Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes.

STUDY QUESTION: Could whole-exome sequencing (WES) be useful in clinical practice for men with maturation arrest (MA) after a first testicular sperm extraction (TESE)? SUMMARY ANSWER: WES in combination with TESE yields substantial additional information and may potentially be added as a test to predict a negative outcome of a recurrent TESE in patients with MA. WHAT IS KNOWN ALREADY: At present, the only definitive contraindications for TESE in men with non-obstructive azoospermia (NOA) are a 46,XX karyotype and microdeletions in the azoospermia factor a (AZFa) and/or AZFb regions. After a first negative TESE with MA, no test currently exists to predict a negative outcome of a recurrent TESE. STUDY DESIGN, SIZE, DURATION: In a cohort study, we retrospectively included 26 patients with idiopathic NOA caused by complete MA diagnosed after a first TESE. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-six men with MA at the spermatocyte stage in all seminiferous tubules, according to a histopathological analysis performed independently by two expert histologists, and a normal karyotype (i.e. no AZF gene microdeletions on the Y chromosome) were included. Single-nucleotide polymorphism comparative genomic hybridization array and WES were carried out. The results were validated with Sanger sequencing. For all the variants thought to influence spermatogenesis, we used immunohistochemical techniques to analyse the level of the altered protein. MAIN RESULTS AND THE ROLE OF CHANCE: Deleterious homozygous variants were identified in all seven consanguineous patients and in three of the 19 non-consanguineous patients. Compound heterozygous variants were identified in another 5 of the 19 non-consanguineous patients. No recurrent variants were identified. We found new variants in genes known to be involved in azoospermia or MA [including testis expressed 11 (TEX11), meiotic double-stranded break formation protein 1 (MEI1), proteasome 26s subunit, ATPase 3 interacting protein (PSMC3IP), synaptonemal complex central element protein 1 (SYCE1) and Fanconi anaemia complementation group M (FANCM) and variants in genes not previously linked to human MA (including CCCTC-binding factor like (CTCFL), Mov10 like RISC complex RNA helicase 1 (MOV10L1), chromosome 11 open reading frame 80 (C11ORF80) and exonuclease 1 (EXO1)]. LARGE SCALE DATA: Data available on request. LIMITATIONS, REASONS FOR CAUTION: More data are required before WES screening can be used to avoid recurrent TESE, although screening should be recommended for men with a consanguineous family background. WES is still a complex technology and can generate incidental findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirmed the genetic aetiology of MA in most patients: the proportion of individuals with at least one pathologic variant was 50% in the overall study population and 100% in the consanguineous patients. With the exception of MEI1 (compound heterozygous variants of which were identified in two cases), each variant corresponded to a specific gene-confirming the high degree of genetic heterogeneity in men with MA. Our results suggest that WES screening could help to avoid recurrent, futile TESE in men with MA in general and in consanguineous individuals in particular, but these results need to be confirmed in future studies before clinical implementation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Fondation Maladies Rares (Paris, France), Merck (Kenilworth, NJ, USA), IRSF (Montigny le Bretonneux, France) and Agence de la Biomédecine (Saint Denis, France). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A MEI1 homozygous missense mutation associated with meiotic arrest in a consanguineous family.

Although meiotic arrest in males is observed in about 25% of azoospermic patients, pure homogeneous arrest in all seminiferous tubules is less frequent, and may be due to mutation of a single gene. However, given the large number of genes involved in meiosis, this gives rises to extensive genetic heterogeneity. Only two genetic abnormalities have been reported on a regular basis: the X-linked exonic TEX11 deletion, and the AZFb microdeletion on the Y chromosome. Other single gene defects were private and found in consanguineous families. Here, we report on a homozygous missense mutation in the gene coding for meiotic double-stranded break formation protein 1 (MEI1; c.C3307T:p.R1103W) observed in two brothers (from a consanguineous Tunisian family) with non-obstructive azoospermia and meiotic arrest. A fertile brother was heterozygous for the mutation. All the queried databases predicted that this mutation is damaging, and it has previously been reported that Mei1 knock-out is associated with meiotic arrest in a murine model. Hence, meiotic arrest in the two brothers was probably caused by an alteration in a gene known to be fundamental for chromosome synapsis.

Impact of fermentation on nitrogenous compounds of cocoa beans (Theobroma cacao L.) from various origins.

Tangential filtration technique was used to separate and quantify three different fractions of nitrogenous compounds depending on their molecular size, during cocoa fermentation. On every phenotype and origin analyzed, protein profile of non-fermented samples was similar. During fermentation course, proteins get degraded with a concomitant increase in amino acids content. Peptides between 3 and 10 kDa were observed at low levels. A strong correlation between amino acids and ammonia nitrogen, a fermentation marker was found. Attention was drawn on each fraction, and enabled to point out other phenomenon occurring during fermentation. The migration of some nitrogenous compounds towards the bean shell during fermentation was demonstrated. Acetone treatment of cocoa powder prior to SDS-PAGE led to losses of nitrogenous compounds. This result gives clues on the tanning phenomenon carried out by polyphenols on nitrogenous compounds, phenomenon which increases during fermentation.

Near infrared spectroscopy as a new tool to determine cocoa fermentation levels through ammonia nitrogen quantification.

Fermentation is a key step in obtaining fine cocoa through the formation of potent aroma precursors. The fermentation level of cocoa beans is traditionally assessed by measuring the amount of ammonia nitrogen (NH3) using the time-consuming Conway technique. Near infrared spectroscopy (NIRS), a rapid and efficient tool, was used to analyze NH3 levels in several hundred cocoa samples at different fermentation levels from six geographical origins. Fermentation levels were expressed as the number of fermentation days and sum of temperatures. The correlation between Conway results and NIRS spectra enabled the development of a reliable and accurate NIRS calibration to determine NH3 content. We confirm that NH3 is produced during fermentation and its amount depends on the fermentation time, sum of temperatures and geographical origin. NIRS could be used by chocolate manufacturers as a routine method to sort cocoa samples according to their level of fermentation.

From irreversibility to participation: towards a participatory foresight for the governance of collective environmental risks.

This paper presents a reflection on the introduction of methods and tools of "participative foresight" for scientific and technology policy as well as environmental policy fields. Future studies have recently made a comeback under the label of foresight. Future technology studies no longer claim to forecast the future, but are presented as a strategic tool for improving interaction between key actors and for anticipatory policy making. They can be defined as a "process by which one comes to a fuller understanding of the forces shaping the long term future which should be taken into account in policy formulation, planning and decision-making" [Foresight in Federal Government Policymaking, Futures Res. Quart. (1985) 29]. We discuss applications of this approach for perspectives on environmental policy and sustainable development. Foresight opens up the possibility of negotiating a new and more fruitful relationship or 'social contract' between science and technology, on the one hand, and society on the other. The focus has moved from merely scientific and industrial insights to social demand, thus emphasizing the importance of both the production and "supply" of innovation, and the "demand" as signaled in the views of citizens.

Optimization of retroviral gene transfer protocol to maintain the lymphoid potential of progenitor cells.

We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.