Exploring Thrips Preference and Resistance in Flowers, Leaves, and Whole Plants of Ten Capsicum Accessions.

Capsicum species grown for pepper production suffer severely from thrips damage, urging the identification of natural resistance. Resistance levels are commonly assessed on leaves. However, Capsicum plants are flower-bearing during most of the production season, and thrips also feed on pollen and flower tissues. In order to obtain a comprehensive estimate of elements contributing to thrips resistance, flower tissues should be considered as well. Therefore, we assessed resistance to Frankliniella occidentalis in flowers, leaves, and whole plants of ten Capsicum accessions. Using choice assays, we found that thrips prefer flowers of certain accessions over others. The preference of adult thrips for flowers was positively correlated to trehalose and fructose concentration in anthers as well as to pollen quantity. Resistance measured on leaf discs and thrips population development on whole plants was significantly and positively correlated. Leaf-based resistance thus translates to reduced thrips population development. Results of the flower assays were not significantly correlated with resistance in leaves or on whole plants. This suggests that both leaves and flowers represent a different part of the resistance spectrum and should both be considered for understanding whole plant resistance and the identification of resistant Capsicum varieties.

High Concentrations of Very Long Chain Leaf Wax Alkanes of Thrips Susceptible Pepper Accessions (Capsicum spp).

The cuticular wax layer can be important for plant resistance to insects. Thrips (Frankliniella occidentalis) damage was assessed on 11 pepper accessions of Capsicum annuum and C. chinense in leaf disc and whole plant assays. Thrips damage differed among the accessions. We analyzed the composition of leaf cuticular waxes of these accessions by GC-MS. The leaf wax composition was different between the two Capsicum species. In C. annuum, 1-octacosanol (C28 alcohol) was the most abundant component, whereas in C. chinense 1-triacotanol (C30 alcohol) was the prominent. Thrips susceptible accessions had significantly higher concentrations of C25-C29 n-alkanes and iso-alkanes compared to relatively resistant pepper accessions. The triterpenoids α- and ß-amyrin tended to be more abundant in resistant accessions. Our study suggests a role for very long chain wax alkanes in thrips susceptibility of pepper.

Slug Feeding Triggers Dynamic Metabolomic and Transcriptomic Responses Leading to Induced Resistance in Solanum dulcamara.

Induced plant responses to insect herbivores are well studied, but we know very little about responses to gastropod feeding. We aim to identify the temporal dynamics of signaling- and defense-related plant responses after slug feeding in relation to induced resistance. We exposed Solanum dulcamara plants to feeding by the gray field slug (GFS; Deroceras reticulatum) for different periods and tested disks of local and systemic leaves in preference assays. Induced responses were analyzed using metabolomics and transcriptomics. GFS feeding induced local and systemic responses. Slug feeding for 72 h more strongly affected the plant metabolome than 24 h feeding. It increased the levels of a glycoalkaloid (solasonine), phenolamides, anthocyanins, and trypsin protease inhibitors as well as polyphenol oxidase activity. Phytohormone and transcriptome analyses revealed that jasmonic acid, abscisic acid and salicylic acid signaling were activated. GFS feeding upregulated more genes than that it downregulated. The response directly after feeding was more than five times higher than after an additional 24 h without feeding. Our research showed that GFS, like most chewing insects, triggers anti-herbivore defenses by activating defense signaling pathways, resulting in increased resistance to further slug feeding. Slug herbivory may therefore impact other herbivores in the community.

Metabolomics of Thrips Resistance in Pepper (Capsicum spp.) Reveals Monomer and Dimer Acyclic Diterpene Glycosides as Potential Chemical Defenses.

The development of pesticide resistance in insects and recent bans on pesticides call for the identification of natural sources of resistance in crops. Here, we used natural variation in pepper (Capsicum spp.) resistance combined with an untargeted metabolomics approach to detect secondary metabolites related to thrips (Frankliniella occidentalis) resistance. Using leaf disc choice assays, we tested 11 Capsicum accessions of C. annuum and C. chinense in both vegetative and flowering stages for thrips resistance. Metabolites in the leaves of these 11 accessions were analyzed using LC-MS based untargeted metabolomics. The choice assays showed significant differences among the accessions in thrips feeding damage. The level of resistance depended on plant developmental stage. Metabolomics analyses showed differences in metabolomes among the Capsicum species and plant developmental stages. Moreover, metabolomic profiles of resistant and susceptible accessions differed. Monomer and dimer acyclic diterpene glycosides (capsianosides) were pinpointed as metabolites that were related to thrips resistance. Sucrose and malonylated flavone glycosides were related to susceptibility. To our knowledge, this is the first time that dimer capsianosides of pepper have been linked to insect resistance. Our results show the potential of untargeted metabolomics as a tool for discovering metabolites that are important in plant - insect interactions.

Thrips Resistance Screening Is Coming of Age: Leaf Position and Ontogeny Are Important Determinants of Leaf-Based Resistance in Pepper.

Capsicum is a genus containing important crop species, many of which severely suffer from thrips infestation. Thrips feeding damages leaves and fruits, and often results in virus infections. Only a few insecticides are still effective against thrips, underlining the importance of finding natural resistance in crops. Capsicum is a perennial plant which is usually cultivated for several months, during which time the fruits are harvested. From the young vegetative stage to the mature fruit bearing stage, the plants are at risk to thrips infestation. Constitutive resistance to thrips over the entire ontogenetic development is therefore a key trait for a more sustainable and successful cultivation of the hot and sweet pepper. In addition to ontogeny, leaf position can affect the level of thrips resistance. Pest resistance levels are known to differ between young and old leaves. To our knowledge, no studies have explicitly considered ontogeny and leaf position when screening for constitutive resistance to thrips in Capsicum. In this study we analyze whether ontogeny and leaf position affect leaf-based resistance to Frankliniella occidentalis and Thrips tabaci, in 40 Capsicum accessions, comprising five different species. Our results show that resistance to both thrips species in Capsicum varies with ontogenetic stage. This variation in resistance among ontogenetic stages was not consistent among the accessions. However, accessions with constitutive resistance in both the flowering and fruit ripening stage could be identified. In addition, we found that thrips resistance is overall similar at different leaf positions within the ontogenetic stage. This implies that resistance mechanisms, such as defense compounds, are constitutively present at sufficient levels on all leaf positions. Finally, we found that resistance to F. occidentalis and resistance to T. tabaci were not correlated. This indicates that leaf-based resistance in Capsicum is thrips species-specific. Because of the variation in resistance over ontogeny, identifying Capsicum accessions with resistance over their entire lifespan is challenging. For resistance screening, accounting for leaf position may be less of a concern. To identify the defense mechanisms responsible for thrips resistance, it is important to further analyze and compare resistant and susceptible accessions.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes.

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay.

STUDY QUESTION: Can a discriminant threshold be determined for human sperm DNA oxidation? SUMMARY ANSWER: A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. WHAT IS KNOWN ALREADY: Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa; optimized consensus protocols are lacking and no thresholds of normality have been established. STUDY DESIGN, SIZE, DURATION: Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. PARTICIPANTS/MATERIALS, SETTING, METHODS: This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). MAIN RESULTS AND THE ROLE OF CHANCE: Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. LIMITATIONS, REASONS FOR CAUTION: The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. WIDER IMPLICATIONS OF THE FINDINGS: Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by institutional grants from the CNRS, INSERM and Université Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

Glycoalkaloid composition explains variation in slug resistance in Solanum dulcamara.

In natural environments, plants have to deal with a wide range of different herbivores whose communities vary in time and space. It is believed that the chemical diversity within plant species has mainly arisen from selection pressures exerted by herbivores. So far, the effects of chemical diversity on plant resistance have mostly been assessed for arthropod herbivores. However, also gastropods, such as slugs, can cause extensive damage to plants. Here we investigate to what extent individual Solanum dulcamara plants differ in their resistance to slug herbivory and whether this variation can be explained by differences in secondary metabolites. We performed a series of preference assays using the grey field slug (Deroceras reticulatum) and S. dulcamara accessions from eight geographically distinct populations from the Netherlands. Significant and consistent variation in slug preference was found for individual accessions within and among populations. Metabolomic analyses showed that variation in steroidal glycoalkaloids (GAs) correlated with slug preference; accessions with high GA levels were consistently less damaged by slugs. One, strongly preferred, accession with particularly low GA levels contained high levels of structurally related steroidal compounds. These were conjugated with uronic acid instead of the glycoside moieties common for Solanum GAs. Our results illustrate how intraspecific variation in steroidal glycoside profiles affects resistance to slug feeding. This suggests that also slugs should be considered as important drivers in the co-evolution between plants and herbivores.

Quantification of Thrips Damage Using Ilastik and ImageJ Fiji.

Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening for host plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Here we present an objective novel method for analyzing thrips damage on leaf disc using the freely available software programs Ilastik and ImageJ. The protocol was developed in order to screen over 40 Capsicum lines for resistance against Frankliniella occidentalis (Western Flower Thrips) and Thrips tabaci (Onion thrips).

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

HAWAIIAN SKIRT controls size and floral organ number by modulating CUC1 and CUC2 expression.

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT (HWS) affects organ growth and the timing of floral organ abscission. The loss-of-function hws-1 mutant exhibits fused sepals and increased organ size. To understand the molecular mechanisms of HWS during plant development, we mutagenized hws-1 seeds with ethylmethylsulphonate (EMS) and screened for mutations suppressing hws-1 associated phenotypes. We isolated the shs1/hws-1 (suppressor of hws-1) mutant in which hws-1 sepal fusion phenotype was suppressed. The shs1/hws-1 mutant carries a G→A nucleotide substitution in the MIR164 binding site of CUP-SHAPED COTYLEDON 1 (CUC1) mRNA. CUC1 and CUP-SHAPED COTYLEDON 2 (CUC2) transcript levels were altered in shs1, renamed cuc1-1D, and in hws-1 mutant. Genetic interaction analyses using single, double and triple mutants of cuc1-1D, cuc2-1D (a CUC2 mutant similar to cuc1-1D), and hws-1, demonstrate that HWS, CUC1 and CUC2 act together to control floral organ number. Loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the CUC1 gene.

Multi-Level Interactions Between Heat Shock Factors, Heat Shock Proteins, and the Redox System Regulate Acclimation to Heat.

High temperature has become a global concern because it seriously affects the growth and reproduction of plants. Exposure of plant cells to high temperatures result in cellular damage and can even lead to cell death. Part of the damage can be ascribed to the action of reactive oxygen species (ROS), which accumulate during abiotic stresses such as heat stress. ROS are toxic and can modify other biomacromolecules including membrane lipids, DNA, and proteins. In order to protect the cells, ROS scavenging is essential. In contrast with their inherent harms, ROS also function as signaling molecules, inducing stress tolerance mechanisms. This review examines the evidence for crosstalk between the classical heat stress response, which consists of heat shock factors (HSFs) and heat shock proteins (HSPs), with the ROS network at multiple levels in the heat response process. Heat stimulates HSF activity directly, but also indirectly via ROS. HSFs in turn stimulate the expression of HSP chaperones and also affect ROS scavenger gene expression. In the short term, HSFs repress expression of superoxide dismutase scavenger genes via induction of miRNA398, while they also activate scavenger gene expression and stabilize scavenger protein activity via HSP induction. We propose that these contrasting effects allow for the boosting of the heat stress response at the very onset of the stress, while preventing subsequent oxidative damage. The described model on HSFs, HSPs, ROS, and ROS scavenger interactions seems applicable to responses to stresses other than heat and may explain the phenomenon of crossacclimation.

Modulating crossover positioning by introducing large structural changes in chromosomes.

BACKGROUND: Crossing over assures the correct segregation of the homologous chromosomes to both poles of the dividing meiocyte. This exchange of DNA creates new allelic combinations thus increasing the genetic variation present in offspring. Crossovers are not uniformly distributed along chromosomes; rather there are preferred locations where they may take place. The positioning of crossovers is known to be influenced by both exogenous and endogenous factors as well as structural features inherent to the chromosome itself. We have introduced large structural changes into Arabidopsis chromosomes and report their effects on crossover positioning. RESULTS: The introduction of large deletions and putative inversions silenced recombination over the length of the structural change. In the majority of cases analyzed, the total recombination frequency over the chromosomes was unchanged. The loss of crossovers at the sites of structural change was compensated for by increases in recombination frequencies elsewhere on the chromosomes, mostly in single intervals of one to three megabases in size. Interestingly, two independent cases of induced structural changes in the same chromosomal interval were found on both chromosomes 1 and 2. In both cases, compensatory increases in recombination frequencies were of similar strength and took place in the same chromosome region. In contrast, deletions in chromosome arms carrying the nucleolar organizing region did not change recombination frequencies in the remainder of those chromosomes. CONCLUSIONS: When taken together, these observations show that changes in the physical structure of the chromosome can have large effects on the positioning of COs within that chromosome. Moreover, different reactions to induced structural changes are observed between and within chromosomes. However, the similarity in reaction observed when looking at chromosomes carrying similar changes suggests a direct causal relation between induced change and observed reaction.

[PRESERVING FERTILITY AND FUTURE DEATH]. / PRÉSERVATION DE LA FERTILITÉ ET LA MORT À VENIR: UNE DOUBLE PEINE?

The fertility preservation when it is threatened is a right enshrined in the french law on bioethics. It is most often performed before gonadotoxic treatments for cancers with a long survival. When the patient has a limited life expectancy, is the preservation of fertility lawful? The authors present the arguments for and against the preservation of fertility in this particular situation and give driving they adopt in their team.

Flooding and fragment size interact to determine survival and regrowth after fragmentation in two stoloniferous Trifolium species.

Clonal plants, which reproduce by means of stolons and rhizomes, are common in frequently flooded habitats. Resilience to disturbance is an important trait enabling plants to survive in such highly disturbed habitats. Resource storage is thought to enable clonal plants to resume growth after clonal fragmentation caused by disturbance. Here we investigated if submergence prior to disturbance reduces survival and regrowth of clonal fragments and whether or not genotypes originating from highly disturbed riverine habitats are more resistant to mechanical disturbance than genotypes from less disturbed coastal dune slack habitats. We further tested if variation in survival and regrowth was affected by internode size. Clones from contrasting habitats of two closely related Trifolium species were first genotypically characterized by amplification fragment length polymorphism and then subjected to soil flooding and subsequent clonal fragmentation. These species differ with respect to their abundance in riverine and dune slack habitats, with Trifolium repens mainly occurring in riverine grasslands and Trifolium fragiferum in coastal dune slacks. Soil flooding decreased survival and regrowth by up to 80 %. Plants originating from riverine grasslands were less negatively affected by fragmentation than plants from dune slack habitats. Surprisingly, ramets did not always benefit from being attached to a larger internode, as internode size was often negatively correlated with survival after fragmentation. Regrowth, on the other hand, was generally positively correlated with internode size. These unexpected results indicate that there may be contrasting selection pressures on internode size in stoloniferous species growing in severely disturbed habitats.

[Cryoconservation of ovarian tissue: indications and outcome of the patients]. / Cryoconservation du tissu ovarien: indications et devenir des patientes.

OBJECTIVES: Ovarian Tissue Cryopreservation (OTC) is a very promising approach of fertility preservation for women and young patients who have to follow gonadotoxic treatments (chemotherapy, radiotherapy…). The aim of this study was to analyse the indications and the outcomes of the patients who had OTC in our center during the last 17 years. PATIENTS AND METHODS: The study is retrospective. Forty-six patients, who underwent OTC in the Laboratory of Reproductive Biology of the University Hospital of Clermont-Ferrand, between January 1997 and December 2009, were included. RESULTS: The average age on the day of ovarian tissue harvesting was 19.5 years. Fifty-two percent of the patients were minor. In order of decreasing frequency, the diseases for which OTC has been proposed were hematologic, ovarian tumors, sarcoma ou PNET and breast neoplasia. In 93.5 %, the harvesting of ovarian cortex was performed by laparoscopy. After OTC, 82.6 % of the patients were treated by chemotherapy. A bone marrow transplant was performed for 48 % of the study patients. At the time of data collection, 57 % of the patients who had evaluation of their ovarian function presented premature ovarian failure. Eight patients had one or more pregnancies after treatment. It was a natural pregnancy for five of them. The three others were obtained by medically assisted procreation (in vitro fertilization and oocyte donation). DISCUSSION AND CONCLUSION: We report a long-term follow-up of patients treated in our center for OTC. The originality of our study is to evaluate all aspects of OTC from the decision to propose the patients an OTC to their outcomes several years after the ovarian tissue harvesting. It is therefore a multidisciplinary approach both oncology, gynecological and pediatric whereas OTC is often considered restrictively in the literature. Finally, it seems to be essential to establish a specific medical care for these patients. This monitoring will allow an adequate assessment of pubertal development and ovarian function, management of estrogen deficiency and secondary infertility, supporting patients in their desire for motherhood.

[Adverse pregnancy outcomes after Assisted Reproduction Technology in women with endometriosis]. / Issues obstétricales des grossesses obtenues par Assistance médicale à la procréation chez les femmes endométriosiques.

OBJECTIVE: While association between endometriosis and infertility is well established, there are few studies about the impact of endometriosis on adverse pregnancy outcomes. The aim of this study was to determine the effect of endometriosis on obstetric outcomes and whether the severity of the disease had an influence on these. PATIENTS AND METHODS: We performed a retrospective study to investigate the obstetric outcomes of a population of 1204 subfertile women, including 258 with endometriosis, who obtained, thanks to assisted reproduction technology, a singleton pregnancy evolving beyond embryonic stage. Two analyzes were performed. The first compared women with endometriosis to women with other causes of infertility. The second observed adverse pregnancy outcomes according to AFS-R stages of endometriosis. RESULTS: The overall rate of live birth children was 95.8%. In case of endometriosis, there was a significant increase of the incidence of preterm delivery, especially before 32 weeks amenorrhea (6.2% vs 3.1% in the group "without endometriosis", P = 0.03), antenatal bleeding (5.3% vs 2.2%, P = 0.01) and placenta previa (4.9% vs 0.9%, P < 0.0001). The incidence of gestational diabetes was significantly decreased (0.4% vs 2.7%, P = 0.04). There was no correlation between endometriosis and cesarean section or preeclampsia, or between the AFS-R stage and adverse pregnancy outcomes. DISCUSSION AND CONCLUSION: Endometriosis is a factor of obstetrical risk, independently of the infertility it causes. The AFS-R score does not seem to be representative of obstetric outcomes beyond first trimester of pregnancy for women with endometriosis.

Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia.

STUDY QUESTION: Can selection of spermatozoa by density gradient centrifugation prior to cryopreservation and/or hypotaurine supplementation improve the post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia? SUMMARY ANSWER: Sperm selection by density gradient centrifugation before freezing and supplementation of the media by hypotaurine is beneficial for the cryopreservation of semen samples of patients with oligoasthenoteratozoospermia. WHAT IS KNOWN ALREADY: Sperm from men with oligoasthenoteratozoospermia are more susceptible than normal to cryoinjury. Density gradient centrifugation before sperm freezing may allow the selection of a subpopulation of spermatozoa more resistant to cryopreservation. Hypotaurine is an antioxidant with a protective effect on sperm functions. STUDY DESIGN, SIZE, DURATION: The experiment was carried out according to a factorial design involving two binary factors resulting in four treatment combinations which were randomly allocated in oligoasthenoteratozoospermia sperm samples from 64 patients recruited between January 2009 and June 2010. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen was provided by 64 men undergoing evaluation for infertility at the Centre for Reproductive Medicine of the University Hospital in Clermont-Ferrand, France, between January 2009 and June 2010. Four treatment combinations were tested: sperm freezing before selection without (F-S/H-; n = 16) and with hypotaurine supplementation (F-S/H+; n = 16); sperm selection before freezing without (S-F/H-; n = 16) and with hypotaurine supplementation (S-F/H+; n = 16). Measurements of sperm recovery rates and markers of apoptosis (externalization of phosphatidylserine (PS), mitochondrial membrane potential and DNA fragmentation) were compared in recovered spermatozoa after each procedure. MAIN RESULTS AND THE ROLE OF CHANCE: Higher recovery rates of progressive and total motile spermatozoa were observed when sperm selection was performed before freezing (P < 0.05). The protective effect of hypotaurine was only observed on the percentage of live spermatozoa with PS externalization among total live spermatozoa (AN+ PI-/((AN+ PI-) + (AN- PI-)) when the sperm selection by density gradient centrifugation was performed before freezing (S-F/H+ versus S-F/H-: 6.8 ± 1.09 versus 11.8 ± 2.03%, P = 0.04). The percentage of mitochondrial membrane potential (DiOC6(3) (high)) spermatozoa was higher (P = 0.001) when sperm selection was done before freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 58.1 ± 3.50 versus 46.7 ± 5.48%) or without (S-F/H- versus F-S/H-: 57.0 ± 5.18 versus 35.4 ± 4.99%) hypotaurine supplementation. The percentages of TUNEL+ spermatozoa were significantly lower (P = 0.001) when sperm selection was done before sperm freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 38.6 ± 9.59 versus 55.7 ± 5.88%) or without hypotaurine supplementation (S-F/H- versus F-S/H-: 37.2 ± 7.91 versus 71.0 ± 5.66%). LIMITATIONS, REASONS FOR CAUTION: The ICSI outcomes were not assessed and the fertility of the spermatozoa remains unknown. WIDER IMPLICATIONS OF THE FINDINGS: Sperm selection by density gradient centrifugation before freezing and hypotaurine supplementation could improve the cryopreservation of sperm from oligoasthenoteratozoospermic men and make a larger number of functional spermatozoa available for ICSI. STUDY FUNDING/COMPETING INTERETS(S): This work was supported by a hospital grant (Projet Hospitalier Recherche Clinique, CHU Clermont Ferrand, France). None of the authors has any conflict of interest to declare.

[Reduction of multiple pregnancies in ART with large SET procedures over the period 2001-2010]. / Réduction des grossesses multiples en AMP par un usage large du SET (Single Embryo Transfer) sur la période 2001-2010.

OBJECTIVE: To evaluate delivery rate and multiple pregnancy rates in ART (assisted reproductive techniques) following introduction of an elective single embryo tranfer (eSET) policy. This strategy was started in 2002 including transfer of one embryo for women less than 35 years with a least two good quality embryo during their first or second attempts. PATIENTS AND METHODS: Retrospective study including all IVF cycles performed in the IVF centre of Clermont-Ferrand University Hospital from 01/01/2001 to 31/12/2010. Main outcome measures were number of embryos transferred, cumulative delivery and multiple pregnancy rates (including fresh and frozen embryo transfers). A subgroup analysis including patients' age was done. RESULTS: Cumulative delivery rate reached 27,3% in 2010 with a significant drop in multiple pregnancy rate: from 30% in 2001 to 7,9% in 2010. The average number of transferred embryo decreased from 2.29 to 1.55 in the same period. In our centre, eSET was performed in 85% of first IVF attempt and in 34,4% of second attempts for women less than 35 years. CONCLUSION: The implementation of an eSET policy does not change the delivery rate but significantly decrease the number of multiple pregnancies compared to double embryo transfer. eSET should be carried out during the 1st and 2nd attempts in patients under 35 years when at least two good quality embryos were obtained.