Biomedical subjects

A Van Steirteghem

Publications and source records attributed to A Van Steirteghem.

At least 19 recordsLinked to original sources

PubMed2006

Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions.

Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.

Cadherins↗

PubMed2006

The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients.

BACKGROUND: Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored. Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells. Therefore, both enrichment and decontamination before transplantation may be necessary. This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions. In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed. METHODS: Murine testicular cells were contaminated with 5% EL4 cells. Fresh and frozen-thawed suspensions were sorted using MACS (CD49f (+)) and FACS (CD49f(+), H-2Kb(-)) and evaluated by FACS, cell culture and transplantation into W/W(v) mice. Human testicular cells were contaminated with 5 or 0.05% CCRF-SB (SB) cells. Frozen-thawed suspensions were sorted using FACS (HLA class I(-)) and evaluated by FACS, cell culture and PCR for the B-cell receptor. RESULTS: In the mouse, the sorted fractions contained 0.39% H-2K(b)-positive and 76.55% CD49f-positive cells. After transplantation, 1 in 20 recipient mice developed a malignancy. In the human experiments, an average of 0.58% SB cells was detected after sorting. In only 1 of 11 samples, there were no SB cells observed. CONCLUSION: MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells. Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important.

Animals↗

PubMed2006

Medical outcome of 8-year-old singleton ICSI children (born >or=32 weeks' gestation) and a spontaneously conceived comparison group.

BACKGROUND: There is little information about the long-term outcome of children born after ICSI. In this study, the eldest cohort of ICSI children worldwide, reaching the age of 8 years, was investigated at the prepubertal stage to monitor subsequent puberty and future fertility. To investigate possible health problems, a thorough medical and neurological examination was performed. METHODS: Medical outcome of 8-year-old singletons (n=150) born through ICSI (>or=32 weeks) was compared with that of 147 singletons of the same age born after spontaneous conception (SC). Information about their general health was obtained from the parents by means of a questionnaire. RESULTS: Fifteen of 150 ICSI children experienced a major congenital malformation compared with 5/147 SC children (P < 0.05). Pubertal staging was similar in both groups. Neurological examination did not show important differences between ICSI and SC children. ICSI children did not require more remedial therapy or surgery or hospitalization than SC children. CONCLUSION: Physical examination including a thorough neurological examination did not reveal important differences between the two groups. Major congenital malformations were significantly more frequent in the ICSI group. However, most of them were corrected by minor surgery. Further monitoring of these children at an older age is recommended.

Belgium↗

PubMed2006

Does PGD for aneuploidy screening change the selection of embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men?

BACKGROUND: An increased incidence of aneuploid embryos has been recently described from azoospermic men. The aim of this study was to assess if embryo selection on day 5, based on morphological criteria, would be different from the selection based on PGD for aneuploidy screening (AS) in couples undergoing ICSI for male azoospermia. METHODS: Sixty-two cycles of testicular sperm extraction (TESE)-ICSI with PGD-AS were included in the analysis. Two embryologists, blinded to the PGD-AS results, retrospectively reviewed the available embryology data from day 5 embryos and selected one, two or three embryos to be transferred. These results were compared with the selected embryos based on PGD-AS. RESULTS: A total of 39 cycles from non-obstructive azoospermia (NOA) and 23 cycles from obstructive azoospermia (OA) were retrospectively analysed. If single embryo transfer (SET) had been performed, in 64.8% of the NOA cycles and 54.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 10.8 and 36.6% of the cycles, respectively, an aneuploid embryo would have been chosen. If double ET (DET) had been performed, in 72.9% of the NOA cycles and 86.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 2.7 and 4.5% of the cycles, respectively, an aneuploid embryo would have been chosen. If triple ET (TET) had been performed, the outcome would have been the same as for DET. DISCUSSION: Our results suggest that under the terms of an SET policy, the performance of PGD-AS in azoospermia would result in a higher chance of success, as the possibility of selecting a euploid embryo is enhanced.

Adult↗

PubMed2006

The role of the testis-specific gene hTAF7L in the aetiology of male infertility.

The X-linked TAF7L gene is homologous to the autosomal transcription factor TAF7. Together with its testis-specific expression pattern, this might point to an important function in spermatogenesis. In order to analyse the involvement of the hTAF7L gene in the aetiology of male infertility, a total of 25 patients with maturation arrest of spermatogenesis have been analysed for the presence of mutations in this gene. Four alterations of the nucleotide sequence, with concomitant changes in the amino acid sequence, have been observed in 12 patients. All sequence alterations were also found either in a control group consisting of men with proven fertility or in a control group with men with normal spermatogenesis. Therefore, these alterations are probably polymorphisms.

Amino Acid Sequence↗

PubMed2006

Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice.

BACKGROUND: Since its introduction in 1994, testicular stem cell transplantation (TSCT) has been widely used for research. This technique may also become important for preserving fertility in pre-pubertal cancer patients. Therefore, it is necessary to investigate the safety aspects of reproduction using spermatozoa obtained after TSCT. In this study, preimplantation development of mouse embryos, using spermatozoa obtained after TSCT, was examined. METHODS: TSCT-derived spermatozoa were used for IVF and ICSI. Embryos were cultured for five days until they reached blastocyst stage and were evaluated by differential staining. RESULTS: IVF revealed significantly lower fertilization and development rates after TSCT-IVF compared to control-IVF. Blastocysts derived from TSCT-IVF had significantly lower inner cell mass numbers (ICMs) and lower ICM/trophectoderm (TE) ratios compared to control-IVF blastocysts. No differences in fertilization and development rates were observed between TSCT-ICSI and control-ICSI, and blastocyst quality in the transplanted group was similar to that of the control blastocysts. CONCLUSION: Our study showed that after TSCT-IVF, fertilization and preimplantation development were disturbed and blastocysts showed reduced ICM and ICM/TE ratio. However, after TSCT-ICSI, both fertilization and preimplantation development were normal and blastocyst formation was comparable to control-ICSI.

Animals↗

PubMed2006

How successful is repeat testicular sperm extraction in patients with azoospermia?

BACKGROUND: Little is known about the extraction rate in repeated sperm retrieval procedures in azoospermic patients. This study aimed to assess the feasibility of repeated sperm recovery in these patients. METHODS: A total of 1066 azoospermic men had their first sperm recovery between 1 January 1995 and 31 December 2003. A total of 381 men had obstructive azoospermia (OA), 628 nonobstructive azoospermia (NOA) and 57 showed hypospermatogenesis. RESULTS: Overall, sperm could be retrieved in all procedures in the 598 cycles performed in OA men (100%). A total of 117, 57, 24, 11, 7 and 1 men underwent, respectively, two, three, four, five, six and seven sperm retrievals; all were successful. Of the 784 procedures performed on the 628 men with NOA, sperm could be retrieved in 384 procedures (49%). During the first testicular sperm extraction (TESE) procedure, sperm could be extracted in 261 men with NOA (41.6%). A total of 103 men had a second attempt, 34 had a third attempt, 11 had a fourth attempt, 6 had a fifth attempt and 2 had a sixth attempt. In these cycles, sperm could be extracted in, respectively, 77 (74.7%), 28 (82.3%), 11 (100%), 5 (83.3%) and 2 (100%) men. CONCLUSION: Repeated TESE ensures a high sperm recovery rate even in patients with NOA. In NOA patients, studies reporting on TESE may therefore overestimate the retrieval rate by reallocating successful patients. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat TESE procedure can be planned.

Humans↗

PubMed2006

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Buffers↗

PubMed2006

Which patients with recurrent implantation failure after IVF benefit from PGD for aneuploidy screening?

Patients with recurrent IVF failure are defined as patients who are younger than 37 years and who had at least three consecutive unsuccessful IVF/intracytoplasmic sperm injection (ICSI) cycles with good quality embryos. These patients might be predisposed to chromosome errors in their embryos and therefore might benefit from preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). This technique is, however, expensive and some normal embryos might be lost due to the error rate. The aim of this retrospective study was to define those patients who would benefit most from it. One hundred and twenty-one first PGD-AS cycles for recurrent IVF failure were analysed. The aneuploidy rate, 'no embryo transfer' rate, live birth rate per embryo transfer and implantation rate were respectively 48.3, 22.3, 29.7 and 19.5%. A multivariate logistic regression analysis gave us a predictive model demonstrating that to have a 90% probability of having an embryo transfer after PGD-AS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes and six embryos for biopsy. This study suggests that most patients with recurrent IVF failure may benefit from PGD-AS. Future studies, however, should more strictly define this heterogeneous group of patients, so that comparison is easier.

Adult↗

PubMed2006

New Belgian embryo transfer policy leads to sharp decrease in multiple pregnancy rate.

Since 1 July 2003, a new transfer policy aiming to reduce multiple pregnancies was brought into law in Belgium. The policy restricts the number of embryos transferred, depending on the patient's age and treatment cycle. This study aimed to evaluate the effect of this policy. Two 15-month periods before and after the start of the new law were compared for the following parameters: positive human chorionic gonadotrophin (HCG), clinical pregnancy rate and multiple pregnancy rate according to the age categories defined by the policy: <36, 36-39 and 40-42 years. HCG rates (34.2 and 32.8%) and clinical pregnancy rates (26.2 and 24.0%) per cycle were similar for the two periods. Overall, the multiple pregnancy rate was reduced from 29.1 to 9.5% (all patients) and from 28.9 to 6.2% in women <36 years. Most twins were observed in the third cycle of patients <36 years and in the first three cycles in women of 36-39 years. It can be concluded that a significant decline (P < 0.001) in multiple pregnancies was mainly observed in patients <36 years of age. Clinical pregnancy rates were not compromised by the new law. Elective single embryo transfer should be considered more seriously for women 36-39 years of age.

Adult↗

PubMed2005

Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients.

BACKGROUND: To try to explain the infertility of chromosomal translocation carrier patients, we compared the expression of two markers of apoptosis in the sperm of patients and of fertile donors, and we studied the meiotic segregation in the ejaculated sperm of these translocation carriers. METHODS: Twenty semen samples of translocation carriers, [reciprocal (n=14) and Robertsonian translocations (n=6)], were compared with the semen samples of donors (n=20). Different tests were applied: annexin V binding assay; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL); and fluorescence in situ hybridization (FISH). RESULTS: The annexin V binding assay in sperm of patients with chromosomal translocation (n=17) showed a significantly increased proportion of sperm with externalized phosphatidylserine (PS) than in the control group (n=20, P<or=0.05). The rates of DNA fragmentation investigated by TUNEL reaction were higher in samples of translocation carriers (n=14) than in donors (n=20, P<0.0001). The measures by FISH technique showed that the proportions of balanced or normal gametes were predominant in the reciprocal translocation group (alternate: n=7; from 33.0 to 58.8%; adjacent I: n=7; from 4.6 to 43.8%) and in the Robertsonian translocation group (normal: n=5; from 76.0 to 88.5%). CONCLUSIONS: Our data show a predominant proportion of balanced gametes in sperm of chromosomal translocation carrier patients. Moreover, PS externalization and DNA fragmentation rates are significantly higher in ejaculated sperm of these patients than in donor sperm. These tests could be used to predict the outcome of ICSI for these patients.

Annexin A5↗

PubMed2005

Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders.

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).

Biomarkers↗

PubMed2005

Is chromosome analysis mandatory in the initial investigation of normovulatory women seeking infertility treatment?

BACKGROUND: There is no agreement about the frequency of chromosomal abnormalities (CAs) in the female partner of an infertile couple and therefore there is no evidence base for determining whether karyotype analysis is mandatory before the initiation of infertility treatment. The aim of this prospective study was to estimate the prevalence of karyotype abnormalities in normovulatory women attending an infertility clinic and compare it to that known to be present in the newborn female population. METHODS: Cytogenetic testing was performed in 1206 women with normal ovulatory cycle seeking infertility treatment. At least 15 GTG-banded metaphases were analysed in each case. In the case of a structural abnormality, fluorescent in situ hybridization (FISH) analysis and high resolution banding (HRB) were performed on a new blood sample to elucidate the aberration. When mosaicism was suspected, the number of analysed metaphases was increased to a total of 115 and an additional analysis of 200 metaphases was done on a second blood sample. RESULTS: A chromosomal abnormality was demonstrated in 0.58% (95% CI: 0.28-1.19) of cases which did not differ significantly from that reported in female newborns (0.79%; 95% CI: 0.68-0.94). Balanced reciprocal translocation was observed in 0.4% of patients (n = 5), paracentric inversion of chromosome X in 0.08% (n = 1) and gonosomal mosaicism in 0.08% (n = 1). However, chromosomal aberrations were less common among females with primary infertility compared to those with secondary infertility (0.25 versus 1.25%, P = 0.04). CONCLUSIONS: The present study suggests that routine cytogenetic analysis cannot be advocated in normovulatory infertile women. Nevertheless, the relatively higher frequency of abnormal karyotypes in women with secondary infertility indicates that this subgroup of patients might benefit from a routine karyotype analysis.

Adult↗

PubMed2005

DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells.

In humans, the Deleted in Azoospermia Like (DAZL) gene is believed to function in the development of primordial germ cells and in germ cell differentiation and maturation because the expression of DAZL is only found in the germ and non-germ lineage of the reproductive system and in embryonic stem (ES) cells. The present study examined the presence of DAZL transcripts in the last stages of oocyte maturation, in ES cells, and throughout the preimplantation development; the link between gametes and ES cells. The finding of DAZL transcripts in the last stages of oogenesis and during the first two cell cycles of the preimplantation development was expected, because DAZL is a germ cell marker and the transcripts present at that time are generally encoded by the maternal genome. During the third cell cycle, DAZL showed a variable expression pattern, which may point to the maternal to embryonic transition. After the third cell cycle, transcripts were again consistently detected, suggesting embryonic DAZL transcription. In blastocysts, DAZL transcripts were only detected in those of good quality and this as well in the inner cell mass (ICM) as in the trophectoderm (TE). The presence of DAZL transcripts in the ICM and in ES cells was not surprising since both can lead to the formation of germ cells, but TE cells cannot. The quality-related expression of DAZL in blastocysts, and especially its trophectodermal expression, might imply other functions for DAZL beyond germ cell development.

Blastocyst↗

PubMed2005

Preimplantation genetic diagnosis for neurofibromatosis type 1.

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.

Adult↗

PubMed2005

Fertility and ageing.

The late 20th century trend to delay birth of the first child until the age at which female fecundity or reproductive capacity is lower has increased the incidence of age-related infertility. The trend and its consequences have also stimulated interest in the possible factors in the female and the male that may contribute to the decline in fecundity with age; in the means that exist to predict fecundity; and in the consequences for pregnancy and childbirth. In the female, the number of oocytes decreases with age until the menopause. Oocyte quality also diminishes, due in part to increased aneuploidy because of factors such as changes in spindle integrity. Although older male age affects the likelihood of conception, abnormalities in sperm chromosomes and in some components of the semen analysis are less important than the frequency of intercourse. Age is as accurate as any other predictor of conception with assisted reproductive technology. The decline in fecundity becomes clinically relevant when women reach their mid-30s, when even assisted reproduction treatment cannot compensate for the decline in fecundity associated with delaying attempts at conceiving. Pregnancies among women aged >40 years are associated with more non-severe complications, more premature births, more congenital malformations and more interventions at birth.

Adult↗

PubMed2005

Oct-4 mRNA and protein expression during human preimplantation development.

The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT-PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.

Blastocyst↗

PubMed2005

Reproductive decisions by couples undergoing artificial insemination with donor sperm for severe male infertility: implications for medical counselling.

Since 1992, ICSI has been introduced as a successful treatment for male infertility, including azoospermia. The present study is aimed at evaluating the practice of insemination with donor sperm (AID) in infertile couples who may benefit from the new developments that ICSI has brought in the last decade. AID was performed in 440 heterosexual couples. Twelve couples were lost for follow-up (3%). In 128 (29.9%) and 229 (53.5%) of the couples the husband was either oligozoospermic (OAT) or azoospermic. In 60 couples (14.0%) the man had a transmissible genetic trait. In 11 couples (2.6%) there were other indications for performing AID. In the OAT group 36 couples never had ICSI treatment (28.1%) because they had already an AID child born before the introduction of ICSI (n = 16), the burden of ICSI treatment was too high (n = 9) or they considered that ICSI success rate was too low (n = 7). Ninety-two couples tried ICSI treatment before opting for AID (71.9%), mainly because ICSI failed (n = 43). In 229 couples the husband had azoospermia (53.5%). In 112 couples (49%) no sperm or too few testicular sperm were found at testicular biopsy (TESE) and 15 couples (6.5%) had more than three failed ICSI-TESE attempts. Eighty-one azoospermic men refused TESE (35.4%) because of an anticipated low success rate (n = 28) or the burden of this approach (n = 23). Although a majority of patients could opt for ICSI, our results show that AID is still an option for many couples for whom these techniques were either not feasible or not successful. A substantial proportion of patients (33%) did not even opt for these advanced fertility treatments.

Counseling↗