[Laser assisted hatching: Rouen University Hospital outcomes]. / Eclosion embryonnaire assistée: évaluation des indications au CHU de Rouen.

OBJECTIVE: Despite technical progress in In Vitro Fertilisation (IVF) procedure, embryo implantation rate remains low. Assisted hatching has been proposed to facilitate natural embryo hatching and implantation. PATIENTS AND METHODS: Our study has evaluated whether laser assisted hatching improves implantation, pregnancy and live birth rates in different cases. We studied retrospectively 143 IVF cycles concerning more than 38 years old women, 166 IVF cycles after two previous implantation failures and 180 frozen-thawed embryo transfers. RESULTS: Population characteristics were comparable in hatched and control groups. Implantation, pregnancy and live birth rates in women more than 38 years old were comparable with or without assisted hatching. Concerning repeated implantation failures, even if implantation, pregnancy and live birth rates were higher in assisted hatching group (FIV or ICSI), the differences were not significant. After frozen-thawed embryo transfers, implantation rate was significantly better with assisted hatching (19.14% vs 8.84% [p=0.02]). DISCUSSION AND CONCLUSION: Assisted hatching improves embryo implantation rate after frozen-thawed embryo transfer.

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital.

BACKGROUND: Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment. METHODS: Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients. RESULTS: Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm. CONCLUSIONS: Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

Medicago species affect the community composition of arbuscular mycorrhizal fungi associated with roots.

Sequencing of the 5' end of the large ribosomal subunit (LSU rDNA) and quantitative polymerase chain reaction (qPCR) were combined to assess the impact of four annual Medicago species (Medicago laciniata, Medicago murex, Medicago polymorpha and Medicago truncatula) on the genetic diversity of arbuscular mycorrhizal (AM) fungi, and on the relative abundance of representative AM fungal genotypes, in a silty-thin clay soil (Mas d'Imbert, France). Two hundred and forty-six Glomeromycete LSU rDNA sequences from the four plant species and the bulk soil were analysed. The high bootstrap values of the phylogenetic tree obtained allowed the delineation of 12 operational taxonomic units (OTUs), all belonging to Glomus. Specific primers targeting Glomeromycetes and major OTUs were applied to quantify their abundance by qPCR. Glomeromycetes and targeted OTUs were significantly more abundant in the root tissues than in the bulk soil, and the frequencies of three of them differed significantly in the root tissues of the different plant species. These differences indicate that, despite the absence of strict host specificity in mycorrhizal symbiosis, there was a preferential association between some AM fungal and plant genotypes.

[Chromosome abnormalities of spermatozoa]. / Anomalies chromosomiques du spermatozoïde.

Chromosome meiotic pairing during male meiosis is a major event for chromosome segregation during anaphase I and spermatogenesis normal process. Chromosome non-disjunctions responsible for aneuploidy in male gametes can be observed during the first and the second meiotic divisions. The analysis of sperm nuclei chromosome constitution is a major and indirect tool for assessing male meiotic non-disjunctions and the genesis of chromosomal abnormalities. This evaluation has been performed initially by the human sperm/hamster oocyte fusion assay and more recently by fluorescence in situ hybridisation (FISH). Therefore, male populations with increased risk of aneuploidy for their progeny could be identified before entering an in vitro fertilization procedure, and depending on the potential risk a preimplantation or prenatal genetic diagnosis could be performed. For males with constitutional chromosome abnormalities, a specific genetic counselling could also be proposed.

Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes.

In this study, we report the cloning and characterization of denitrifying gene clusters of Pseudomonas fluorescens C7R12 containing the narXLDKGHJI, nirPOQSM, norCB and nosRZDFYL genes. While consensus sequences for Fnr-like protein binding sites were identified in the promoter regions of the nar, nir, nor and nos genes, consensus sequences corresponding to the NarL binding sites were identified only upstream the nar genes. Monitoring by mRNA analysis the expression of the narG, nirS, norB and nosZ structural genes suggests a sequential induction of the denitrification system in P. fluorescens.

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake.

Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.

Relationship between clinical phenotype, semen parameters and aneuploidy frequency in sperm nuclei of 50 infertile males.

The purpose of this study was to analyse the frequency of disomy for chromosomes 1, 13, 14, 18, 21, 22, X and Y in sperm nuclei of 50 infertile men and 10 healthy probands of proven fertility. Semen parameters (sperm count, global motility and morphology), urological clinical examination, genital ultrasound and lymphocyte karyotyping were performed for each patient. Disomy frequency was established by fluorescence in situ hybridization by using whole chromosome paint probes. The mean rate of disomy for the various autosomes studied was higher in infertile males than in subjects of proven fertility. Interchromosomal and interindividual differences in the disomy frequency were observed between the 50 patients. The mean frequency of homodisomy YY and heterodisomy XY was increased in spermatozoa of patients with low semen quality parameters (0.24% and 0.54%, respectively). The disomy frequency in infertile males was directly correlated with the severity of oligospermia. However, no relationship was established between aneuploidy rate, sperm motility, morphology or clinical phenotype. These results support the hypothesis that, during spermatogenesis of males with sperm parameter alterations, a decreased frequency of meiotic chromosome pairing and crossing over may lead to spermatogenesis arrest at the meiosis stage and/or to an increase of meiotic nondisjunctions. Meiotic arrest in some germ cells may be responsible for oligospermia and nondisjunctions in other cells for aneuploidy in mature male gametes.

Assessment of autosome and gonosome disomy in human sperm nuclei by chromosome painting.

Disomy and diploidy frequencies for autosomes 1-22 and the gonosomes were assessed in 299,442 sperm nuclei from four normal fertile men by chromosome painting. This novel approach allowed us to perform a specific and sensitive detection of each chromosome. A minimum of 5000 sperm nuclei per subject were evaluated for each chromosome by dual colour fluorescence in situ hybridization. The disomy rate proved to be similar for all the autosomes (0.24%) and the diploidy rate varied from 0.12% to 0.15%. No interchromosomal or interindividual differences in the frequency of disomic and diploid sperm nuclei were observed between the four subjects. The mean frequency of XX-, YY- and XY-bearing spermatozoa was estimated to 0.17%, 0.17% and 0.32%, respectively. This strategy constitutes a new approach for detecting aneuploidy in human sperm nuclei and suggests an equal repartition of non-disjunction among chromosomes in male gametes.

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae.

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.

Typing of Listeria strains by random amplification of polymorphic DNA.

The polymerase chain reaction (PCR) was used to obtain randomly amplified polymorphic DNA (RAPD) profiles for typing of Listeria strains. In this procedure, whole cells were incubated in the reaction mixture. The discriminating ability of a randomly designed 10-mer primer, HLWL74, was assessed. A total of 60 collection strains of Listeria, encompassing all 7 Listeria species and all known serovars was submitted to PCR with the primer HLWL74. Upon agarose gel electrophoresis, 29 different banding profiles were reproducibly obtained. No common profiles were recorded for strains from different Listeria species. For various groups of strains sharing the same serotype (e.g. 4b, 1/2a, 1/2b), RAPD analysis could generate further subdivision. On the other hand, some strains from different serotypes produced identical RAPD profiles with the primer HLWL74. The RAPD typing method from whole cells is proposed as an attractive alternative for other Listeria typing systems, and the 10-mer HLWL74 as a primer to include in a forthcoming set of standard primers for RAPD typing of Listeria isolates.

A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates.

The analysis of RAPD profiles generated by PCR with a single 10-mer, HLWL74, was compared to bacteriophage susceptibility data for epidemiological typing of Listeria monocytogenes strains. A total of 104 L. monocytogenes strains was screened, all from serogroup 1 or serotype 4b. Of these, 53 had been isolated during 6 different listeriosis outbreaks. The remaining 51 strains were chosen randomly from our collection. A total of 38 RAPD types were observed, although each epidemic group of strains isolated during one of these outbreaks displayed a specific RAPD profile. For 98% of the strains isolated during outbreaks, the correlation between RAPD typing and phage typing was complete. Only one strain, typed as epidemic by phage typing, was clearly distinguishable from the others by RAPD analysis. Among the 51 strains not related to an outbreak, 12 were linked to epidemic groups by RAPD analysis. Two of these rearrangements were supported by phage typing. The remaining 10 strains could be excluded by phage typing from any of the epidemic groups studies. Considering all 104 isolates, the decision to relate a strain to a particular epidemic group or to exclude a strain from any epidemic group was the same for 92 isolates, using either phage typing or RAPD analysis. The RAPD analysis, which is quick, simple and suited for automation, is proposed as an attractive alternative for phage typing in epidemiological studies of listeriosis.

Study on the epidemiology and control of Campylobacter jejuni in poultry broiler flocks.

Broiler flocks are frequently infected with Campylobacter jejuni. The origin of the infection is still unclear. The question of whether colonization of flocks results from transmission of C. jejuni from breeder flocks to progeny (vertical transmission) or from environmental sources (horizontal transmission) remains to be answered. Therefore, in this study samples were taken from successive broiler flocks in two broiler houses (house A on farm A and house B1 on farm B) as well as from the environment of the houses. All C. jejuni isolates were typed by using the Penner serotyping system, and part of the isolates from farm B were typed by using a randomly amplified polymorphic DNA-typing system. In poultry house A, C. jejuni was isolated from the first flock but not from subsequent flocks. In poultry house B1, C. jejuni strains of the same Penner serotypes and exhibiting identical DNA profiles were isolated from successive flocks. Infection of the flocks from a common source via horizontal pathways is suspected, while a vertical route of infection is not likely to exist. Application of measures to control horizontal transmission of C. jejuni on farm B was successful.

RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA.

A method was developed to obtain reproducible DNA fingerprints from Campylobacter by PCR-based amplification, without the need to isolate total DNA. Randomly amplified polymorphic DNA (RAPD) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. A range of C. jejuni serotypes could be typed by RAPD analysis. Depending on the primer, the analysis of RAPD profiles resulted in different levels of discrimination between the strains. Clear correlations were observed between results of RAPD analysis and serotyping. Two of the primers tested generated RAPD profiles which allowed discrimination of strains within given Penner and Lior serotypes.