/COMPETING INTERESTS: No funding was received for the study. None of the authors have any conflict of interest to declare.

Clinical implications of measuring prolactin levels in males of infertile couples.

Although in females of infertile couples abnormal prolactin (PRL) has a definitive role in the medical flowchart, its role in males is less clear. Animal models suggest that PRL does not play a major role in male reproduction, although its trophic action on male accessory glands was often observed. Studies in humans are scanty. We systematically evaluated possible clinical and ultrasound correlates of PRL in males of infertile couples. Of 288 consecutive males of infertile couples, 269 (36.6 ± 4.4 years) without genetic abnormalities were studied. All men underwent physical, biochemical, seminal evaluation and scrotal and transrectal ultrasound before and after ejaculation. Ejaculatory and erectile functions were assessed by Premature Ejaculation Diagnostic Tool (PEDT) and International Index of Erectile Function (IIEF)-15 respectively; prostate-related symptoms by National Institutes of Health-Chronic Prostatitis Symptom Index and International Prostate Symptom Score; psychological symptoms by Middlesex Hospital Questionnaire. Among semen parameters, only the positive association between PRL and ejaculate volume was significant, even adjusting for age, total testosterone and thyroid-stimulating hormone (adj. r = 0.126, p < 0.05). In a logistic ordinal model, adjusting for the aforementioned confounders and ejaculate volume, PRL was negatively associated with delaying ejaculation according to PEDT#1 score (Wald = 4.65, p < 0.05). In an age- and ejaculate volume-adjusted, iterative binary logistic model, low PRL was associated with a fivefold risk of any failure in controlling ejaculation (HR = 5.15 [1.15-23], p < 0.05). Among scrotal and transrectal ultrasound features, we found a significant positive association between PRL and seminal vesicles (SV) volume and inhomogeneity, before and after ejaculation, and with deferential ampullas diameter. Associations with PRL were confirmed in nested 1 : 1 case-control analysis. No significant associations were found between PRL and other clinical parameters. For the first time, this study extends the concept of a trophic effect of PRL on male accessory glands from animals to humans. We report a positive association among PRL and ejaculate and SV volume, before and after ejaculation. Low PRL is associated with a lessened ability to control ejaculation.

High variability in results of semen analysis in andrology laboratories in Tuscany (Italy): the experience of an external quality control (EQC) programme.

We report the results of the first three trials of an external quality control (EQC) programme performed in 71 laboratories executing semen analysis in Tuscany Region (Italy). At the end of the second trial, participants were invited to attend a teaching course illustrating and inviting to adhere to procedures recommended by WHO (V edition). Results of the first three trials of the EQC documented a huge variability in the procedures and the results. The highest variability was found for morphology (CV above 80% for all the trials), followed by count (CV of about 60% for all the trials) and motility (CV below 30% for all the trials). When results of sperm count and morphology were divided according to the used method, mean CV values did not show significant differences. CV for morphology dropped significantly at the third trial for most methods, indicating the usefulness of the teaching course for morphology assessment. Conversely, no differences were observed after the course for motility and for most methods to evaluate count, although CV values were lower at the second and third trial for the laboratories using the Burker cytometer. When results were divided according to tertiles of activity, the lowest mean bias values (difference between each laboratory result and the median value of the results) for count and morphology were observed for laboratories in the third tertile (performing over 200 semen analysis/year). Of interest, mean bias values for concentration dropped significantly at the third trial for low activity laboratories. In conclusion, lack of agreement of results of semen analysis in Tuscany is mainly because of the activity and the experience of the laboratory. Our study points out the importance of participating in EQC programmes and periodical teaching courses as well as the use of WHO recommended standardized procedures to increase precision and to allow the use of WHO reference values.

Semen apoptotic M540 body levels correlate with testis abnormalities: a study in a cohort of infertile subjects.

STUDY QUESTION: What are the associations between semen apoptotic M540 bodies and other parameters of semen quality and sonographic alterations of the male genital tract in a cohort of infertile subjects? SUMMARY ANSWER: In infertile subjects, semen M450 bodies are highly correlated with ultrasound and clinical signs of testis abnormalities but not with alterations of other parts of the male genital tract, suggesting a testicular origin of M540 bodies. WHAT IS KNOWN ALREADY: We have reported the presence in semen of round anucleate elements, named 'M540 bodies', resembling apoptotic bodies as they contain several apoptotic markers. STUDY DESIGN AND SIZE: A consecutive series of 130 males with couple infertility were evaluated, during the same day session, for clinical, scrotal and transrectal color-Doppler ultrasound characteristics, and hormonal and semen parameters, including interleukin 8 (sIL-8) and M540 body levels. PARTICIPANTS/MATERIALS, SETTING METHODS: Semen parameters were analyzed by WHO recommended procedures. CDU was performed using the ultrasonographic console Hitachi H21. sIL-8 and serum hormones were evaluated by ELISA methods. MAIN RESULTS AND THE ROLE OF CHANCE: The average percentage value of M540 bodies was 24.6 ± 18.3. After adjusting for possible confounders (age, waist, calculated free testosterone and smoking habit), M450 body levels negatively correlated with sperm number/ejaculate, progressive motility, normal morphology and sIL-8 levels (adj.r = -0.455, P < 0.0001; adj.r = -0.464, P < 0.0001; adj.r = -0.430, P < 0.001; adj.r = -0.236, P < 0.05, respectively). In a subset of patients with a history of cryptorchidism (n = 8), M540 bodies were higher than in non-cryptorchid men (40.5 ± 14.8 versus 23.6 ± 18.2%; P < 0.02). A negative correlation was found between M540 and ultrasound testis volume (adj.r = -0.241, P < 0.05), whereas a positive association was found with testis inhomogeneity [HR = 1.06 (1.02-1.09); P = 0.002], hypoechogenicity [HR = 1.05 (1.01-1.08); P < 0.02] and FSH levels (adj.r = 0.309, P < 0.01). No relationships were found with CDU characteristic of the prostate, seminal vesicles, epididymis and vas deferens. In a multivariate model, testis inhomogeneity and history of cryptorchidism were independently associated with M540 body levels (adj.r = 0.355, P < 0.01 and adj.r = 0.223, P < 0.05, respectively). Receiver operating characteristic analysis demonstrated that at the threshold of 27%, M540 bodies discriminate subjects with testis inhomogeneity with a sensitivity of 72% and specificity of 73%. LIMITATIONS, REASONS FOR CAUTION: The increased M540 body semen levels in men with a history of cryptorchidism should be confirmed in a larger number of patients. WIDER IMPLICATIONS OF THE FINDINGS: M540 bodies may be considered a semen marker of altered testis function and thus their evaluation may be helpful in the diagnosis of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Ministry of University and Scientific Research (Prin project to E.B. and FIRB project to S.M.) and Regione Toscana (to G.F.).

Vitamin D deficiency in septic patients at ICU admission is not a mortality predictor.

BACKGROUND: Vitamin D is involved in immune regulation in humans. Vitamin D serum deficiency is reported to be common in hospitalized patients, especially among Intensive Care Unit (ICU) patients. Our aim was to evaluate the relationship between vitamin D levels in septic patients and outcome. METHODS: A total of 170 patients were studied, of which 92 were severe sepsis/septic shock patients, and 72 were major trauma patients, as an age-matched control group. Exclusion criteria were: age <18 years (y), malnutrition state, pregnancy, breast feeding, chemotherapy, immunotherapy, pathologies affecting bone and calcium metabolism, vitamin D metabolism derangement for therapy, hematological and solid malignancies, and HIV. Vitamin D levels were measured by radioimmunoassay at admission. RESULTS: Median vitamin D levels at admission to ICU were 10.1 ng/mL in the sepsis group and 18.4 ng/mL in the trauma group (P<0.0001). In univariate analysis, mortality rate in septic patients was significantly correlated with age, gender, SAPS II, vitamin D level at admission, duration of mechanical ventilation, and ICU/hospital length of stay, however, the multivariate logistic regression model confirmed significance only for age. CONCLUSION: In our cohort, septic patients showed a significantly lower vitamin D level than trauma patients in comparison to age cohort patients with the same demographic/clinical characteristics, but no clear relationship between vitamin D level and outcome was found. Further studies with larger samples are needed to clarify the prognostic role of vitamin D and nutraceutical interventions in critically ill patients.

Proopiomelanocortin gene expression and beta-endorphin localization in the pituitary, testis, and epididymis of stallion.

Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), beta-endorphin (beta-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, beta-EP, is produced locally in the testis, thus playing effects in a paracrine/autocrine fashion. To investigate this hypothesis the POMC gene expression was analyzed using 3' RACE-PCR and Northern Blot approaches in the testis and epididimys of stallion; moreover, immunocytochemical localization for beta-EP was also performed through confocal laser microscopy. The immunofluorescence results showed a positive beta-EP reaction not only in cellular nest of pituitary but also in the testis and genital tract of stallion, which function could be related with sperm mobility. Such role seem not to be no dependent on the peptide synthesized locally, because the molecular biology approach demonstrated the presence of POMC transcript in the pituitary only. In fact the Northern Blot analysis showed the presence of a single POMC transcript in the pituitary while no signal was detected in the testis and epididimys. The same results were obtained by applied 3' RACE-PCR analysis. In conclusion, opioid-derived peptide beta-EP is present in the genital tract of stallion, but is not locally produced as in other mammalian, and nonmammalian models; its possible biological function at testicular level could be linked to a long-loop feed-back mechanisms.

DAZL polymorphisms and susceptibility to spermatogenic failure: an example of remarkable ethnic differences.

Polymorphisms in genes involved in spermatogenesis are considered potential risk factors for male infertility. Recently a polymorphism in the deleted in azoospermia-like (DAZL) gene (T54A) was reported as susceptibility factor to oligo/azoospermia in the Chinese population. DAZL is an autosomal homologue of the Y chromosomal DAZ (deleted in azoospermia) gene cluster and both are considered master regulators of spermatogenesis. The aim of the present study was to screen (i) for mutations of the entire coding sequence of the DAZL gene in patients lacking of the DAZ gene cluster, in order to evaluate if DAZL polymorphisms may influence the AZFc deletion phenotype; (ii) for the two previously described (and eventually newly identified) single nucleotide polymorphisms (SNPs) in a large group of infertile and normospermic men of Italian origin. We failed to detect new mutations. We confirmed previous results showing no evidence for a functional role of the T12A mutation. Surprisingly, the T54A polymorphism, which was present in 7.4% of the Chinese patients was absent in our Caucasian population. This remarkable difference represent an example of how ethnic background is important also for polymorphisms involved in spermatogenesis and contributes to better select clinically relevant tests, specifically based on the ethnic origin of the infertile patients.

The clinical significance of the POLG gene polymorphism in male infertility.

Based on association studies, an increasing number of gene polymorphisms have been proposed as modulators of spermatogenesis. Interestingly, a clear cause-effect relationship between a polymorphism of the POLG gene and oligo(astheno)zoospermia was recently described. The POLG gene contains a polymorphic CAG repeat, and the presence of a homozygous mutant (not10/not10 CAG) genotype was found only in infertile men. In the present study, a large number of infertile patients and normospermic men of Italian origin were studied to define the effect of POLG genotypes on spermatogenic potential and whether the homozygous mutant is specific for spermatogenic disturbances. The mutated genotype was found at the same frequency in both infertile and normospermic men. Mean values of sperm parameters such as sperm count, motility, and morphology did not differ significantly between carriers of the three different genotypes. Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis. More importantly, considering that the homozygous mutant genotype has been found in normospermic fertile men, the analysis of the CAG repeat tract of the POLG gene does not appear to have any clinical diagnostic value.

Mutagenicity of pesticides containing 1,3-dichloropropene.

In a systematic study of the mutagenic effect of chemical compounds used as pesticides, we found that D. D. soil fumigant and Telone are mutagenic. The test was performed using the bacterial tester strains following the procedure developed by Ames. The active principle of D. D. soil fumigant and Telone is a mixture of the cis and trans isomers of 1,3-dichloropropene. Both isomers are mutagenic in Salmonella strains TA 1535 and TA 100. 2,3-Dichloro-1-propene, a minor component (5%) of the commercial preparation Telone, was also found to be mutagenic in strains TA 1535 and TA 100. Mutagenesis of these tester strains is an indication of a base-pair substitution event causing a missense mutation. 1,3-Dichloropropene is widely used in agriculture all over the world. In Italy 2,187,100 kg were produced in 1972. In California over 1,000,000 kg of 1,3-dichloropropene-containing pesticides were used in 1971.