Gustavo Spadetta

Novel Stage Classification of Human Spermatogenesis Based on Acrosome Development

ABSTRACT 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.

Transcranial Doppler for brain death after decompressive craniectomy: persistence of cerebral blood flow with flat EEG

Dear Editor, Brain death (BD) diagnosis is made based upon clinical criteria—unresponsive coma with absence of brainstem reflexes and persistent apnea—and upon neurophysiologic observation of persistent ‘‘flat EEG.’’ Cerebral circulatory arrest (CCA) must also be assessed with ‘‘ancillary tests,’’ e.g. conventional angiography, transcranial Doppler (TCD), and other neuroimaging techniques, in infants younger than 12 months, when EEG flattening may be related to sedative treatment, and in BD of uncertain origin [1]. CCA may indeed happen both when intracranial pressure (ICP) overrides mean arterial blood pressure (MAP) as well as in diseases affecting cerebral tissue at a cellular level, with ICP not exceeding MAP. TCD is a sensitive, specific, and noninvasive technique to detect CCA in BD by identifying specific patterns [2]. However, it may lead to false-negative results in cases of skull defects (decompressive craniectomy, external drains, and in infants), because in these cases the increase in ICP may partially be compensated for. For these reasons, we recently described the TCD modifications of the CCA patterns in infants with BD, confirming that CCA detection for BD confirmation should be done cautiously in these cases [3]. Here we describe the different TCD findings observed in two adults with BD who were subjected to decompressive craniectomy. Case 1 was a female, 62 years old, with intracerebral right temporo-parietal hemorrhage, middle cerebral artery aneurism rupture. She underwent large right frontotemporal craniectomy and presented with unresponsive coma and flat EEG (Fig. 1). TCD, performed under stable hemodynamic conditions (BP 140/70 mmHg), showed the typical CCA pattern. Short compression of the dural expansion induced a further reduction of the signal, promptly returning to the basal conditions at the end of compression (Fig. 1). Case 2 was female, 56 years old, with a small deep right basal ganglia hemorrhage and aneurysm of the right intracranial internal carotid artery. She was treated with an endovascular procedure. Following sedation withdrawal, 2 days after the procedure, she was conscious but agitated and uncooperative, and she was again sedated. TCD performed under stable hemodynamic conditions (BP 150/ 100 mmHg) (Fig. 2a) showed a very high-resistive pattern with diastolic reduction in both the middle cerebral arteries and expression of elevated ICP, confirmed by invasive

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. Summary: Marker protein expression pattern analysis improves understanding of human spermatogonial development and stem cell renewal, facilitating further research and providing insight into fertility problems.

Optic Nerve Sheath Diameter Ultrasound Evaluation in Intensive Care Unit: Possible Role and Clinical Aspects in Neurological Critical Patients’ Daily Monitoring

Background. The increase of the optic nerve sheath diameter (ONSD) is a reliable, noninvasive sonographic marker of intracranial hypertension. Aim of the study was to demonstrate the efficacy of ONSD evaluation, when monitoring neurocritical patients, to early identify malignant intracranial hypertension in patients with brain death (BD). Methods. Data from ultrasound ONSD evaluation have been retrospectively analyzed in 21 sedated critical patients with neurological diseases who, during their clinical course, developed BD. 31 nonneurological controls were used for standard ONSD reference. Results. Patients with neurological diseases, before BD, showed higher ONSD values than control group (CTRL: RT 0.45 ± 0.03 cm; LT 0.45 ± 0.02 cm; pre-BD: RT 0.54 ± 0.02 cm; LT 0.55 ± 0.02 cm; p < 0.000) even without intracranial hypertension, evaluated with invasive monitoring. ONSD was further significantly markedly increased in respect to the pre-BD evaluation in neurocritical patients after BD, with mean values above 0.7 cm (RT 0.7 ± 0.02 cm; LT 0.71 ± 0.02 cm; p < 0.000), with a corresponding dramatic raise in intracranial pressure. Logistic regression analysis showed a strong correlation between ONSD and ICP (R 0,895, p < 0.001). Conclusions. ONSD is a reliable marker of intracranial hypertension, easy to be performed with a minimal training. Routine ONSD daily monitoring could be of help in Intensive Care Units when invasive intracranial pressure monitoring is not available, to early recognize intracranial hypertension and to suspect BD in neurocritical patients.

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.