Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.     

Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.     

Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine

The psychotropic beta-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for the O-demethylations mediated by each isozyme and by pooled HLMs. K(cat) (min(-1)) and K(m) Awake M) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline.     

Continuous fluid resuscitation for treatment of uncontrolled hemorrhagic shock following massive splenic injury in rats

We have previously observed that bolus fluid resuscitation in uncontrolled hemorrhagic shock induced by solid organ injury leads to increased blood loss and mortality. In the present investigation, we studied the effect of continuous fluid resuscitation on the hemodynamic response and survival following massive splenic injury (MSI) in rats. The animals were randomized into 11 groups: group 1, sham-operated; group 2, MSI untreated; group 3, MSI treated with 17.5 mL/kg/h of Ringers lactate (RL) solution (RL-17.5); group 4, MSI treated with 35 mL/kg/h RL (RL-35); group 5, MSI treated with 70 mL/kg/h RL (RL-70); group 6, MSI treated with 7.5 mL/kg/h of 7.5% NaCl (HTS-7.5); group 7, MSI treated with 15 mL/kg/h of 7.5% NaCl (HTS-15); group S, MSI treated with 30 mL/kg/h of 7.5% NaCl (HTS-30); group 9, MSI treated with 7.5 mL/kg/h 6% hydroxyethyl starch (HES-7.5); group 10, MSI treated with 15 mL/kg/h 6% hydroxyethyl starch (HES-15); and group 11, MSI treated with 30 mL/kg/h 6% hydroxyethyl starch (HES-30). MSI in untreated group 2 was followed by a fall of mean arterial pressure (MAP) to 50.1 +/- 6.7 mmHg (P < 0.001) in 15 min. Mean survival time (MST) was 99.5 +/- 16.6 min, and total blood loss (TBL) was 37.8% +/- 2.6% of blood volume. Fluid treatment with increasing volumes of RL in groups 3, 4, and 5 was followed by a gradual increase in TBL compared with untreated animals, and MST remained unchanged. Increasing volumes of HTS infusion in groups 6, 7, and 8 was also followed by incease in TBL, but MST remained unchanged except for an increase to 123.0 +/- 20.5 min (P < 0.05) in group 6. Increasing volumes of HES in groups 9, 10, and 11 was also followed by increase in TBL, but MST remained unchanged. In conclusion, continuous infusion of LR, HTS, and HES following massive splenic injury resulted in a significant increase in intra-abdominal bleeding, but survival time in the first hour following injury remained unchanged in contrast to bolus fluid infusion, which increases early mortality.     

Nephrogenic systemic fibrosis associated with stromal and vascular calcification, report of two cases

Nephrogenic systemic fibrosis (NSF) is a systemic fibrosing disorder characterized by the development of large indurated plaques on the skin, primarily in patients with end-stage renal disease (ESRD). Skin biopsy reveals an increased frequency of CD34⁺ and Factor XIIa+ cells. Microscopic calcification has been reported in the skin biopsies of rare cases of NSF. The etiology and significance of this calcification is not clear. We present two cases of typical NSF, for which marked stromal and vascular calcification was identified on skin biopsy. In both cases, the calcification was within typical NSF lesions and was intimately associated with aggregates of CD34⁺, Factor XIIIa+ spindle and stellate cells. This particular pattern of calcification strongly argues against either nonspecific dystrophic calcification or coincidental metastatic calcification. These findings suggest that calcification may be intrinsic to the pathophysiology of NSF, at least in a subset of NSF patients. In addition, the vascular calcification involving small arteries and arterioles in these two cases morphologically resembled calciphylaxis, which is another poorly understood complication of ESRD. This resemblance raises the possibility that NSF may predispose to or develop in the vicinity of indolent lesions of early-stage calciphylaxis. We propose that skin biopsies performed as part of a diagnostic workup for suspected NSF should preferably include the deep dermis and subcutis, in order to assess possible vascular calcification.     

Analysis of Trabecular Microstructure and Vascular Distribution of Capital Femoral Epiphysis Relevant to Legg–Calve–Perthes Disease

Legg–Calve–Perthes disease is characterized by the capital femoral epiphyseal collapse, which occurs more reliably in the anterior quadrant than the more weight‐bearing lateral quadrant. The purpose of this study was to determine whether there is a vascular or microstructural predisposition for anterior femoral epiphyseal collapse in Perthes disease. Thirty‐two cadaveric proximal femoral epiphyses from 17 subjects (age 4–14 years old) underwent micro‐computed tomography at 10‐μm resolution. Each quadrant was analyzed for four markers of trabecular architecture: bone volume fraction (BV/TV), trabecular thickness, trabecular separation (TbSp), and trabecular number (TbN). Vascular channels were then mapped in each quadrant, identified by correlating surface topography with cross‐sectional imaging. One‐way analysis of variance revealed an overall difference between quadrants (p < 0.001) in BV/TV, TbN, and TbSp. However, post hoc analysis revealed there was no significant difference between the anterior and lateral quadrants for any of the four markers of trabecular architecture. Vascular channel mapping illustrated a predominance of vessels in the posterior half of the epiphysis compared to the anterior half (8.7 ± 4.0 vs. 3.4 ± 3.1 vascular channels, p < 0.001). The lack of microstructural differences between the anterior and lateral quadrants, and the predominance of vascular channels in the posterior half of the epiphysis with posteriorly‐based medial femoral circumflex and ligamentum teres vessels suggests that the anterior femoral epiphysis may be a relative vascular watershed region, which predisposes it to collapse after the vascular insult of Perthes disease. Clinical significance: Improved understanding of the pathophysiology of anterior femoral epiphyseal collapse may inform future treatments aimed at revascularization. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1784–1789, 2019