Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis.

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach - results of a second EUROFORGEN / EDNAP collaborative exercise.

In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category.

Lack of association between Tenascin-C gene and spondyloarthritis.

OBJECTIVES: We previously identified a new susceptibility region linked to SpA in 9q31-34. Tenascin-C (TNC) appears as one of the best positional and functional candidate genes lying within this SPA2 locus. The objectives of the present study were to identify TNC polymorphisms, and to examine their putative association with SpA. METHODS: We first performed variants screening in 20 independent SpA patients from families with high linkage score to the SPA2 locus, and three unrelated controls: TNCs coding regions (28 exons), intron-exon boundaries and 5'- and 3'-flank regions were fully re-sequenced to identify polymorphisms. Then we genotyped selected variants in 183 independent trios, and assessed their intrafamilial association with SpA by transmission disequilibrium test. RESULTS: Variants screening allowed us to identify 26 polymorphisms, 7 of which were selected for further study, in addition to an intronic polymorphism previously reported as associated with Achilles tendon injuries. In intrafamilial association test, none of the variants showed significant transmission disequilibrium. Results from analysis restricted to AS were not different from those obtained on the whole SpA group. CONCLUSIONS: TNC was one of the best positional and functional candidate genes within the SPA2 locus. Nevertheless, we found no association between polymorphisms in this gene and SpA. However, we cannot exclude that variants located in intronic regions or in the vicinity of TNC, which were not tested in the present study, could be implicated in the predisposition to SpA.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Patient satisfaction regarding outpatient mastectomy in Saint-Nazaire hospital center.

OBJECTIVES: To assess patient satisfaction with outpatient mastectomy and their postoperative quality of life. MATERIALS AND METHODS: A retrospective descriptive study was conducted of patients who had a mastectomy as outpatients at the hospital of Saint-Nazaire between January 2013 and May 2015. Patients were contacted by phone to answer a survey assessing their satisfaction. RESULTS: Out of 16 eligible patients for an outpatient mastectomy, 11 patients were included in our study (4 [25%] had to remain hospitalized after their intervention and one patient could not be contacted). One patient (9%) presented a seroma requiring aspiration. No patients were readmitted. All patients, except one, were satisfied with the overall care. CONCLUSION: The management of outpatient mastectomies seems to be a good option, provided that patients are motivated, properly selected and well-informed prior to surgery.

[Total laparoscopic hysterectomy and same-day discharge: Satisfaction evaluation and feasibility study]. / Prise en charge ambulatoire de l'hystérectomie laparoscopique : évaluation de la faisabilité et de la satisfaction des patientes.

OBJECTIVE: To evaluate the safety and patients' satisfaction after outpatient laparoscopic hysterectomy (LH). METHODS: A retrospective case series of 47 patients discharged home the same day after total LH. Outcomes include the same-day discharge rate, complication and readmission rates, patients' short-term satisfaction and an evaluation of patients' opinion several months later. RESULTS: Among the 47 patients, 42 (89%) were discharged the same day. Five patients remained hospitalized due to postoperative complications (4 patients) and the patient's decision (1 patient). No patient required emergency consultation, readmission or reoperation in the first 72hours after discharge. Six patients (13%) had postoperative complications within the first 3 months after the surgery. Four patients (8%) were readmitted, three of them (6%) had to undergo a second surgery. In the short-term assessment, patients were satisfied with the management in 93% of cases and would recommend it to others, with a median satisfaction rate of 3.8 out of 4. CONCLUSION: Ambulatory laparoscopic hysterectomy is feasible in a well-selected population, with a high level of patients' satisfaction. This management could be an alternative to traditional long hospital stays proposed in France.

[Immunological treatment of spontaneous repeated abortions. The value of transfusing the partner's leukocytes in the third week of gestation]. / Traitement immunologique des fausses couches spontanées itératives. Intérêts des transfusions de leucocytes de conjoint durant la troisième semaine de gestation.

Twenty-two nulli- all primipara who had had previous repeated spontaneous abortions and who did not have anti-HLA antibodies for the partner, received immunological treatment consisting of a single transfusion of the partner's lymphocytes in the third week of pregnancy, and giving natural progesterone supplements after the kinetic of Beta-HCG in the plasma had been assessed. The number of pregnancies which went to terme (94% success) was significantly better than those obtained in our first protocol which was to give one to three transfusions of the partner's lymphocytes before the pregnancy started (58% success rare after 24 treatments). Apart from obtaining much better results the second protocol made it possible to avoid giving a significant number of useless transfusions (22% of pre-conceptual transfusions were not followed by a pregnancy at all).

[Menopausal hormone replacement therapy and prevention of neurologic complications]. / Traitement hormonal substitutif de la ménopause et prévention des complications neurologiques.

Aging of the population in industrialized countries, as a result of increased life expectancy, is associated with an increased risk of neurological diseases. Prevention of disability is one of the objectives of hormone replacement therapy of menopause (HRTM). Based on a review of the literature, the authors present the benefits of HRTM in terms of prevention of neurological complications. Estrogens directly and indirectly influence the neurological status. A decreased incidence of cerebrovascular accidents and dementia of the Alzheimer type and improvement of cognitive function in cases of dementia have been reported in several clinical and epidemiological papers. These data need to be confirmed by large-scale prospective studies conducted by epidemiologists and neurologists.

[Pelvic pain and endometriosis]. / Douleurs pelviennes et endométriose.

Endometriosis is defined by the presence outside the uterine cavity of tissue histologically and functionally similar to the endometrium. Endometriosis consists of glands and an underlying cytogenic stroma. This ectopic tissue can react to hormonal stimulation: estrogens and progesterone (growth, decidualisation, withdrawal bleeding). Pain is correlated with the depth of implantation of endometriosis lesions. Deeply infiltrating endometriosis exceeds 6 mm in depth, with an incidence of between 6 and 20% of women complaining of pelvic pain. The authors review the various types of treatment and mention the special role of celio-surgery and prolonged hormone therapy.

[Cancer of the ovary and medically assisted reproduction]. / Cancer de l'ovaire et procréation médicalement assistée.

Case reports and epidemiologic studies have recently indicated a possible link between ovarian stimulation, ovarian cancer and ovarian tumours. We sent a questionnaire to 116 French IVF centers to determine whether ovarian cancer had occurred in women treated during the last 10 years. The 19 cases of ovarian cancer reported have since been studied more thoroughly in a follow up questionnaire. Six can now be excluded, and the 13 remaining cases are compared. Five of these patients had fewer than 4 stimulation cycles here. Imputability was considered probable for 2 patients, possible for 5 patients, improbable for 3 and highly improbable for 3. Histologically, 6 (46%) cases involved borderline lesions. These results indicate that there is no need to change medical practice, but we are convinced that prospective epidemiologic research should be carried out immediately.