Search PubMed⌕ Search

Biomedical subjects

A De Vos

Publications and source records attributed to A De Vos.

At least 19 recordsLinked to original sources
PubMed2003

Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser.

BACKGROUND: Zona pellucida opening for blastomere removal can be done by mechanical or chemical means, or by laser. So far, only limited data on the use of laser systems for zona drilling in cases of PGD are available. METHODS: Results of embryo biopsy and outcome of PGD in two periods were compared. In the first period, acid Tyrode medium was used for zona drilling. In the second period, zona drilling was performed by a 1.48 micro m infrared laser. RESULTS: In the first period, 59 cycles resulted in 53 biopsy procedures with 356 biopsied embryos. In the second period, these numbers were 69 cycles, 69 biopsy procedures and 402 biopsied embryos. Fewer blastomeres were intact (95.2%) after zona drilling with acid Tyrode than after laser zona drilling (98.3%, P = 0.02). Rates of positive HCG (37.5% versus 35.5%), ongoing pregnancy rates (31.3% versus 25.0%) and ongoing implantation rates (18.9% versus 14.9%) did not differ. CONCLUSIONS: The use of a laser for zona drilling in cases of PGD is an easier procedure and results in more intact blastomeres. Since similar pregnancy rates are obtained, it is advantageous to use a laser for zona drilling. Further follow-up is necessary to prove the safety of this procedure.

Biopsy↗
PubMed2003

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.

Alleles↗
PubMed2003

Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the DeltaF508 mutation.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.

Biopsy↗
PubMed2001

PGD in the lab for triplet repeat diseases - myotonic dystrophy, Huntington's disease and Fragile-X syndrome.

Myotonic dystrophy (DM), Huntington's disease (HD) and Fragile X syndrome (FRAXA) are three monogenic disease which are caused by so-called dynamic mutations. These mutations are caused by triplet repeats inside or in the vicinity of the gene which have the tendency to expand beyond the normal range thus disrupting the normal functioning of the gene. We describe here our experiences from 1995 to May 2000 with PGD for these three triplet repeat diseases.

Embryo Transfer↗
PubMed2001

Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.

We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%.

Adult↗
PubMed2001

Aspects of biopsy procedures prior to preimplantation genetic diagnosis.

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca2+/Mg2+-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos.

Adult↗
PubMed2001

Preimplantation genetic diagnosis for spinal and bulbar muscular atrophy (SBMA).

X-linked spinal and bulbar muscular atrophy is characterized by adult onset motor neuron disease and results from a defect in the androgen receptor. The disease is caused by a dynamic mutation in the first exon of the androgen receptor gene, involving a CAG trinucleotide repeat. We have developed a single-cell polymerase chain reaction assay for the androgen receptor gene and describe the application of this assay for preimlantation genetic diagnosis (PGD) in a couple at risk, where the female partner is a carrier of 47 repeats. Diagnosis was based on the detection of both normal and expanded alleles. Allele dropout of the expanded allele was observed in only 1 of 25 lymphoblasts of the carrier and of a non-expanded allele in 1 of 20 research blastomeres tested before the actual PGD. One contraction of four repeats was also found in the carrier's lymphoblasts. Neither expansions nor contractions were observed in the blastomeres biopsied from 11 embryos. Two embryos were unaffected, eight were female carriers and one was an affected male embryo.

Alleles↗
PubMed2001

Involvement of 5-HT(2C) receptors in the anti-immobility effects of antidepressants in the forced swimming test in mice.

Several recent studies have demonstrated that 5-HT(1A), 5-HT(1B) and 5-HT(3) receptors were implicated in the mechanism of action of antidepressants in the mouse forced swimming test. Despite extensive evidence for a role of 5-HT(2C) receptors in depression, the precise role of these receptors in the effects of clinically established antidepressants was not directly investigated in the mouse forced swimming test. This work was aimed at exploring interactions between several doses of Ro 60-0175, a recently available, full and selective 5-HT(2C) agonist, and antidepressant drugs in the mouse forced swimming test. Spontaneous locomotor activity was measured as an index of intact sensorimotor functions and the dose-effect of Ro 60-0175 alone, as well as interactions with several antidepressants, such as tricyclic antidepressants (imipramine, desipramine and maprotiline) and selective serotonin reuptake inhibitors (paroxetine, citalopram, fluoxetine, fluvoxamine and sertraline), were studied in the mouse forced swimming test. There was no intrinsic antidepressant-like effect of Ro 60-0175, but an impairment in locomotor function was detected when using doses higher than 4 mg/kg in the mouse. There was a synergistic effect of low doses of Ro 60-0175 with sub-active doses of imipramine, paroxetine, citalopram and fluvoxamine; an antagonism between the highest dose of Ro 60-0175 and the active doses of paroxetine and fluoxetine was also detected. There is evidence that 5-HT(2C) receptors may be involved in the action of antidepressants which are able to boost the concentration of serotonin in the synapse, i.e. SSRIs and imipramine

Animals↗
PubMed2001

Comparison between light induced and chemically induced oxidation of rhVEGF.

PURPOSE: The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. METHODS: A liquid formulation of rhVEGF was exposed to fluorescent light (2 x 10(4) lux for up to 4 weeks), hydrogen peroxide (H2O2), or t-butythydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. RESULTS: Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78.81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%. possibly due to the role of Met 18in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H2O2, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H2O2 and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. CONCLUSIONS: Methionine residues with exposed surface areas greater than 65 A2 and sulfur surface areas greater than 16 A2 were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H2O2 to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 A2, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.

Amino Acid Sequence↗
PubMed2000

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the delta F508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non-affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born.

Adult↗
PubMed2000

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X-linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X-linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore 'better quality' embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a > or =7-cell stage embryo in order to render the diagnosis more accurate and reliable.

Biopsy↗
PubMed2000

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV.

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.

Adult↗
PubMed2000

Preimplantation genetic diagnosis for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in fatty acid oxidation. The disease is inherited in an autosomal recessive fashion (carrier frequency around 1 in 70) and probably affects as many as 1 in 10000 new-borns. Affected children usually present within the two first years of life with recurrent episodes of hypoketotic hypoglycaemia and lethargy leading to death in approximately 25% of the cases. One mutation (c985A-->G) accounts for approximately 90% of the carrier chromosomes. We developed a preimplantation genetic diagnosis (PGD) strategy for MCAD for a couple who had already lost two affected children. When tested on heterozygous lymphoblasts, the amplification efficiency was 67 out of 71 (94%) and the allele drop-out rate was 0 out of 67. The patient became pregnant after one PGD cycle during which two embryos were replaced. The twin pregnancy was checked by chorionic villus sampling (CVS) and was shown to be unaffected. The twins have been born and are healthy.

Acyl-CoA Dehydrogenase↗
PubMed2000

Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction.

Prior to fertilization, the zona pellucida surrounding the mammalian oocyte acts as a species-specific sperm barrier and is involved in sperm binding. After fertilization, the zona plays a role in blocking polyspermic fertilization, it protects the integrity of the preimplantation embryo during early embryonic development, and also helps its oviductal transport. Zona hardening occurs naturally after fertilization in order to ensure this threefold function. A combination of lysins produced by the cleaving embryo or the uterus and physical expansion then reduces the zona thickness in preparation for hatching. Zona hardening, although not readily quantifiable, may also be induced by in vitro culture and by in vivo aging. Indeed, prolonged exposure of human oocytes and embryos to artificial culture conditions seems to impair their ability to implant. Implantation rates are also inversely correlated with advanced female age. Recently, failure of the embryonic zona pellucida to rupture following blastocyst expansion has been put forward as a possible contributing factor in implantation failure. In order to help embryos escape from their zonae during blastocyst expansion, different types of assisted hatching have been developed. Zona drilling involves the creation of an opening in the zona with acidified medium, whereas zona slitting is carried out in the same manner as partial zona dissection. In zona thinning, the zona is just made thinner over a certain area without a hole or a slit being created. More recently, laser-assisted hatching has been introduced. In vitro studies with both mouse and human embryos have indicated that an artificial gap in the zona pellucida significantly improves the hatching ability of blastocysts grown in vitro as compared to non-micromanipulated embryos. However, the clinical relevance of assisted hatching within an assisted reproduction program remains controversial and elusive. Very few randomized studies are available. Most reports are of retrospective analyses which report either no differences in implantation and pregnancy rates between assisted hatching and control embryos or better results after assisted hatching. Five randomized controlled studies suggest that assisted hatching - of no benefit to the overall patient population - might be of value in increasing embryo implantation rates only in selected cases. No further evidence exists for an age-related benefit from assisted hatching in patients with advanced maternal age.

Animals↗
PubMed1999

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2.

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.

Amino Acid Substitution↗
PubMed1999

Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen.

An auto-controlled study was conducted in couples with tubal infertility and normozoospermic semen. The fertilization rates and embryonic development in sibling oocytes treated, using the same semen sample, either by conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at the same time were compared. Sibling oocyte-cumulus complexes (OCC) of 56 different couples with tubal infertility and normozoospermic semen were randomly divided in order of retrieval into two groups inseminated either by conventional IVF or by ICSI. Of the retrieved OCC in the same cohort, 53.0 +/- 31.2 and 62.0 +/- 26.6% showed two distinct pronuclei after conventional IVF and ICSI respectively (not significant). Complete fertilization failure occurred after conventional IVF in 12.5% (7/56 couples). After ICSI, the comparable figure was 3.6% (2/56). The number of cases was too small to apply a statistical test to this difference. Total cleavage rates were quite similar: 86.7 +/- 28.0 and 90.1 +/- 21% of the zygotes developed into transferable embryos after IVF and ICSI respectively (not significant). Similarly, no difference in embryo quality was observed. Although injection and insemination of the oocytes were performed at the same time in the two groups, at 42 h post-insemination more embryos were at the four-cell stage after ICSI (P < 0.001) than after conventional IVF, where more embryos were still at the two-cell stage (P < 0.02). Embryo transfer was possible in all 56 couples, resulting in 16 positive serum human chorionic gonadotrophin tests (28.6% per embryo transfer), from which a clinical pregnancy resulted in 15 couples. The best embryos were selected for transfer independently of the insemination procedure, but preferably from the same origin. There appeared to be no difference in implantation potency of the embryos obtained with either technique after the non-randomized transfers.

Adult↗