OBJECTIVE: To evaluate the effects on human spermatozoa of freeze-drying, also known as lyophilization, and of cryopreservation in liquid nitrogen. DESIGN: Prospective experimental study. SETTING: ...Reproductive medicine unit and a private IVF center. PATIENT(S): Thirty healthy male donors. INTERVENTION(S): Sperm samples from 30 donors divided as two aliquots, one to be lyophilized and the other to be cryopreserved in liquid nitrogen. MAIN OUTCOME MEASURE(S): Assessment of count, motility, morphology, viability, DNA integrity, chromosomal status, and birefringence properties of lyophilized and cryopreserved human spermatozoa compared with the same parameters in the fresh sample. RESULT(S): Although sperm viability and motility were totally compromised after freeze-drying, the sperm chromatin structure was not altered in comparison with fresh samples, which demonstrated that the procedure did not affect DNA integrity. The sperm-head inner protoplasmic structures were also preserved, which was estimated by assessing the corresponding birefringence characteristics. After cryopreservation with liquid nitrogen, the motility, viability, and DNA integrity of spermatozoa were statistically significantly reduced compared with the fresh samples; the proportion of sperm cells with abnormal head birefringence increased meaningfully. CONCLUSION(S): The process of freeze-drying deeply damages cell membranes; however, unlike with liquid nitrogen preservation, it does not affect DNA integrity.
Radial deficiencies (RDs), defined as under/abnormal development or absence of any of the
structures of the forearm, radial carpal bones and thumb, occur with a live birth incidence ranging
from 1 ...out of 30,000 to 1 out 6,000 newborns and represent about one third/one fourth of all the congenital
upper limb anomalies. About half of radial disorders have a mendelian cause and pattern of
inheritance, whereas the remaining half appears sporadic with no known gene involved. In sporadic
forms certain anomalies, such as thumb or radial hypoplasia, may occur either alone or in association
with systemic conditions, like vertebral abnormalities or renal defects. All the cases with a mendelian inheritance are syndromic
forms, which include cardiac defects (in Holt-Oram syndrome), bone marrow failure (in Fanconi anemia), platelet
deficiency (in thrombocytopenia-absent-radius syndrome), ocular motility impairment (in Okihiro syndrome). The
genetics of radial deficiencies is complex, characterized by genetic heterogeneity and high inter- and intra-familial clinical
variability: this review will analyze the etiopathogenesis and the genotype/phenotype correlations of the main radial deficiency
disorders in humans.
Branchio-oto-renal syndrome combines branchial arch defects, hearing impairment and renal malformations or hypoplasia. Due to the high phenotypic variability, prenatal diagnosis has a limited ...prognostic value in mutation-positive cases. We report the first branchio-oto-renal syndrome molecular prenatal diagnosis and ultrasonographic follow-up, showing a normal renal growth until the 24th week of pregnancy, a growth deceleration during the third trimester and a renal volume recovery during the first months of life.
We describe the identification and characterization of
Trim43a,
Trim43b, and
Trim43c genes, whose expression are restricted to preimplantation stages and peak at the 8-cell to morula stage. We ...identified a 5
kb DNA fragment that covers upstream region of
Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos.
Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a ...gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix–loop–helix ubiquitous kinase gene (
Chuk, also known as IκB kinase α, IKKα or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes (
Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by
Chuk and other housekeeping genes (
Actb, Gapdh, Eef1a1, and
H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that
Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.
Obesity is a multifactorial disease caused by the interaction of genetic, environmental, and behavioral factors. Currently, only a small number of obese children undergo genetic analysis, usually ...when obesity is associated with dysmorphic features. The aim of this study was to identify genomic rearrangement causing obesity.
We analyzed the DNA of children and adolescents by single-nucleotide polymorphism-array (platform CytoScan HD, Affymetrix). Patients included in this study were obese with dysmorphic features and/or intellectual disabilities and/or neuropsychomotor signs.
Ninety-four children and adolescents with obesity (9.25 ± 4.04 years old, 60 males) were enrolled in the study. Dysmorphic features were found in 64 out of 94 subjects (68.1%), intellectual disability was found in 23 subjects (24.5%), and other neuropsychomotor signs in 31 (32.9%). Copy number variations (CNVs) were identified in 43 out of 94 patients (45.7%): among these 14 subjects showed at least 1 deletion, 22 duplication, whereas 7 patients showed both deletion and duplication. In 20 subjects (13 males), CNVs were linked or possibly related with obesity; in 23 subjects, this correlation cannot be inferred.
A genetic origin of obesity was detected in about half of our obese children and adolescents with associated dysmorphic features and/or intellectual disability and/or neuropsychomotor signs. In these children, array-CGH analysis can be useful to identify causative genetic mutations, with consequent advantage in therapeutic management and follow-up of these patients.
The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to ...the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named
Zscan4a-Zscan4f, three –
Zscan4c,
Zscan4d, and
Zscan4f – encode full-length ORFs with 506 amino acids.
Zscan4d is a predominant transcript at the late 2-cell stage, whereas
Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any
Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development.
To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable ...tetracycline-repressible locus. Of the 50 TFs,
Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by
Esx1,
Sox9,
Tcf3,
Klf4, and
Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice.
Abstract National legislations represent one of the main factors influencing access to assisted reproduction treatment. The Italian situation in the last decade is an example of how the treatment of ...patients for preimplantation genetic diagnosis (PGD) was more dependent on regulators than on medical choices. This report analysed how the changes in Italian regulation affected the number of PGD referrals to this study centre, as well as their decision to opt for cross-border reproductive care (CBRC). The analysis showed that during the period in which PGD was actually not performed because of the restriction imposed by the Italian law on IVF (from 24 February 2004 to 7 May 2009) there was a significant decrease in the number of referrals asking for PGD (2.5% of total referrals) compared with the previous years (3.3%; P < 0.025) and following years when PGD was legalized (5.1%; P < 0.001). The number of couples opting for CBRC had an opposite trend, reaching a maximum when PGD was banned from Italian centres (55 couples), whereas after the readmission of PGD, only eight couples went abroad for treatment. Concomitantly, since May 2009, the proportion of couples performing a PGD cycle in this centre has constantly increased. National restrictions in the Italian law on IVF affect dramatically the access of preimplantation genetic diagnosis (PGD) for couples carrying a monogenic disease and/or a translocation. These couples are often forced to travel abroad to receive an IVF treatment (so-called ‘cross-border reproductive care’), and this often entails a series of discomforts for patients, including the interruption of employment and high expense, which are related to travel and long-term accommodation as well as to the PGD treatment itself. Even more critical is the situation of couples that already have children, especially if affected by a severe pathology. This study analyses the impact that the Supreme Court Decision on 8 May 2009 (which declared the law to be partly unconstitutional) had to the flux of PGD couples coming to our centre. This figure was significantly higher compared with two previous periods: from January 1998 to 23 February 2004 when no regulation on IVF matters existed and from 24 February 2004 to 7 May 2009 during which the law banned any kind of treatment. The number of couples opting for receiving treatment abroad is also reported, showing a substantial decrease after PGD was readmitted in Italy. Results indicate that if patients have the possibility of performing a treatment cycle in their own country, they opt for this choice, and, hopefully, the phenomenon of cross-border reproductive care come to an end.
Abstract In the present study we describe the exome sequencing and analysis of a patient with Catel-Manzke-like phenotype showing bilateral hyperphalangism of the second finger and thumb clinodactyly ...due to a unilateral delta phalanx, associated with growth, cardiac and vertebral defects. The exome sequencing analysis excluded pathogenetic mutations in the genes known to cause syndromes with hyperphalangism and did not identify any alteration in the X-chromosome or de novo mutations in likely candidate genes. Under the assumption of an autosomal recessive mode of inheritance and based on the frequency of the single nucleotide variants found in homozygous or double heterozygous states and the results of computer prediction programs, only one gene, DNAH10 , emerged as a candidate in the pathogenesis of the disease in our patient. However, the differences among the known biological functions of DNAH10 and the genes involved in the other syndromes with hyperphalangism, suggest caution in the interpretation of the results.
PDF
