The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region.

BACKGROUND: Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/KCNQ1OT1 in Beckwith-Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain. AIM: To analyse the methylation status of various imprinted genes (IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11-13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally). RESULTS: 3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1. CONCLUSIONS: Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre-implantation development.

Bisulfite genomic sequencing of microdissected cells.

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes.

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

Histopathology of bone marrow after allogeneic bone marrow transplantation for chronic myeloid leukaemia.

The histopathology of bone marrow after allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) was studied in 20 bone marrow biopsy specimens from 14 patients. Biopsies were performed at day 30 post-transplantion (5 biopsies), between day 90 and day 100 (14 biopsies), and one year post-transplantation (1 biopsy). Eight biopsies taken at day 30 post-transplantation for lymphoproliferative disorder were studied as controls. Granulopoiesis was predominant in all day 30 bone marrows in patients treated for CML. In 2 cases, rejection was suspected because of marrow hypoplasia at day 30: one case developed acute leukaemia soon after persistant hypoplasia at day 90 while the evolution of the other case was good. Two day 30 biopsies showed granulocytic hyperplasia but follow-up proved complete remission of CML. Thus, in our series, early biopsies at day 30 did not have any prognostic value. Twelve cases at day 90-100 showed little modifications, such as minor dyserythropoiesis and dysmegakaryopoiesis. One case showed granulocytic hyperplasia at day 100 but bone marrow was normal one year later and the patient in complete remission. The haematopoietic reconstitution after allogeneic bone marrow transplantation may present a transient granulocytic hyperplasia that must not be diagnosed as a persistence of CML.

[Management of upper digestive hemorrhage occurring in the community: patterns of patient care in 4 French administrative areas]. / Prise en charge des hémorragies digestives hautes communautaires. Trajectoires des malades dans 4 départements français.

AIMS: To describe patterns of health care management in patients with upper gastrointestinal hemorrhage and to identify factors linked to the different patterns. PATIENTS AND METHODS: We conducted a prospective study of patients over 18 with upper gastrointestinal hemorrhage (inpatients excluded) among all public hospitals and private practice gastroenterologists in 4 French administrative areas (3 in Northern France and one in the South West). RESULTS: One thousand six hundred and two patients were included over a six-month period (1996). An endoscopic procedure was performed in 1532 patients in public (70%) or private (20.5%) hospitals, or at private office (9.5%). Hospitalization was necessary in 78.8% of the patients in university, non university public or private hospitals (38.9, 45.5 and 15.6%, respectively) with a median duration of 6.5 days. Admission was associated to old age, short delay between hemorrhage and endoscopic procedure, previous gastrointestinal bleeding, cirrhosis or cancer, bleeding from peptic ulcer or esogastric varices. Endoscopic hemostasis was performed in 21.4% of the patients, more often in university and no university public hospitals. Surgery was necessary in 4% of the patients. Death rate was 10.7%. Important geographical variations were observed concerning referral patterns. Patients' characteristics did not differ between the 4 areas. On the other hand, health care supply provided in the management of upper gastrointestinal hemorrhage was different in the four French geographical areas. CONCLUSION: a) An initial endoscopic procedure is nearly always performed in patients with an upper gastrointestinal hemorrhage in France; in 1 patient out of 10, endoscopy was performed in a private gastroenterologist office; b) hospital admission was strongly related to epidemiological and clinical criteria of severity; c) the geographical variations observed in referral patterns depend in part on health care supply; d) upper gastrointestinal haemorrhage status could be used as an indicator of the quality of health care organizations.

[A 1993-1995 epidemiological survey of home parenteral nutrition in approved centers for adults in France]. / Données épidémiologiques 1993-1995 de la nutrition parentérale à domicile en centres agréés chez l'adulte en France.

OBJECTIVES: A 1993-1995 three year epidemiological survey of home parenteral nutrition was performed through in France in approved centers for adults. METHODS: Data were retrospectively collected each year on a standardized questionnaire focussing on indications and short term outcome. RESULTS: All centers (n = 14) participated in the study and 524 new adult patients were recruited. The overall incidence was unchanged at 3.75 patients/10(6) adults. Indications for AIDS rose (8 to 18%) whereas other indications were stable. Prevalence increased by 19%: 4.40 adults/10(6) patients at 01.01.1996. At six months, the probability to stay on treatment was 19.5% for AIDS and cancer indications but 52% for others, whereas death rates were 59% and 9% respectively. CONCLUSIONS: For both cancer and AIDS indications, short-term treatment was due to a poor prognosis. For other diagnosis, complicated with a short bowel in 51% of cases, prognosis was excellent but associated with treatment dependency. The latter point focuses on the need for additional treatments in irreversible intestinal failure.

[Hemorrhaging eso-gastro-duodenal ulcers: epidemiology and management. A multicenter prospective study]. / Les ulcères oeso-gastro-duodénaux hémorragiques: épidémiologie et prise en charge. Etude prospective multicentrique.

UNLABELLED: The aim of this study was to estimate the incidence, and to describe the characteristics and medical care in patients with bleeding upper gastrointestinal ulcers in the general population. PATIENTS AND METHODS: A study was performed over six months in 1996 in 4 French geographical areas: Finistère, Gironde, Seine-Maritime, and the Somme (3 million people minimum 18 years). All public or private hospitals, and specialist gastroenterologists in private practice participated in the study, based on a standardized questionnaire. RESULTS: Over 6 months 793 patients with bleeding ulcers were identified, corresponding to 27 per 100,000 inh./year or 24,000 cases in France. Most patients were men (60%) and 40.1% were 75 years and older. The ulcer was oesophageal (6%), gastric (47%), or duodenal (69%). In 406 patients (51.2%) a chronic disease was present (cancer, cirrhosis, circulatory, respiratory or cardiac disease). In 237 cases (29.9%) the ulcer occurred in patients, 453 patients (57.1%) were admitted and 103 patients (13%) were managed as outpatients. Gastrotoxic drugs were taken by 349 patients (44%): non steroidal anti-inflammatory drugs (18.7%), aspirin (21.2%, including 2/3 with doses under 330 mg/day), corticosteroids (7.8%) and 24.3% had anticoagulant therapy. Patients were managed in university hospitals (39.3%), other public or non profit hospitals (44.2%) or private hospital (16.5%) with geographical differences between the 4 areas. Therapeutic endoscopy was performed in 16.9% and a surgical procedure was performed in 5.9%. The mortality rate (outpatients excluded) was 13.5% (n = 93), but only 2% (n = 16) of death were associated with a bleeding ulcer: mortality was higher in inpatients (24.1%) than in out patients (8.1%). A chronic disease was also associated with higher mortality (17.9% versus 8.1%). CONCLUSION: Bleeding ulcers are frequent and severe, especially in inpatients or associated with chronic conditions. A gastrotoxic drug used is found in about fifty percent of the cases.

Effect of in-utero diethylstilboestrol exposure on human oocyte quality and fertilization in a programme of in-vitro fertilization.

Genital tract abnormalities and adverse pregnancy outcome are well known in women exposed in utero to diethylstilboestrol (DES). Data about adverse reproductive performance in women exposed to DES have been published, including controversial reports of menstrual dysfunction, poor responses after ovarian stimulation, oocyte maturation and fertilization abnormalities. We compared oocyte quality, in-vitro fertilization results and embryo quality for women exposed in utero to DES with a control group. Between 1989 and 1996, 56 DES-exposed women who had 125 in-vitro fertilization (IVF) attempts were retrospectively compared to a control group of 45 women with tubal disease, who underwent 73 IVF attempts. Couples suffering from male infertility were excluded. The parameters compared were oocyte quality (maturation abnormalities, immature oocyte, mature oocyte), fertilization and cleavage rate (per treated and metaphase II oocytes), and embryo quality (number and grade). We found no significant difference in oocyte maturational status, fertilization rates, cleavage rates, embryo quality and development between DES-exposed subjects and control subjects. These results suggest that in-utero exposure to DES has no significant influence on oocyte quality and fertilization ability as judged during IVF attempts.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

Tolerance, effectiveness, and acceptability of sulfate-free electrolyte lavage solution for colon cleaning before colonoscopy.

BACKGROUND AND STUDY AIMS: The unpleasant taste of the solution used for preparation before colonoscopy may limit patients' compliance with the procedure. However, the published results concerning the acceptability of sulfate-free electrolyte lavage solution (SF-ELS) for colon cleansing before colonoscopy are conflicting. The aim of this study was to compare SF-ELS with the standard polyethylene glycol (PEG) solution with regard to tolerance, effectiveness, and acceptability. PATIENTS AND METHODS: In the first part of the study, 24 patients were assigned to receive either one liter of SF-ELS or one liter of the standard PEG solution. After two hours, the patients had to choose two further liters (of either the first or second solution), and preparation for colonoscopy was completed. In the second part, fifty further patients were randomized into two groups: 25 patients received four liters of standard solution, and 25 patients received four liters of SF-ELS. The patients' opinions regarding the preparation and their willingness to repeat the use of the same preparation were recorded by questionnaire. The quality of the colon preparation was assessed by the endoscopists. RESULTS: Seventeen patients (71%; P < 0.05) preferred SF-ELS. The compliance rate in the two groups was 96%, and the frequency of occurrence of adverse effects was also similar. Colonoscopy was completed in 24 of the 25 patients in the SF-ELS group and 22 of the 25 patients in the standard PEG group (the difference was not significant). Visualization of the mucosa in the areas explored was perfect in 20 of the 25 patients in the SF-ELS group and in 17 of the 25 patients in the PEG group (not significant). Patients had a significant preference for SF-ELS. Eighteen of the 25 patients in the SF-ELS group were willing to accept the same preparation for a further colonoscopy, compared with 11 of the 25 in the other group (P < 0.05). CONCLUSIONS: Improving the acceptability of colonic preparation before colonoscopy could improve patients' compliance and the quality of the follow-up. The results of this study justify further investigation of SF-ELS.

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer.

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.