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Biomedical subjects

I Liebaers

Publications and source records attributed to I Liebaers.

At least 37 records · Page 2Linked to original sources
PubMed2003

Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF.

BACKGROUND: Since the introduction of ICSI in 1991, medical outcome studies on ICSI children have been performed, but few have addressed developmental outcome. Hence, this outcome was assessed by performing a standard developmental test on children born after ICSI as compared with children born after IVF, at the age of 2 years. METHODS: In a prospective study, the medical and developmental outcome of 439 children born after ICSI (378 singletons, 61 twins) were compared with those of 207 children born after IVF (138 singletons, 69 twins), at the age of 24-28 months. These children were part of a cohort of children followed since birth. Of children reaching the age of 24-28 months between May 1995 and March 2002, 44.3% (2375/5356) were examined by a paediatrician who was unaware of the type of treatment used for each couple. Of all the children born, 12.2% (439/3618) in the ICSI group and 11.9% (207/1738) in the IVF group underwent a formal developmental assessment using the Bayley Scale of Infant Development (mental scale) by a paediatrician blinded to the type of treatment. RESULTS: There was no significant difference in maternal educational level, maternal age, gestational age, parity, birthweight, neonatal complication rate or malformation rate at 2 years between ICSI and IVF singletons, or between ICSI and IVF twins. No significant difference was observed in the developmental outcome using the Bayley scale at the age of 24-28 months (raw scores or test age) between ICSI children or IVF children. A multivariate regression analysis for the singleton children indicated that parity, sex (boys had lower scores than girls) and age had a significant influence on the test result, but that the fertility procedure (ICSI versus IVF) did not influence the test result. ICSI children from fathers with low sperm concentration, low sperm motility or poor morphology had a similar developmental outcome to that of children from fathers with normal sperm parameters. There were no significant differences between the initial cohort and the group lost to follow-up, nor between the psychologically tested and the non-tested group for a number of variables such as maternal educational level, birthweight in singletons and neonatal malformation rate. Although only some of the cohort of ICSI children were evaluated, a representative sample of both ICSI and IVF children was compared. CONCLUSIONS: There is no indication that ICSI children have a lower psychomotor development than IVF children. Paternal risk factors associated with male-factor infertility were found not to play a role in developmental outcome.

Aging↗
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↗
PubMed2002

Intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) with ejaculated, epididymal or testicular spermatozoa was first successful in 1992 and has since become the widely accepted treatment for couples with severe male-factor infertility. The outcome of several thousands of ICSI cycles in terms of fertilization, embryo cleavage and implantation is similar to that for conventional in-vitro fertilization in couples with tubal or idiopathic infertility. To evaluate the important issue of safety of the new technique of ICSI, a prospective follow-up study of children born after ICSI was carried out. The aim was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones. Parents' agreement to genetic counseling was obtained, as well as prenatal diagnosis, followed by a physical examination of the children at 2 months, 1 and 2 years. Important outcome data to be examined comprise information on major and minor congenital malformations obtained prenatally or after birth, as well as on the further development of the children.

Adult↗
PubMed2002

Citrate infusion test in the diagnosis of hypocalcemia due to a mutation in the calcium-sensing receptor gene.

The calcium-sensing receptor (Ca-R) is a G-protein-coupled surface receptor that plays a crucial role in calcium homeostasis via parathyroid hormone secretion. Mutations of this receptor can cause a gain in, or loss of, function, leading to hypo- or hypercalcemia, respectively. We report here a family with hypocalcemia in whom a heterozygous missense mutation in exon 4 was demonstrated, predicting a proline to leucine substitution (P221L) in the extracellular part of the Ca-R. Clinical symptoms were limited to fatigue. When serum calcium was further lowered via a citrate infusion, a significant increase in circulating iPTH was observed, although with lower peak values than in normal controls, suggesting a gain in function of the Ca-R. Treatment with calcium supplements and calcitriol led to prohibitive hypercalciuria without normalizing serum calcium. The aims of this case report are: (1) to present a mutation in the Ca-R with a gain in function at a codon where previously loss of function was described, and (2) to suggest that measuring circulating iPTH during a citrate infusion in the presence of familial hypocalcemia is an additional test to diagnose this particular form of hypoparathyroidism.

Journal Article↗
PubMed2002

Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance.

A broad spectrum of assisted reproductive technologies has become available for couples with fertility problems. Follow-up studies of children born as a result of assisted reproduction have shown that neonatal outcome and malformation rates are not different from those of the general population, except for a low birthweight and a slight increase in chromosomal abnormalities. The safety aspect of assisted reproduction at the epigenetic level has not been well studied. Epigenetics refers to phenomena where modifications of DNA methylation and/or chromatin structure underlie changes in gene expression and phenotype characteristics. This article intends to analyse epigenetic risks related to assisted reproduction on the basis of an overview of epigenetic reprogramming events in the gamete and early embryo. Two epigenetic modifications, methylation and imprinting, are considered in more detail. The interference of in-vitro embryo culture, immature sperm cells and nuclear transfer with epigenetic reprogramming is discussed, as well as the possibility of epigenetic inheritance.

Animals↗
PubMed2002

Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling.

Recently, several requests were made by couples with an affected child who wanted preimplantation genetic diagnosis (PGD) to select embryos in the hope of conceiving an HLA identical donor sibling. This article considers the ethical arguments for and against the application of PGD for this goal. Only embryos HLA matched with an existing sibling in need of a compatible donor of haematopoietic stem cells would be transferred. The main arguments are the instrumentalization of the child, the best-interests standard, the postnatal test for acceptability and the experience of the donor child. It is argued that conceiving a child to save a child is a morally defensible decision on the condition that the operation that will be performed on the future child is acceptable to perform on an existing child. The instrumentalization of the donor child does not demonstrate disrespect for its autonomy or its intrinsic worth.

Ethics, Medical↗
PubMed2002

FISH analysis of chromosome X, Y and 18 abnormalities in testicular sperm from azoospermic patients.

BACKGROUND: Sperm extracted from testicular biopsies of azoospermic men can successfully be used for ICSI. The concern exists that testicular sperm from azoospermic men suffering from severe testicular failure may have a higher frequency of aneuploidy, which may lead to an increased risk for chromosomally abnormal offspring. METHODS: Testicular sperm from patients showing spermatogenic failure (n = 17) and from patients with normal spermatogenesis (n = 26) were analysed by fluorescence in-situ hybridization (FISH). Numerical chromosomal abnormalities for chromosomes X, Y and 18 were evaluated by FISH in a total of 1697 testicular sperm derived from 43 azoospermic patients. RESULTS: No difference was observed between the frequency of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis (5.6%) and from patients with spermatogenic failure (8.2%). However, the frequency of aneuploidy for chromosome 18 was higher in the group of azoospermic patients with spermatogenic failure than in the group with normal spermatogenesis (3.2 versus 1.3%). Within the obstructive group, sex chromosome aneuploidy (4.5%) occurred more frequently than chromosome 18 aneuploidy (1.3%; P < 0.001). Among testicular sperm derived from patients with spermatogenic failure, sex chromosomal aneuploidy (5.8%) was similar to that for chromosome 18 (3.2%). CONCLUSIONS: So far, no difference in the total frequency of chromosomal abnormalities has been observed between patients with normal spermatogenesis and patients with severe testicular failure. However, aneuploidy for chromosome 18 was higher in the group with spermatogenic failure.

Adult↗
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

Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype--phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and II by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.

Alleles↗
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

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

Imprinting analysis in spermatozoa prepared for intracytoplasmic sperm injection (ICSI).

Genetic imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed. This process is mediated by the methylation of DNA. As spermatozoa represent exclusively the paternal contribution to a future individual, they are expected to carry the paternal imprint only. For intracytoplasmic sperm injection (ICSI), spermatozoa mostly have to be selected from samples with pathological semen parameters. Correct establishment of the paternal imprint in these spermatozoa has not yet been demonstrated. In the present study, imprinting analysis was undertaken using DNA extracted from spermatozoa from men with normal semen analysis (group A: n=30 patients) and from men with an abnormal sperm count (B: n=30 patients with 5--20 million spermatozoa/mL and C: n=30 patients with < or =5 million spermatozoa/mL) from the ICSI program. It was performed using firstly a conventional methylation-specific polymerase-chain-reaction (M-PCR) and secondly a more sensitive modified hemi-nested M-PCR technique. In addition, a single cell PCR was performed on a total of 88 single spermatozoa (collected from nine males) and on 25 leucocytes (control group). With the conventional M-PCR, exclusively paternal imprints were found in all groups. Using the more sensitive hemi-nested M-PCR, additional maternal imprints were found in 63% of the samples in A, 57% in B and 60% in C. In the single cell PCR, exclusively paternal imprints were detected. Because of the very small amount of DNA (3 pg), a complete amplification failure occurred in 43% of spermatozoa. The correct paternal and maternal imprints were found in 56% of the analysed leucocytes (complete amplification failure in the other 44%). In conclusion, ejaculated spermatozoa from males with medium or high-grade semen pathology proved to have the same imprinting status as those from males with normal semen parameters. As the additional maternal imprints were never found at the single cell level, they were classified as contamination by diploid cells such as leucocytes or immature germ cells in the processed and purified semen samples, which can be detected by a more sensitive PCR method in contrast to the conventional standard PCR.

Adult↗
PubMed2001

Characterization of the genomic organization, localization and expression of four PRY genes (PRY1, PRY2, PRY3 and PRY4).

PRY (PTP-BL related on the Y chromosome) has been proposed as a candidate spermatogenesis gene. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of the cDNA sequence with the genomic sequence, five exons were identified. Analysis of GenBank-derived clones on the Y chromosome revealed the presence of two full-length copies in azoospermia factor region b (AZFb) (PRY1 and PRY2) and two shorter versions of the PRY gene containing exons 3, 4 and 5 in AZFc (PRY3 and PRY4). A clone containing sequences homologous to exons 3, 4 and 5 is located in area 5L (between AZFa and AZFb), a clone containing a sequence homologous to exon 5 is located in area 5M (in AZFb) and a clone containing a fragment homologous to exon 3 is located in 6F. A repeat structure of exons 1 and 2 is present on the short arm of the Y chromosome as well as on the long arm. PRY1 and PRY2, two gene copies that are located in AZFb, a region often deleted in patients with severe male infertility, were shown to be expressed in the testis. PRY may therefore play an important role in spermatogenesis.

Base Sequence↗
PubMed2001

DNA methylation analysis in immature testicular sperm cells at different developmental stages.

Testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) is a frequently used therapeutic option in azoospermic males. Genetic imprinting is a mechanism of gene regulation - mediated by the methylation of deoxyribonucleic acids (DNA) - by which only one of the parental copies of a gene is expressed. Whether the establishment of the genetic paternal imprint in this immature haploid sperm cell is complete and stable at this stage of maturation has never been analysed. In the present study, a highly sensitive heminested methylation-specific polymerase chain reaction for the target region 15q11-13 was developed for imprinting analysis at the single-cell level. Imprinting analysis was then carried out on DNA extracted from single diploid leucocytes (n = 25), ejaculated spermatozoa (n = 88), elongated testicular spermatids (n = 30), and round spermatids (n = 25). Amplification was obtained in 57% of the ejaculated spermatozoa, in all elongated spermatids and in 20% of the round spermatids. In the amplified samples, only the paternal imprint was detected. The maternal imprint was not found at all in any of the sperm cells; in 56% of the diploid leucocytes, the maternal and paternal imprints were detected at the same time (complete failure in the other 44%). The data reveal the completed establishment of the correct paternal imprint in ejaculated spermatozoa, elongated spermatids and amplified round spermatids. However, the high rate of amplification failure in round spermatids remains a factor of uncertainty.

Adult↗
PubMed2000

Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency.

Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.

Amino Acid Sequence↗