[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction]. / Microscopia de luz polarizada para evaluación de ovocitos como factor pronóstico en la evolución de un ciclo de reproducción asistida.

BACKGROUND: Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. OBJECTIVE: To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. MATERIAL AND METHODS: Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. RESULTS: Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). CONCLUSIONS: Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Somatostatin analogues improve health-related quality of life in polycystic liver disease: a pooled analysis of two randomised, placebo-controlled trials.

BACKGROUND: Polycystic liver disease is associated with impaired health-related quality of life (HRQL). Somatostatin analogues reduce hepatomegaly in polycystic liver disease. AIM: To determine whether somatostatin analogues improve HRQL and to identify factors associated with change in HRQL in polycystic liver disease. METHODS: We pooled data from two randomized, double-blind, placebo-controlled trials that evaluated HRQL using the Short-Form 36 (SF-36) in 96 polycystic liver disease patients treated 6-12 months with somatostatin analogues or placebo. The SF-36 contains a summarizing physical and mental component score and was administered at baseline and at the end of treatment. We used random effect models to delineate the effect of somatostatin analogues on HRQL. We determined the effect of demographics, height-adjusted liver volume, change in liver volume, somatostatin analogue-associated side effects with change in HRQL. In patients with autosomal dominant polycystic kidney disease, we estimated the effect of height-adjusted kidney volume and change in kidney volume in relation to HRQL. RESULTS: Physical component scores improved with somatostatin analogues, but remained unchanged with placebo (3.41 ± 1.29 vs. -0.71 ± 1.54, P = 0.044). Treatment had no impact on the mental component score. Large liver volume was independently associated with larger HRQL decline during follow up (-4.04 ± 2.02 points per logarithm liver volume, P = 0.049). In autosomal dominant polycystic kidney disease, patients with large liver and kidney volumes had larger decline in HRQL (5.36 ± 2.54 points per logarithm liver volume; P = 0.040 and -4.00 ± 1.88 per logarithm kidney volume; P = 0.039). CONCLUSION: Somatostatin analogues improve HRQL in symptomatic polycystic liver disease. Halting the progressive nature of polycystic liver disease is necessary to prevent further decline of HRQL in severe hepatomegaly.

Therapeutic benefit of melatonin in refractory central serous chorioretinopathy.

PURPOSE: To evaluate the efficacy and safety of melatonin for the treatment of chronic central serous chorioretinopathy (CSCR). METHODS: Prospective comparative case series. A total of 13 patients with chronic CSCR were treated for 1 month: 8 patients were treated orally with 3 mg melatonin t.i.d., and 5 with placebo. All patients had 20/40 or worse Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) in the affected eye or presented an incapacitating scotoma. Most of the patients had previous failed treatments for their condition. Observational procedures included ETDRS BCVA, and complete ophthalmic examination. Optical coherence tomography (OCT) was performed at day 1 and week 4. Fluorescein angiography was performed at baseline only for diagnostic purposes. Data were subjected to two-sample t-test statistical analysis. P-values of <0.05 were considered statistically significant. RESULTS: At 1-month follow-up, BCVA significantly improved in 87.5% of patients treated with melatonin (7 of 8 patients, P<0.05). All patients showed a mean significant reduction (P<0.01) of central macular thickness (CMT) when compared with the baseline, with 3 patients (37.5%) exhibiting complete resolution of subretinal fluid at 1-month follow-up. No significant side effects were observed. No changes in BCVA or CMT were noted in the control group. CONCLUSIONS: These results suggest that melatonin is safe, well tolerated, and effective in the treatment of chronic CSCR, as it significantly improved BCVA and CMT in patients with this pathology. Further evaluations with longer follow-up and a larger patient population are desirable.

Formation and deposition of stable silver nanoparticles encapsulated in solid spheres.

We prepared solid spheres, with diameters between 100 and 400 nm, encapsulating silver nanocrystallites (about 20 ± 10 nm in diameter), by heating colloidal solutions of silver in methoxyethanol at 120 °C. Stable coatings were obtained using spin coating and rapid thermal processing on optical glass substrates. UV-Vis spectroscopy measurements demonstrated the remarkable stability against aggregation and particle growth, even after prolonged heating at 120 °C and exposed at direct sunlight, of the solutions, even using high concentrations of silver (1 M), and coatings of spheres encapsulating silver.

Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63.

Polycystic liver disease (PCLD) is characterized by intralobular bile duct cysts in the liver. It is caused by mutations in PRKCSH, encoding hepatocystin, and SEC63, encoding Sec63p. The main goals of this study were to screen for novel mutations and to analyze mutations for effects on protein structure and function. We screened 464 subjects including 76 probands by direct sequencing or conformation-sensitive capillary electrophoresis. We analyzed the effects of all known and novel mutations using a combination of splice site recognition, evolutionary conservation, secondary and tertiary structure predictions, PolyPhen, and pMut and sift. We identified a total of 26 novel mutations in PRKCSH (n = 14) and SEC63 (n = 12), including four splice site mutations, eight insertions/ deletions, six non-sense mutations, and eight missense mutations. Out of 48 PCLD mutations, 13 were predicted to affect splicing. Most mutations were located in highly conserved regions and homology modeling for two domains of Sec63p showed severe effects of the residue substitutions. In conclusion, we identified 26 novel mutations associated with PCLD and we provide in silico analysis in order to delineate the role of these mutations.

Determinants of renal volume in autosomal-dominant polycystic kidney disease.

The Consortium of Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) recently showed that renal enlargement in autosomal-dominant polycystic kidney disease mimicked exponential growth. We determined the effects of cyst initiation rate, total number, and growth rate on the time-dependent change of total cyst volume (TCV). Mathematical models with equations integrating cyst surface area, volume, and an invariant growth rate constant were used to compute the time-dependent change in volume of solitary and multiple cysts. Multiple expanding cysts increased TCV in an exponential-like pattern even when individual cysts formed at different rates or exhibited different but constant growth rates. TCV depended on the rate of cyst initiation and on the total number of cysts; however, the compounding effect of exponential-like growth was the most powerful determinant of long-term cyst expansion. Extrapolation of TCV data plots for individual subjects back to an age of 18 predicted TCV values within an established range. We conclude that cysts started early in life were the main contributor to eventual TCV while their growth rate primarily determined renal size; although the rate of formation and the ultimate number of cysts also contributed. The good fit between the exponential models and the extrapolated CRISP data indicates that the TCV growth rate is a defining trait for individual patients and may be used as a prognostic marker.

A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1(del2/del2) mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1(del2/del2) mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities.

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

Update on autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent, potentially lethal monogenic disorder. Over the past two decades, its study has yielded remarkable progress. The mutated genes have been identified by positional cloning, the function of a novel class of conserved proteins encoded by these genes has been partially elucidated, and a neglected organelle, the primary cilium, has become the focus of investigation. The large inter- and intra-familial variability, explained to a large extent by genetic heterogeneity and modifier genes, is now better appreciated. Increased understanding of the progression of the disease and of its underlying genetic, molecular and cellular mechanisms have laid the foundations for the development of potentially effective therapies and clinical trials.

Long-term ammonium chloride or sodium bicarbonate treatment in two models of polycystic kidney disease.

Administration of ammonium chloride aggravates, while short-term administration of sodium or potassium bicarbonate lessens the development of polycystic kidney disease in Han:SPRD rats. We have conducted studies to determine whether the protection afforded by the administration of sodium bicarbonate is sustained and prevents development of uremia during chronic administration and whether the effects of the administration of ammonium chloride and sodium bicarbonate are also observed in a different model of polycystic kidney disease, the CD1-pcy/pcy mouse. We found that chronic administration of 200 mM sodium bicarbonate to Han:SPRD rats inhibited cystic enlargement and prevented the subsequent development of interstitial inflammation, chronic fibrosis, and uremia. We also found that, while the administration of ammonium chloride has similar effects in Han:SPRD rats and CD1-pcy/pcy mice, the administration of sodium bicarbonate is only protective in the Han:SPRD rats. This probably reflects differences in these models (predominately involvement of proximal tubules in Han:SPRD rats and of collecting ducts and distal tubules in pcy/pcy mice) and the different location and nature of the renal metabolic responses to the administration of acid or alkaline load.

Treatment prospects for autosomal-dominant polycystic kidney disease.

An increased understanding of the molecular genetic and cellular pathophysiologic mechanisms responsible for the development of autosomal-dominant polycystic kidney disease (ADPKD), made possible by the advances in molecular biology and genetics of the last three decades, has laid the foundation for the development of effective therapies. As the concept that a polycystic kidney is a neoplasm in disguise is becoming increasingly accepted, the development of therapies for ADPKD may benefit greatly from the expanding body of information on cancer chemoprevention and chemosuppression. This review summarizes the observations that already have been made and discusses therapies for PKD that deserve investigation.

The pck rat: a new model that resembles human autosomal dominant polycystic kidney and liver disease.

BACKGROUND: The pck rat is a recently identified model of polycystic kidney disease (PKD) and liver disease (PLD) that developed spontaneously in the rat strain Crj:CD/SD. Its pattern of inheritance is autosomal recessive. METHODS: To characterize this new model, we studied pck rats derived from F9 breeding pairs from Charles River Japan and control Sprague-Dawley rats. Blood and tissues (kidneys, liver, and pancreas), obtained from these rats at 1, 7, 21, 70, and 182 days of age, were used for biochemical determinations, light and electron microscopy, and immunohistochemistry. RESULTS: The pck rats develop progressive cystic enlargement of the kidneys after the first week of age, and liver cysts are evident by day 1. The renal cysts developed as a focal process from thick ascending loops of Henle, distal tubules, and collecting ducts in the corticomedullary region and outer medulla. Flat and polypoid epithelial hyperplasia were common in dilated tubules and cysts. Apoptosis was common and affected normal, as well as dilated tubules, but less frequently cysts lined by flat epithelium. The basement membranes of the cyst walls exhibited a variety of alterations, including thinning, lamellation, and thickening. Focal interstitial fibrosis and inflammation were evident by 70 days of age. Segmental glomerulosclerosis and segmental thickening of the basement membrane with associated effacement of the podocyte foot processes were noted in some rats at 70 days of age. The PKD was more severe in male than in female pck rats, as reflected by the higher kidney weights, while there was no gender difference in the severity of the PLD. Mild bile duct dilation was present as early as one day of age. With age, it became more severe, and the livers became markedly enlarged. Even then, however, there was only a mild increase in portal fibrosis, without formation of fibrous septae. Slight elevations of plasma blood urea nitrogen levels were detected at 70 and 182 days of age. CONCLUSIONS: The pck rat is a new inherited model of PKD and PLD with a natural history and renal and hepatic histologic abnormalities that resemble human autosomal dominant PKD. This model may be useful for studying the pathogenesis and evaluating the potential therapies for PKD and PLD. The identification of the pck gene may provide further insight into the pathogenesis of autosomal dominant PKD.

Matched comparison of radical nephrectomy vs nephron-sparing surgery in patients with unilateral renal cell carcinoma and a normal contralateral kidney.

OBJECTIVE: To report the long-term follow-up of a matched comparison of radical nephrectomy (RN) and nephron-sparing surgery (NSS) in patients with single unilateral renal cell carcinoma (RCC) and a normal contralateral kidney. PATIENTS AND METHODS: Between August 1966 and March 1999, 1492 and 189 patients with unilateral RCC and a normal contralateral kidney underwent RN and NSS, respectively. Patients with renal impairment, previous nephrectomy, bilateral or multiple RCCs, metastasis, and familial cancer syndromes were excluded. A total 164 patients in each cohort were matched according to pathological grade, pathological T stage, size of tumor, age, sex, and year of surgery. The Kaplan-Meier method and stratified Cox proportional hazards model were used to estimate and compare overall, cancer-specific, local recurrence-free, and metastasis-free survival and survival free of chronic renal insufficiency. The 2 groups were evaluated for early (< or = 30 days) complications and proteinuria at last follow-up. RESULTS: At last follow-up, 126 RN patients (77%) and 130 NSS patients (79%) were alive with no evidence of disease. There was no significant difference observed between patients who had RN and those who had NSS with respect to overall survival (risk ratio, 0.96; 95% confidence interval [CI], 0.52-1.74; P = .88) or cancer-specific survival (risk ratio, 1.33; 95% CI, 0.30-5.95; P = .71). At 10 years, similar rates of contralateral recurrence (0.9% for RN vs 1% for NSS) and metastasis (4.9% for RN vs 4.3% for NSS) were seen in each group, whereas the rate of ipsilateral local recurrence for patients who underwent RN and NSS was 0.8% and 5.4%, respectively (P = .18). There was no significant difference in the early complications between the RN and NSS groups. However, patients who underwent RN had a significantly higher risk for proteinuria as defined by a protein/osmolality ratio of 0.12 or higher (55.2% vs 34.5%; P = .01). At 10 years, the cumulative incidence of chronic renal insufficiency (creatinine > 2.0 mg/dL at least 30 days after surgery) was 22.4% and 11.6%, respectively, for the RN and NSS groups (risk ratio, 3.7; 95% CI, 1.2-11.2; P = .01). CONCLUSIONS: This retrospective study of patients with unilateral RCC and a normal contralateral kidney suggests that NSS is as effective as RN for the treatment of RCC on long-term follow-up. The increased risk of chronic renal insufficiency and proteinuria after RN supports use of NSS.

Identification of a locus for autosomal dominant polycystic liver disease, on chromosome 19p13.2-13.1.

Polycystic liver disease (PCLD) is characterized by the growth of fluid-filled cysts of biliary epithelial origin in the liver. Although the disease is often asymptomatic, it can, when severe, lead to complications requiring surgical therapy. PCLD is most often associated with autosomal dominant polycystic kidney disease (ADPKD); however, families with an isolated polycystic liver phenotype without kidney involvement have been described. The clinical presentation and histological features of polycystic liver disease in the presence or absence of ADPKD are indistinguishable, raising the possibility that the pathogenetic mechanisms in the diseases are interrelated. We ascertained two large families with polycystic liver disease without kidney cysts and performed a genomewide scan for genetic linkage. A causative gene, PCLD, was mapped to chromosome 19p13.2-13.1, with a maximum LOD score of 10.3. Haplotype analysis refined the PCLD interval to 12.5 cM flanked by D19S586/D19S583 and D19S593/D19S579. The discovery of genetic linkage will facilitate diagnosis and study of this underdiagnosed disease entity. Identification of PCLD will be instrumental to an understanding of the pathogenesis of cyst formation in the liver in isolated PCLD and in ADPKD.

Gender-dependent effect of L-NAME on polycystic kidney disease in Han:SPRD rats.

To determine whether the renal nitric oxide (NO) system has a role in the pathogenesis of polycystic kidney disease (PKD) in Han:Sprague-Dawley (SPRD) rats, the NO synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), 70 mg/L, or L-arginine, 0.5 g/L, was administered to heterozygous diseased (cy/+) and homozygous normal animals. Urine nitrate and nitrite excretion was reduced by L-NAME treatment and, in the male cy/+ rats, increased by L-arginine administration. The administration of L-NAME significantly increased blood pressure in all groups, whereas L-arginine had no effect. L-NAME and L-arginine had a modest but significant overall effect on the severity of cystic disease in male rats, reflected by relative kidney weights and cyst volume densities. This effect was gender dependent because it was not observed in female animals. The administration of L-NAME resulted in a significant increase in plasma creatinine concentration of the cy/+ rats, which was more marked in male than female animals. These observations support the recently reported gender differences in the renal NO system and a small role for NO synthesis that can be inhibited by L-NAME in the pathogenesis of PKD in Han:SPRD rats. These observations do not exclude a more important role for the endogenous renal NO production in the pathogenesis of PKD in view of a recent report of a major NOS resistant to conventional inhibitors in the rat kidney.

Chronic subdural hematoma in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) has been associated with an increased incidence of aneurysmal subarachnoid hemorrhage and intracerebral hematomas. We describe five patients with chronic subdural hematomas, a previously unrecognized complication of ADPKD. In four of the five cases, no trigger was apparent. Clinical presentation was subtle, with mild hemiparesis, headache, or both in four patients and transient neurological deficits mimicking transient ischemic attacks in one patient. In three of the five patients, a retrocerebellar arachnoid cyst was found, suggesting a plausible causal relation between the intracranial arachnoid cysts and the subdural hematomas. In one patient, subdural hematoma was in close proximity to the frontally located arachnoid cyst.