Presence of an extra chromosome alters meiotic double-stranded break repair dynamics and MLH1 foci distribution in human oocytes.

Studies performed on human trisomic 21 oocytes have revealed that during meiosis, the three homologues 21 synapse and, in some cases, achieve what looks like a trivalent. This implies that meiotic recombination takes place among the three homologous chromosomes 21, and to some extent, crossovers form between them. To see how meiotic recombination is in the presence of an extra chromosome 21, we analyzed the distribution of three recombination markers (γH2AX, RPA, and MLH1) on trisomic 21 oocytes at pachynema and, in particular, on chromosomes 21. Results clearly show how the presence of an extra chromosome 21 alters meiotic recombination progression, leading to the presence of a higher number of early recombination markers at pachynema. Moreover, the distribution on these chromosomes 21 of some of these markers is different in aneuploid oocytes. Finally, there is a substantial increase in the number of MLH1 foci, a marker of most crossovers in mammals, which is related to the number of synapsed chromosomes in pachynema. Thus, bivalents 21 had fewer MLH1 foci than partial or total trivalents, suggesting a close relationship between synapsis and crossover designation. All of the data presented suggest that the presence of an extra chromosome alters meiotic recombination globally in aneuploid human oocytes.

Notch-mediated induction of N-cadherin and α9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. Recently, several works have demonstrated a connection between Notch pathway activation and the regulation of cell motility and invasiveness. However, the molecular mechanisms of this possible relationship remain unclear. METHODS: The Notch pathway was manipulated pharmacologically and genetically. The mRNA changes were analysed by quantitative PCR and protein variations by western blot and immunofluorescence. Finally, the capabilities of RMS cells to adhere, heal a wound and invade were assessed in the presence of neuronal cadherin (N-cadherin)- and α9-integrin-blocking antibodies. RESULTS: Cells treated with γ-secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-α9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. CONCLUSION: Neuronal cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS.

Gene expression is altered after bisphenol A exposure in human fetal oocytes in vitro.

Bisphenol A (BPA) is a 'weak' endocrine disruptor. The effect of BPA on human reproduction is controversial but has been related to meiotic anomalies, recurrent spontaneous abortion, abnormal karyotypes, the diminishing of oocyte survival, delay in meiotic progression and an elevated rate of MLH1 foci in vitro. The aim of this study is to characterize the gene expression of human fetal oocytes in culture as well as to evaluate the effect of BPA in cultured human oocytes. To accomplish our objective, 12 ovaries from 6 euploid fetuses were used. The ovarian fetal tissue was cultivated in two groups: control group and BPA group (BPA30 µM). The cultures were analyzed at T0 and after 7 (T7), 14 (T14) and 21 (T21) days of culture. Evaluation of gene expression was performed by real-time PCR (RT-PCR), with the evaluated genes being: Smc1ß, Sycp1 (pairing-synapsis), Spo11, Rpa, H2ax, Mlh1 and Blm [double-strand break (DSBs) generation, signaling and repair], Erα, Erß and Errγ (estrogen receptors), Stra8 and Nalp5 (markers of meiotic progression). Oocytes from ovaries cultured and treated with BPA show changes in the expression of Spo11, H2ax and Blm genes, with a significant increase from 3- to 5-fold (P≤ 0.05). Finally, Rpa, showed a 100-fold increment (P≤ 0.01). Erα, Erß and Errγ genes showed a BPA up-regulation of 2-4-fold in all of the culture times (P≤ 0.05). Oocytes exposed to BPA showed an up-regulation of genes involved in DSB generation, signaling and repair except by Mlh1. Thus, BPA can modify the gene expression pattern, which may explain the effects of BPA on female germ cells.

SRD5A2 gene mutations and polymorphisms in Spanish 46,XY patients with a disorder of sex differentiation.

One hundred and forty-six index patients with 46,XY DSD in whom gonads were confirmed as testes were consecutively studied for a molecular diagnosis during the period 2002-2010. AR gene was analysed in all patients as the first candidate gene, yielding a mutation in 42.5% of cases and SRD5A2 gene was analysed as the second candidate gene, resulting in the characterization of 10 different mutations (p.Y91D, p.G115D, p.Q126R, p.R171S, p.Y188CfsX9, p.N193S, p.A207D, p.F219SfsX60, p.R227Q and p.R246W) in nine index patients (6.2% of the total number of 46,XY DSD patients). One of the mutations (p.Y188CfsX9) has never been reported. In addition, we genotyped SRD5A2 gene p.V89L and c.281+15T>C polymorphisms in 46,XY DSD and in 156 normal adult males and found that patients with SRD5A2 mutations or without a known molecular diagnosis presented a higher frequency of homozygous p.L89, homozygous TT and combined CCTT genotypes compared with controls. This result suggests that 46,XY DSD patient phenotypes may be influenced by SRD5A2 polymorphism genotypes. SRD5A2 gene mutations may not be as infrequent as previously considered in 46,XY DSD patients with variable degrees of external genitalia virilization at birth and normal T production and appears to be the second aetiology in our series.

A histological study of fetoscopic membrane defects to document membrane healing.

OBJECTIVE: To evaluate the rate of spontaneous healing in human fetal membranes after fetoscopy. STUDY DESIGN: Membranes from patients that had undergone fetoscopic interventions and delivered in one of the two treatment centers were included in the study. The membranes were examined macroscopically for any remaining defects and if present, the size of the defect in chorion and amnion was measured. Subsequently, the defect was excised and stained with HE for histological evaluation. Additional immunohistochemical staining was performed with Ki-67, cytokeratin and vimentin. The proliferation index (percentage of proliferating cells) was calculated in amnion and chorion. RESULTS: Nineteen membrane defects were included in the study. The median time interval between invasive procedures and delivery was 60 days (range 3-112). All fetoscopic defects (n=19) could be identified in the gestational sac and in none spontaneous closure had occurred. Proliferation indices as measured by inmunohistochemistry were very low (median 2.8%, range 0-7%) in the chorion and 0% in the amnion. CONCLUSION: No evidence of spontaneous membrane healing was found after fetoscopic procedures, suggesting that the membrane defect normally persists until delivery. Absence of amniotic fluid leakage after invasive procedures may be based on mechanisms other than histologic membrane repair.

[New surgical strategy in gastroschisis: treatment simplification according to its physiopathology]. / Nueva estrategia quirúrgica en la gastrosquisis: simplificación del tratamiento atendiendo a su fisiopatología.

INTRODUCTION: Gastroschisis (GS) is a congenital abdominal wall defect that permits bowel exposure to amniotic fluid (AF). Intestinal damage is related to the chemical action of AF and constriction. After birth at term, a thickened intestinal wall with inflammation and, in some cases, intestinal atresias were observed. Surgical repair and intestinal reubication may be difficult, and thus staged silo repair could be necessary. These patients require a long hospital stay owing to bowel damage causing severe intestinal hypoperistalsis and poor absorptive capacity. Total parenteral nutrition (TPN) is required for a long period. OBJECTIVE: The aim of this prospective study is to evaluate the benefits of a preterm delivery to avoid bowel damage and its post-natal consequences. PATIENTS AND METHODS: Six cases of prenatally-diagnosed GS have been treated following a new strategy since July 2002. A preterm Cesarean section (c-section) delivery was programmed at 34-35 weeks of gestational age (GA). Some hours after birth, at bedside in the NICU, bowel reduction through the defect hole was performed under general anesthesia. This preterm group (PT) was compared the past 6 cases at term (AT) from January 1998 to July 2002. Macroscopic appearance, atresia existence, surgical technique, silo requirement, neonatal outcome, TPN and hospital stay were analyzed. RESULTS: All six cases AT (mean GA: 36.3 weeks) presented bowel inflammation and thickened wall. Only 2/6 cases allowed the intestine to be housed in a primary closure after laparotomy. 4/6 cases required staged silo repair. 1 patient presented intestinal atresia and other had perforations who died at 17 days of life from intestinal sepsis. Mean postoperative intubation period was 16.2 days. Mean TPN was 41.2 days and mean hospital stay 69.8 days. PT group was monitored by prenatal sonography seeking bowel sonolucency. After programmed PT c-section delivery (mean GA: 34.8 weeks) in all 6 cases, bowel loops presented normal appearance and intestinal thickening was absent, except in one case. No prematurity-related respiratory complications were observed. Easy bowel reduction without abdominal compression was performed in all cases. 1/6 cases required surgical release of occlusive intestinal adherence. Mean postoperative intubation period was 0.4 days (9.6 hours). Oral feeding was started at 6 days. Mean TPN was 13.4 days and mean hospital stay 28.6 days. CONCLUSIONS: The third trimester is a critical period for fetal bowel development. Intestinal damage rises with increasing exposure time to amniotic fluid. This strategy of preterm delivery for the treatment of GS avoids intestinal damage, prevents "peel" and intestinal atresia, renders surgical reduction easier, reduces the hypoperistalsis, need for TPN and hospital stay. Multidisciplinary coordination between obstetricians, neonatologists and pediatric surgeons is required.

Vascular endothelial growth factor is expressed in human fetal growth cartilage.

Angiogenesis is a crucial event in endochondral ossification. Chemoattractants and mitogens for endothelial cells (such as basic fibroblast growth factor [bFGF] and transforming growth factor beta [TGF-beta]), which act as local regulators of the process, are synthesized by chondrocytes under several stimuli and in relation to the differentiation stage of the cartilage. Vascular endothelial growth factor (VEGF) is a 44-kDa protein well known as a potent angiogenic molecule owing to its mitogenic and permeability-causing properties. In this work, VEGF was located by immunohistochemistry in growth plate cartilage of human fetuses (20-22 weeks old) and its expression was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR). Primary culture of human fetal epiphyseal chondrocytes (HFEC) maintained VEGF expression at protein and messenger RNA (mRNA) levels and this expression was stimulated by cartilage-promoting growth factors incorporated into the culture media (rFGF-b, rTGF-beta1, and insulin-like growth factor [rFGF-b] at 50 ng/ml). The conditioned medium (CM) of HFEC stimulated the proliferation of endothelial cells, and this was partially blocked by anti-VEGF antibody. These studies showed VEGF production by chondrocytes of the epiphyseal growth cartilage and suggested a role of this factor in cartilage physiology and the angiogenic process.

Meiotic arrest and germ cell apoptosis in androgen-binding protein transgenic mice.

The fundamental role of androgen-binding protein (ABP) in spermatogenesis remains obscure after nearly 25 yr since its first characterization. In the present investigation, we used a transgenic mouse model that overexpresses rat ABP to examine the potential involvement of this protein in the regulation of processes occurring during spermatogenesis. Specifically, homozygous or heterozygous transgenic mice were analyzed in terms of spermatogenic progression, DNA fragmentation pattern, and germinal cell ploidy status. All animals homozygous for transgenic ABP exhibited an increased accumulation of primary spermatocytes and cells at metaphase with abnormal morphology and localization within the seminiferous epithelium. Analysis of DNA fragmentation by in situ techniques and agarose gel electrophoresis provided evidence for an increased occurrence of apoptosis in the transgenic animals, principally involving pachytene spermatocytes and cells at metaphase. Flow cytometric analysis of the DNA content of isolated germ cells revealed a reduction in the number of haploid cells, an increase in the number of tetraploid cells, and the appearance of a hypotetraploid cell population, consistent with degenerating primary spermatocytes. In mice heterozygous for the transgene, the effects were less prominent, and the degree to which spermatogenesis was compromised correlated with the levels of ABP messenger RNA in individual animals. The present results are interpreted to suggest that ABP can act as a modulator of spermatogenesis by regulating completion of the first meiotic division of primary spermatocytes.

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages.

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.

Giant-cell chondrodysplasia in a male infant with clinical and radiological findings resembling the Piepkorn type of lethal osteochondrodysplasia.

We report on a patient whose clinical, radiologic, and histopathologic findings are compatible with the Piepkorn type of lethal short-limb osteochondrodysplasia, but who also showed multinucleated giant chondrocytes in cartilage. Multinucleated giant cells are an unusual finding in osteochondrodysplasias, having been reported in atelosteogenesis type I and boomerang dysplasia. This uncommon histopathologic finding and the clinical and radiographic findings strongly support the diagnosis of boomerang dysplasia in the present patient. Our patient supports the previously suggested existence of an entity including atelosteogenesis and boomerang dysplasia. If this is so, the current patient and that described by Piepkorn et al. [1977: Teratology 16:345-350] could represent the most severe clinical expression of that condition.

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice.

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.

Use of human central nervous system cell cultures in neurotoxicity testing.

The nervous system is highly sensitive to toxic damage. Many environmental contaminants can produce acute or chronic neurological effects, and contribute to neural damage and cell death in neurodegenerative diseases. The utilization of primary cultures of neurons and glial cells is an essential step in investigating the specificity of the effects and mechanisms of action of the test chemical. If we take into account interspecies differences, cultures of human central nervous system (CNS) cells would be the best-suited test models for in vitro neurotoxicity testing. For practical and ethical reasons, human neuronal and glial cultures cannot be used for routine neurotoxicity testing, but they may be very useful for validating results from murine cultures and to address specific toxicity questions. For instance, we are investigating the action of agents producing oxygen radical damage in CNS cells. Oxidative stress is known to trigger apoptotic death of neurons and lead to neurodegeneration. A useful model in which to study these processes could be neuronal cultures obtained from CNS tissue with trisomy 21, since these cells suffer oxidative stress and apoptotic cell death in vitro. Besides primary cultures, human-derived clonal cell lines such as neuroblastoma SH-SY5Y can offer a first-step approach in neurotoxicity testing.

Brown adipose tissue changes in anencephalic infants. Image analysis study.

Perirenal brown adipose tissue was studied in 49 liveborn anencephalic infants of appropriate weight, whose lifespan ranged from 2 hours to 6 days. An additional series of 187 full-term infants was used as control. Morphological evidence of active and marked BAT lipolysis was found in large number of anencephalic infants suggesting that activation of BAT metabolism leading to lipolysis in the newborn does not depend on a diencephalic pathway but on peripheral mechanisms. Image analysis study did not sustain significant differences in the intrauterine storage of lipids in brown adipose tissue cells for the anencephalic and control infants who died within 24 hours of birth.

Morphogenesis of pulmonary veno-occlusive disease in a newborn. Image analysis study.

A 9-day-old girl was admitted to hospital because of respiratory distress. The girl died on the 11th day of life shortly after an haemodynamic study and the autopsy disclosed a pulmonary veno-occlusive disease with lesions in relatively early stages of development. Morphological pattern and computerized image analysis data induce to rule out a thrombosis as a previous event to the development of fibrotic changes and point to a "primary" intimal involvement, presumably triggered by an endothelial injury, determining a myxoid change of the intima and leading to a cellular proliferation and ultimate fibrotic occlusion of the small pulmonary veins. Apparently the lesions progress from the smaller pulmonary veins to the larger ones, in which the involvement is focal in character and related to the openings of collateral branches. The homogeneous character of the more recent lesions in the larger veins is in contrast to the heterogeneity of the fibrotic process in the smaller ones.

Pancreatectomy extension in persistent hyperinsulinaemic hypoglycaemia: a new strategy.

INTRODUCTION: Persistent neonatal and infantile hyperinsulinaemic hypoglycaemia (PNHH) is a rare entity which remains to be elucidated but is associated with severe lesions in paediatric patients. The aim of this study was to present our current surgical strategy with this disease, based on our pathologic findings and clinical experience. MATERIALS AND METHODS: This is a retrospective study of 29 patients treated, medically and surgically, at our centre. In 15 surgical patients, morphologic, morphometric and immunohistochemical studies for insulin, somatostatin and glucagon were performed and consequently it was possible to establish a focal and different forms of a diffuse type. RESULTS: Of 29 patients studied, 25 were diagnosed before one year of age and 4 between the first and second year of infancy. Of the first 25 patients, one died 7 hours post partum. Twelve patients received medical treatment alone: one died at 45 days of life and the remaining 11 had a good outcome. Another 12 patients additionally received surgical treatment. In 2 of these, adenoma were observed and removed and the patients cured. Subtotal pancreatectomy was performed in the remaining 10. (One case was normal and cured and the other 9 had the diffuse type.) Of these 9 patients with diffuse type, 4 died, 3 were cured and 2 underwent repeat surgery. Of the 4 patients diagnosed later, 3 underwent surgery (2 with adenomas and 1 diffuse type) and the other received medical treatment alone. CONCLUSIONS: We currently give medical treatment for all types and forms of PNHH. If the patient is resistant to therapy, adenoma is ruled out. If adenoma is diagnosed, it is removed. If the type is diffuse, near-total pancreatectomy is performed with a perioperative biopsy. In cases of hyperplasia or mixed forms we recommend total pancreatectomy and in cases of nesidioblastosis, partial pancreatectomy.

[Hypoplasia and pulmonary hypertension in Down syndrome: prognostic evaluation using pulmonary biopsy]. / Hipoplasia e hipertensión pulmonar en un síndrome de Down: valoración pronóstica mediante la biopsia pulmonar.

The pulmonary hypoplasia, in Down's syndrome with congenital cardiac malformation, probably explains the poor behaviour of these patients, leading to early vascular pulmonary lesions, which progress quickly. We present a case, in which the lung biopsy, done before cardiac surgery, showed a lung hypoplasia and also enabled us to establish the grading of the pulmonary vascular disease. The pathologic evaluation is necessary to decide upon the intracardiac repair and to predict the child's outcome.

[A quantitative study of the structural anomalies in the pulmonary vessels by pulmonary biopsy in children with an interventricular communication]. / Estudio cuantitativo de las anomalías estructurales de los vasos pulmonares, mediante la biopsia pulmonar, en niños con comunicación interventricular.

INTRODUCTION: Pulmonary vascular disease occurs in patients with cardiac anomalies and left to right shunt as the cases of interventricular heart defects. The hemodynamic study of the structural changes and their relationship with the pathological lesions are useful to know the state of the pulmonary vessels and to establish the postsurgical patients outcome. METHODS: We analyzed the morphologic and morphometric data in 17 lung biopsies from children under three years with ventricular septal defect, focused the histologic study in grading the structural changes of the intima, media and adventitial vascular walls and the morphometric evaluation on the thickening of the media and the number of the intracinar arteries. RESULTS: There are significant statistical correlation among the pulmonary pressure and the age, the vascular resistances and the age, the cocient between the pulmonary arterial pressure and the systemic pressure and the external vascular diameter, the cocient of the pulmonary and systemic fluxes and the thickening of the media and finally between the pulmonary vascular resistances and the external arterial diameter. CONCLUSIONS: Our results suggest that the intracardiac repair is desirable within 6 months of life and lung biopsy should be undertaken because is the only way to determine the medial thickening and his muscular of fibrous proliferation that will clarify the post-surgical patients outcome.

Orbital metastasis, by transitional cell carcinoma of the bladder.

We report a new case of a patient with transitional cell carcinoma of the urinary bladder and a solitary metastasis to the orbita. A review of the literature shows two cases described previously. This case is interesting for the clinical features and evolution.

[Androgen insensitivity syndrome. Diagnosis and therapy]. / Sndrome de insensibilidad a los andrógenos. Diagnóstico y terapéutica.

Ten patients with complete or incomplete androgens insensitivity syndrome (A.I.S.) is reported. Diagnosis of A.I.S. is presented on the basis: clinical, phenotype, studies of sexual steroids specially androgens, gonadotropins, 5-alpha-reductase activity and possible abnormality of androgen target cells from the genital skin. Histology of the testes is shown. Surgical treatment of these patients is reported, depending of the abnormality of the genitalia.