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The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.

Study Question: What are the consequences of ageing on human Leydig cell number and hormonal function?, Summary Answer: Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age., What Is Known Already: There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer., Study Design, Size, Duration: This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis., Participants/materials, Setting, Methods: Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age., Main Results and the Role of Chance: Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men., Limitations, Reasons for Caution: In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables., Wider Implications of the Findings: This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans., Study Funding/competing Interest(s): This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Time to final brain death (BD) determination is fundamental to rapidly identify donors without organ deterioration. Guidelines for BD determination are different from country to country and, through years, they have been subjected to several revisions, to simplify the procedure. The aim of this study was to describe a one-year experience according to the latest Italian Guidelines for BD in our University hospital and to focus on timing of final BD declaration according to the ancillary tests executed., Methods: Sixty-six consecutive inpatients with BD diagnosis were enrolled. Etiological factors, ancillary tests and timing to final declaration were analyzed., Results: Electroencephalogram (EEG) could be performed without artifacts in all the patients. Time to BD procedure starting depended on whether the demonstration of cerebral circulatory arrest was required, being shorter with EEG only (40±17 min), longer with cerebral blood flow evaluation (175±95 min), minimal with transcranial Doppler (83±32 min), maximal with angiography (165±20 min). None of the patients who initiated BD procedure were found to recover cerebral or brainstem function at the second observation., Conclusions: In Italy, the same guidelines ensure the same approach in every hospital, with multi-specialist cooperation. The EEG is mandatory and prompt recognition of the first, flat EEG is fundamental to reduce time to the final procedure. A multimodal neurophysiological approach with trained specialists, neurosonologists and monitoring devices in intensive care units may represents a valid help to further reduce time for BD diagnosis.

Status epilepticus (SE) is a neurological emergency associated with a high morbidity and mortality. A prospective 3-year study was conducted in our hospital on 56 consecutive inpatients with SE. Demographic and clinical data were collected. EEG and clinical SE features were considered for the SE classification, both separately and together. The etiology of SE was determined. Patients were treated according to international standardized protocols of guidelines for the management of epilepsy. Response to treatment was evaluated clinically and electrophysiologically. Outcome at 30 days was considered as good, poor or death. Convulsive SE (CSE) was observed in 35 patients and non-convulsive SE (NCSE) in 21. Patients with CSE, in particular focal-CSE, were older than those with NCSE. As regards etiology, patients with SE secondary to cerebral lesions were the oldest, followed by patients with anoxic SE and those with toxic dysmetabolic SE. A first-line treatment was usually sufficient to control seizure activity in lesional and epileptic SE, while more aggressive treatment was necessary in all anoxic SE patients. Outcome was good in 35 patients, poor in 12, while 9 died. A prompt neurophysiological EEG evaluation, combined with the clinical evaluation, helps to make a rapid prognosis and take therapeutic management decisions. First-line treatments may be sufficient to control electro-clinical status in lesional and epileptic SE, while intensive care unit management, a more aggressive therapeutic approach and continuous EEG monitoring are recommended for refractory SE., (Copyright © 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess the feasibility of remifentanil-based sedation in hypoxemic acute respiratory failure (HARF) patients refusing to continue noninvasive ventilation (NPPV) for intolerance to two different interfaces-helmet and total face mask., Design and Setting: Prospective uncontrolled clinical investigation in a 14-bed ICU of an university hospital in Italy., Patients: Thirty-six patients with persistent severe HARF who complained of discomfort and asked for interruption of NPPV session., Intervention: Patients started sedation with remifentanil (0.025 μg kg(-1) min(-1)) and the infusion rate was increased by 0.01 μg kg(-1) min(-1) every minute to a maximum of 0.12 μg kg(-1) min(-1) to obtain patient comfort., Measurements and Results: Twenty-two out of 36 patients (61%) with median (IQR) SAPS II score of 32 (30, 38) continued the NPPV treatment after the introduction of remifentanil infusion. In this success group, median (IQR) respiratory rate decreased from 34 (31, 37) to 24 (20, 26) min(-1) (p < 0.0001) and PaO(2)/FiO(2) ratio increased from 156 (144, 176) to 270 (210, 300) mmHg (p < 0.0001) after 1 h of NPPV with remifentanil-analgosedation either with helmet or total face mask. Fourteen patients failed to continue the noninvasive treatment and were intubated after a mean of 2.5 ± 2.3 h; they showed a respiratory rate decrease from 35 (30, 38) to 27 (25, 35) min(-1) (p = 0.02) and an inability to increase the PaO(2)/FiO(2) ratio above 180 mmHg. The ICU mortality in the failure group patients was 50 versus 14% in the NPPV success group (p < 0.05). The mean remifentanil dose administered was 0.07 ± 0.03 μg kg(-1) min(-1)., Conclusion: This clinical study suggests that a remifentanil-based sedation protocol can decrease the rate of failure in patients with intolerance to NPPV.

Objective: Transcranial Doppler (TCD) is a sensitive technique for circulatory arrest diagnosis in brain death when patterns such as reverberant flow and short systolic spikes are observed. In infants, the nonossified fontanelles compensate for intracranial hypertension. We describe TCD patterns in infants with brain death, different from adults, with the hemodynamic modifications induced by anterior fontanelle compression., Method: TCD was performed in 2 infants with diagnosed brain death admitted to the neonatal intensive care unit., Results: TCD showed a large peak 'reverberant' flow, with a high peak systolic velocity and a consistent retrograde component, away from the brain. Compression of the anterior fontanelle induced, at first, a reduction in systolic flow with the subsequent appearance of the characteristic short systolic spikes. Upon compression removal, a brief increase in the systolic flow was observed before the prompt reappearance of the reverberant flow., Conclusion: TCD for brain death diagnosis should be done cautiously in infants. In these cases, reverberating flow may be indicative of circulatory arrest even if with a large peak and with a high peak systolic velocity. Heavy fontanelle compression may reproduce the classical adult TCD patterns of brain death, thus supporting the diagnosis of cerebral circulatory arrest., (Copyright 2010 S. Karger AG, Basel.)

Objective: To evaluate the reliability and clinical value of partial noninvasive CO2 (NICO2) rebreathing technique for measuring cardiac output compared with standard thermodilution in a group of intensive care nonpostoperative patients., Design and Setting: Clinical investigation in a university hospital ICU., Patients: Twelve mechanically ventilated patients with high (n=6) and low (n=6) pulmonary shunt fractions., Measurements and Results: Thirty-six paired measurements of cardiac output were carried out with NICO2 and thermodilution in patients ventilated in pressure-support mode and sedated with a sufentanil continuous infusion to obtain a Ramsay score value of 2. The mean cardiac output was: thermodilution 7.27+/-2.42 l/min; NICO2 6.10+/-1.66 l/min; r2 was 0.62 and bias -1.2 l/min+/-1.5. Mean values of cardiac output were similar in the low shunt group (Qs/Qt < 20), with r2=0.90 and a bias of 0.01 l/min+/-0.4; conversely, in the high pulmonary shunt group (Qs/Q > 35%) the mean was 9.32+/-1.23 l/min with thermodilution and a mean NICO2CO value was 6.97+/-1.53 l/min, with r2 of 0.38 and a bias of -2.3 l+/-1.2 min., Conclusions: The partial CO2 rebreathing technique is reliable in measuring cardiac output in nonpostoperative critically ill patients affected by diseases causing low levels of pulmonary shunt, but underestimates it in patients with shunt higher than 35%.

Objective: We conducted a randomized prospective study comparing noninvasive positive pressure ventilation (NPPV) with conventional mechanical ventilation via endotracheal intubation (ETI) in a group of patients with chronic obstructive pulmonary disease who failed standard medical treatment in the emergency ward after initial improvement and met predetermined criteria for ventilatory support., Design and Setting: Prospective randomized study in a university hospital 13-bed general ICU., Patients: Forty-nine patients were randomly assigned to receive NPPV (n=23) or conventional ventilation (n=26)., Results: both NPPV and conventional ventilation significantly improved gas exchanges. The two groups had similar length of ICU stay, number of days on mechanical ventilation, overall complications, ICU mortality, and hospital mortality. In the NPPV group 11 (48%) patients avoided intubation, survived, and had a shorter duration of ICU stay than intubated patients. One year following hospital discharge the NPPV group had fewer patients readmitted to the hospital (65% vs. 100%) or requiring de novo permanent oxygen supplementation (0% vs. 36%)., Conclusions: The use of NPPV in patients with chronic obstructive pulmonary disease and acute respiratory failure requiring ventilatory support after failure of medical treatment avoided ETI in 48% of the patients, had the same ICU mortality as conventional treatment and, at 1-year follow-up was associated with fewer patients readmitted to the hospital or requiring for long-term oxygen supplementation. An editorial regarding this article can be found in the same issue (http://dx.doi.org/10.1007/s00134-002-1503-3).

Infection surveillance in ICU is fundamental to monitor endemic rates, to identify outbreaks on-time in order to activate control procedures implementing a correct empirical antibiotic treatment. The data collection surveillance software CIN-20002 was established in the Teaching Hospital Umberto I ICU to monitor the following site-specific infection rates: urinary tract infections (UTI), pneumonia (PNE), blood stream infections (BSI), surgical site infections (SSI). According to CDC definitions all patients developing infection 48 hours or more after ward admission were included. Furthermore risk factors (i.e. age, sex, SAPS II), invasive procedures (i.e. endotracheal intubation, vascular and urinary catheterisation), microbiological isolates and their antibiotic susceptibility were screened. Overall 279 patients (183 men and 96 women) were admitted; age 54,8 20,3 years (mean), SAPS II 44,4 17,1 (mean) and average ward stay 13,3 17,8 days. Results showed a total of 121 infection episodes (56 PNE, 51 BSI, 10 UTI e 4 SSI) in 80 patients (28,7%). Standardized infection rates associated to invasive procedures were: Urinary catheter-associated UTI rate (2.9/1000), Ventilator-associated PNE rate (20.4/1000), Vascular catheter-associated BSI rate (19.1/1000). Among the infected patients the most common microrganisms isolated were P. aeruginosa (31,8%), MRSA (14,8%), A. baumanni (12,5%) e S. maltophilia (8,5%). Considering site-specific infections: PNE (P. aeruginosa 38,3% and MRSA 18,1%), BSI (MR CNS 21,9% and P. aeruginosa 17,2%), UTI (P. aeruginosa 70,0%). The surveillance software CIN-2000 proved to be very accurate, usefull and easy to use. The results showed a high incidence of infections associated to invasive procedures and the presence of multiresistant bacteria

Background: The aim of the present study was to evaluate the effects of hyperbaric oxygenation on lipid peroxidation, on the release of circulating cytokines (TNFa, IL6, IL1b) and endothelin-1 (ET1)., Experimental Design: single arm, prospective study., Setting: ICU hyperbaric division of a University Hospital., Patients: fifteen healthy volunteers (10 male and 5 female, mean age 32+/-7 years) studied during hyperbaric oxygenation divided at random into two groups: group A (7 subjects) and group B (8 subjects)., Interventions: Both groups were consecutively pressurized at 2 atmospheres (2 atm abs) and 2.8 atm abs, with a constant descending rate of 1 m/min; in accordance with the experimental design, group A breathed pure oxygen continuously through facial masks and group B breathed chamber air during pressurization., Measures: Twenty millilitres of blood were drawn from all individuals at the following times: 1) basal, before HBO; 2) after 10 min at 2 atm abs; 3) after 10 min at 2.8 atm abs; 4) 30 min after the end of HBO. In all collected samples thiobarbituric reacting substances were evaluated, using the spectrophotometric technique, IL1 TNF and IL6 serum levels by ELISA and endothelin 1 plasma levels by radioimmunoassay., Results: In both groups, TBARS levels showed a twofold increase (p<0.05) in relation to the baseline, during and after hyperbaric oxygenation. Serum IL6 and IL1b values did not significantly change over the study in any of the volunteers. TNFa amounts significantly increased (p<0.05) during HBO, at 2 atm abs and 2.8 atm abs in both groups, with almost twofold increments. ET1 plasma values increased (p<0.05) in all volunteers during and after HBO: at 2 atm abs (range 7 to 24 pg/ml), 2.8 atm abs (range 7 to 19 pg/ml) and 30 min after (range 8 to 17 pg/ml) in relation to baseline (range 4 to 12 pg/ml). All the studied compounds had a similar trend in the two groups., Conclusions: Hyperbaric oxygenation in healthy volunteers can induce not only lipid peroxidation, but also liberation of compounds such as TNFa and endothelins, no matter whether pure oxygen is breathed or not. These results suggest that the phenomenon behind this release might be leukocyte activation as induced by HBO. The possible role of ET1 in determining vasoconstriction occurring during HBO is also suggested.

2-deoxy-5-fluorouridine, also known as floxuridine (FUdr) is a fluoropyridine antimetabolite, used in the treatment of metastatic renal cell carcinoma. We report the first case of lesional pulmonary oedema developed after receiving Fudr, recently treated in our unit. The patient refused endotracheal intubation, and was successfully treated associating noninvasive ventilation (NIV) with full-face mask to steroid treatment. The authors conclude that mechanical ventilation via face-mask can be an effective, comfortable, dignified method of support for patients with end-stage disease and acute respiratory failure.

Objective: To verify and quantify lipidic peroxidation by means of tiobarbituric-acid reactive substance (TBARS) dosage in patients treated daily with HBO. To verify if a potentiated glutathione enzymatic system, with N-acetylcisteine (NAC) treatment, may determine higher HBO tolerance and reduced lipidic peroxidation., Design: Randomised study on patients treated with 20 HBO 2.2 ATA (90' oxygen) sessions., Setting: Hyperbaric Medical Centre., Patients: Seventeen patients divided, at random, into two groups; group A: 10 patients treated with only HBO; group B: 7 patients treated with NAC antioxidant therapy (Fluimucil, Zambon Group, Italy) 1800 mg/day in addition to HBO., Interventions: None., Measurements and Main Results: TBARS on blood sample at T0 (basal) T1 (at the end of the 1st HBO session) T2 (at the beginning of the 20th HBO session) T3 (at the end of the 20th). The group A TBARS analysis at the different study time has shown significant data (p < 0.01) as the difference between TBARS values of the two groups at T2 (p < 0.01)., Conclusions: HBO induces a lipidic peroxidation even if the therapeutical protocol cannot determine lung or cerebral oxygen toxicity symptoms. The NAC administration, during HBO treatment, determines a protection against the HBO radicalic stress.