Cerebral Palsy

Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.

Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity.

Immunology plays a crucial role in establishing successful pregnancy. This is primarily due the recognition of fetal antigen and alteration of the immune response mounted by the mother. It is therefore easy to understand the importance of testing for immune causes of reproductive failure and offer immune therapy to those with abnormal immune response. The main difficulty with this model is the understanding of what is normal, which is still under study, and then to test for abnormality. It is only after these two questions are answered that we can consider the use of immunotherapy in women with immune causes of reproductive failure. This review looks at the current available information on the immunology surrounding reproductive failure and the current therapies offered to rectify these abnormalities. Till date numerous studies have looked into the role of various immune suppressant modulatory therapies in women with recurrent miscarriage or implantation failure after assisted conception and have suggested that there is a place for immune therapy in reproductive failure. However the search for best evidence is still on and randomized, blinded data with adequate sample size is lacking. Till such time that it becomes available, immune testing and immunotherapy should be offered within the realms of a research setting.

AIM: The aim of this study was to evaluate the performance of the combined test (nuchal translucency, NT) and maternal serum free-beta human chorionic gonadotropin (free beta-hCG) and pregnancy-associated plasma protein-A (PAPP-A), compared to the NT measurement alone, in fetal aneuploidy screening in the general population and in pregnant women aged 35 years and over. In addition, the association between increased NT and presence of cardiac defects in fetuses with normal karyotype was evaluated. METHODS: Screening at 11-14 weeks of gestation by NT measurement and combined test was carried out in 1521 pregnant women. The estimated risk for trisomy 21 and trisomy 13+18 was calculated (risk cut-off 1/300 and 1/750 respectively) and the outcomes was evaluated. RESULTS: Ten cases of trisomies (21 and 18) occurred, seven of which among the older group of pregnant women. The detection rate (DR) for the combined test was 80% in the general population and 85.7% in older pregnant women, which resulted higher rate than NT measurements alone. Detection rate of cardiac defects using NT measurements was 66.6%. CONCLUSION: The combined test is an effective screening for aneuploidies and reduces at 14% the need of invasive testing in the older obstetric population, detecting all the trisomies occurred in this group. The association between increased NT and cardiac defects is confirmed but it seems too weak to consider NT as a single screening strategy for these abnormalities.

Steroidogenic factor 1 (SF1, NR5A1, Ad4BP) is a nuclear receptor and regulator of multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Complete deletion of Nr5a1 in XY mice results in adrenal and gonadal agenesis, female external genitalia and presence of Müllerian structures. These findings were first reported in the early 1990s. Subsequently, NR5A1 mutations were found in two 46,XY phenotypic females with Müllerian structures and adrenal failure and in one 46,XX female with adrenal failure. More recently, heterozygous NR5A1 mutations have been identified in a substantial proportion of patients with 46,XY disorders of sex development (46,XY DSD) without adrenal insufficiency. Most of these individuals display severe underandrogenization with ambiguous genitalia at birth, partial gonadal dysgenesis, and absence of Müllerian structures or remnants. Some of the patients have a milder phenotype such as hypospadias and cryptorchidism, due to less severe defects in androgen synthesis. Testosterone, inhibin B and AMH are usually low indicating a partial (or sometimes progressive) form of gonadal dysgenesis in most cases. However, normal testosterone production at birth might also be present. The frequency of NR5A1 mutations in otherwise unexplained 46,XY DSD with underandrogenization and partial testicular dysgenesis has been estimated to be about 15%. Furthermore, NR5A1 mutations have now been found in women with familial and sporadic 46,XX primary ovarian insufficiency without adrenal failure. These human phenotypes associated with NR5A1 mutations show that SF-1 is a key factor involved in both human testis and ovarian development, but that human adrenal development seems to be more resistant to the effects of SF-1 haploinsufficiency than gonadal development. Patients with 46,XY DSD and mild underandrogenization due to partial testicular dysgenesis should possibly be assigned to the male sex, as small testes with Leydig, Sertoli and germ cells are present in almost all cases. Additionally, spontaneous virilization in puberty might be possible in patients with NR5A1 mutations. However, fertility options and the risk of testicular malignancy and adrenal insufficiency in adulthood are unknown and need to be investigated in long-term outcome studies.

AIM: There are no common guidelines to identify the population at risk to develop hypocalcemia preoperatively or early in the postoperative course in thyroidectomized patients, therefore the authors suggest to examine the PTH value preoperatively. METHODS: We divided 391 patients in two groups according to the preoperative PTH level (normal, pound72 pg/mL vs. increased >73 pg/mL). RESULTS: In 92/391 cases (23.52%) preoperative PTH was increased (mean PTH level 112.4+/-24.8 pg/mL; normal range 12-72 pg/mL). Out of these, 43 (46.7%) had hypocalcaemia postoperatively. In 18 out of the 43 patients clinical hypocalcemia also developed. The mean follow-up was of 148+/-13 days. Of the 299 patients with normal preoperative PTH, 127 (42.47%) developed postoperative hypocalcemia (mean calcium level 7.4+/-0.33 mg/dL). In 30 patients it was also clinically evident. The difference in terms of incidence of symptomatic hypocalcemia was statistically significant (increased preoperative PTH 19.5% vs. normal preoperative PTH 10.03% , P=0.036). CONCLUSION: All candidates to thyroidectomy should be investigated for preoperative PTH abnormalities.

BACKGROUND AND OBJECTIVES: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Most childhood cases are caused by Shiga toxin-producing bacteria. The other form, atypical HUS (aHUS), accounts for 10% of cases and has a poor prognosis. Genetic complement abnormalities have been found in aHUS. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: We screened 273 consecutive patients with aHUS for complement abnormalities and studied their role in predicting clinical phenotype and response to treatment. We compared mutation frequencies and localization and clinical outcome in familial (82) and sporadic (191) cases. RESULTS: In >70% of sporadic and familial cases, gene mutations, disease-associated factor H (CFH) polymorphisms, or anti-CFH autoantibodies were found. Either mutations or CFH polymorphisms were also found in the majority of patients with secondary aHUS, suggesting a genetic predisposition. Familial cases showed a higher prevalence of mutations in SCR20 of CFH and more severe disease than sporadic cases. Patients with CFH or THBD (thrombomodulin) mutations had the earliest onset and highest mortality. Membrane-cofactor protein (MCP) mutations were associated with the best prognosis. Plasma therapy induced remission in 55 to 80% of episodes in patients with CFH, C3, or THBD mutations or autoantibodies, whereas patients with CFI (factor I) mutations were poor responders. aHUS recurred frequently after kidney transplantation except for patients with MCP mutations. CONCLUSIONS: Results underline the need of genetic screening for all susceptibility factors as part of clinical management of aHUS and for identification of patients who could safely benefit from kidney transplant.

Rationale: The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. Objective: We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. Methods and Results: We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and taudecay in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+i) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+i) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R4496C/+) mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Conclusions: Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca(2+) handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca(2+) release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remains unclear. We investigated potential Ca(2+) abnormalities in skeletal muscles comprised with different fiber composition and whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5 month-old wild type (WT) and congenic alpha(2A)/alpha(2C) ARKO mice in a C57Bl6/J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of 8 weeks running session of 60 min, 5 days/wk (from 5 to 7 months of age). After completion of exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography and Ca(2+) handling related protein expression by Western blot. alpha(2A)/alpha(2C) ARKO mice displayed decreased ventricular function, exercise intolerance and muscle weakness paralleled by decreased expression of sarcoplasmic Ca(2+) release-related proteins [alpha1, alpha2 and beta1 subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca(2+) reuptake-related proteins [sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA1/2), and Na(+)/Ca(2+) exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance, muscle function and re-established the expression of proteins involved in sarcoplasmic Ca(2+) handling toward WT levels. We provide evidence that Ca(2+) handling-related protein expression is decreased in this heart failure model and that exercise training improved skeletal muscle function associated to changes in the net balance of skeletal muscle Ca(2+) handling proteins.

OBJECTIVE: To identify and define the imaging characteristics of children with auditory neuropathy spectrum disorder (ANSD). DESIGN: Retrospective medical records review and analysis of both temporal bone computed tomographic (CT) and magnetic resonance images (MRI) in children with a diagnosis of ANSD. SETTING: Tertiary referral center. Patients: One hundred eighteen children with the electrophysiologic characteristics of ANSD with available imaging studies for review. INTERVENTIONS: Two neuroradiologists and a neurotologist reviewed each study, and consensus descriptions were established. MAIN OUTCOME MEASURES: The type and number of imaging findings were tabulated. Results: Sixty-eight (64%) MRIs revealed at least 1 imaging abnormality, whereas selective use of CT identified 23 (55%) with anomalies. The most prevalent MRI findings included cochlear nerve deficiency (n = 51; 28% of 183 nerves), brain abnormalities (n = 42; 40% of 106 brains), and prominent temporal horns (n = 33, 16% of 212 temporal lobes). The most prevalent CT finding from selective use of CT was cochlear dysplasia (n = 13; 31%). CONCLUSION: Magnetic resonance imaging will identify many abnormalities in children with ANSD that are not readily discernable on CT. Specifically, both developmental and acquired abnormalities of the brain, posterior cranial fossa, and cochlear nerves are not uncommonly seen in this patient population. Inner ear anomalies are well delineated using either imaging modality. Because many of the central nervous system findings identified in this study using MRI can alter the treatment and prognosis for these children, we think that MRI should be the initial imaging study of choice for children with ANSD.