Interplay of spermatogonial subpopulations during initial stages of spermatogenesis in adult primates.

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.

The Propensity of the Human Liver to Form Large Lipid Droplets Is Associated with PNPLA3 Polymorphism, Reduced INSIG1 and NPC1L1 Expression and Increased Fibrogenetic Capacity.

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.

Spermatogonial kinetics in humans.

The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1High, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.

Age-related changes in human Leydig cell status.

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.

Novel stage classification of human spermatogenesis based on acrosome development.

To date, in the human seminiferous epithelium, only six associations of cell types have been distinguished, subdividing the epithelial cycle into six stages of very different duration. This hampers comparisons between studies on human and laboratory animals in which the cycle is usually subdivided into 12 stages. We now propose a new stage classification on basis of acrosomal development made visible by immunohistochemistry (IHC) for (pro)acrosin. IHC for acrosin gives results that are comparable to periodic acid Schiff staining. In the human too, we now distinguish 12 stages that differ from each other in duration by a factor of two at most. B spermatogonia are first apparent in stage I, preleptotene spermatocytes are formed in stage V, leptonema starts in stage VII, and spermiation takes place at the end of stage VI. A similar timing was previously observed in several monkeys. Stage identification by way of IHC for acrosin appeared possible for tissue fixed in formalin, Bouin fixative, diluted Bouin fixative, Cleland fluid, and modified Davidson fixative, indicating a wide applicability. In addition, it is also possible to distinguish the 12 stages in glutaraldehyde/osmium-tetroxide fixed/plastic embedded testis material without IHC for acrosin. The new stage classification will greatly facilitate research on human spermatogenesis and enable a much better comparison with results from work on experimental animals than hitherto possible. In addition, it will enable a highly focused approach to evaluate spermatogenic impairments, such as germ cell maturation arrests or defects, and to study details of germ cell differentiation.

Noninvasive ventilation by helmet or face mask in immunocompromised patients: a case-control study.

OBJECTIVE: To compare the efficacy of noninvasive positive pressure ventilation (NPPV) by helmet and face mask in immunocompromised patients with hypoxemic acute respiratory failure (ARF). DESIGN: Case-control study. SETTING: The general ICU of a university hospital. PATIENTS: Nineteen immunocompromised patients (hematologic malignancies [n = 8], solid-organ recipients [n = 8], AIDS [n = 3]) with hypoxemic ARF, fever, and lung infiltrates were treated with NPPV delivered by a helmet. Nineteen immunocompromised patients matched for diagnosis, age, simplified acute physiology score II, and Pao(2)/fraction of inspired oxygen (Fio(2)) receiving NPPV through a facial mask served as case-control subjects. RESULTS: The use of NPPV delivered via helmet was as effective as NPPV delivered via face mask in avoiding endotracheal intubations (intubation rate, 37% vs 47%, respectively; p = 0.37) and improving gas exchange; 14 patients (74%) in the helmet group showed a sustained improvement in Pao(2)/Fio(2) ratio (ability to increase Pao(2)/Fio(2) ratio > 200, or an increase > 100 from the baseline) in comparison with 7 patients (34%) in the mask group (p = 0.02), whose Pao(2)/Fio(2) at treatment discontinuation was higher (p = 0.02) and had fewer complications related to NPPV (ie, skin necrosis, p = 0.01). Moreover, the patients receiving ventilation via helmet required significantly less NPPV discontinuations in the first 24 h of application (p < 0.001) than patients receiving ventilation via face mask. CONCLUSIONS: The helmet may represent a valid alternative to a face mask in immunocompromised patients with lung infiltrates and hypoxemic ARF, increasing the patient's tolerance (ie, the number of hours of continuous NPPV use without interruptions) and decreasing the rate of complications directly related to the administration of NPPV.

A comparative evaluation of thermodilution and partial CO2 rebreathing techniques for cardiac output assessment in critically ill patients during assisted ventilation.

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%.

[Microbiological surveillance in an intensive care unit of a large Roman hospital]. / Sorveglianza microbiologica presso la terapia intensiva di un grande ospedale romano.

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

An observational electro-clinical study of status epilepticus: from management to outcome.

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.

Rescue treatment for noninvasive ventilation failure due to interface intolerance with remifentanil analgosedation: a pilot study.

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.