Another look at human sperm morphology.

STUDY QUESTION: Can a standardized assessment of abnormal human sperm morphology provide additional useful information by identifying men with more severe disturbances in different types of abnormalities? SUMMARY ANSWER: Definition-based categorization of sperm head, midpiece and tail defects has shown how differently these abnormalities are distributed in fertile men and other groups of men, thus providing high and low thresholds, a starting point for diagnosis or research purposes. WHAT IS KNOWN ALREADY: Several recent studies have reported indisputable genetic origins for various sperm defects. A few studies have also identified associations between environmental factors and low percentages of morphologically normal spermatozoa. Nevertheless, with the exception of rare situations in which the vast majority of spermatozoa have specific, easily characterized defects, such as 'globozoospermia', little attention has been paid to the description and precise quantification of human sperm abnormalities. The lack of standardization in the phenotyping of sperm morphological defects by conventional microscopy is a limiting factor for diagnosis and for intra- or inter-observer or centre consistency in studies investigating the causal factors and possible functional consequences of the abnormalities detected. There are currently no baseline data for abnormalities of sperm morphology based on a standardized classification, in the general population, among fertile or other groups of men. STUDY DESIGN, SIZE, DURATION: This study is based on detailed sperm abnormality datasets acquired by a standardized classification method, from several groups of men, over the same 5-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied cross-sectional data from fertile men (n = 926), male partners from infertile couples (n = 1747) and testicular cancer patients (n = 239). We used a standardized classification to analyse Shorr-stained slides, taking into account all the abnormalities encountered. MAIN RESULTS AND THE ROLE OF CHANCE: Most sperm defects were significantly more frequent in infertile than in fertile men, with 20-30% of infertile men having frequencies of abnormalities above the 95th percentile in fertile men for 9 out of the 15 categories of abnormalities. Interestingly, several head abnormalities were significantly more frequent in patients with testicular cancer than in infertile men, highlighting the particular impact of this condition on sperm morphogenesis. We used the 95th percentile in fertile men as the lower threshold and the 99th percentile in infertile men as an extreme upper threshold, for the classification of morphological abnormality frequencies into three levels: low, intermediate and high. The assessment of several semen samples, with or without a genetic background, for abnormal sperm morphology, based on the percentage of normal spermatozoa, a teratozoospermia index, and the detailed profile of abnormalities categorized according to the three levels proposed, has highlighted the value of detailed phenotyping for diagnosis and research purposes. LIMITATIONS, REASONS FOR CAUTION: The thresholds proposed for the various categories of sperm abnormality should be considered relative rather than absolute, owing to the known sampling error related to the limited number of spermatozoa assessed per sample, or when studying the general population or populations from regions other than Western Europe. The standardized assessment of abnormal sperm morphology requires time and experience. We therefore suggest that this assessment is carried out during a first andrological check-up or for epidemiological or research studies, rather than in the routine management of infertile couples for assisted reproductive technologies. WIDER IMPLICATIONS OF THE FINDINGS: The study design used for the fertile group of men was similar to that previously used for the WHO reference values, providing a rationale for considering the 95th percentile in fertile men as the level below which abnormalities may be considered to occur at a frequency representing random background variations of a normal spermiogenesis process. The crude frequencies obtained, and the three levels of abnormality frequency proposed for each standardized category of sperm defect, provide baseline data useful for diagnosis and a starting point for future studies aiming to identify associations with genetic or environmental factors. STUDY FUNDING/COMPETING INTERESTS: Part of this study was supported by contract BMH4-CT96-0314 from the European Union. The authors have no competing interests to declare.

Identification of genital tract markers in the human seminal plasma using an integrative genomics approach.

STUDY QUESTION: Can protein biomarkers of the male genital tract be identified in human seminal plasma? SUMMARY ANSWER: We identified potential biomarkers for each of the organs participating in the secretions of the human seminal plasma. WHAT IS KNOWN ALREADY: The seminal plasma fulfills critical functions for fertility by providing spermatozoa with a protective milieu, promoting their final maturation and modulating the immune responsiveness of the female reproductive tract. It is also considered to be a promising source of biomarkers of male infertility and/or pathologies of the male genital tract. STUDY DESIGN, SIZE, DURATION: This study combines proteomic analyses of normal seminal plasma together with transcriptomic gene expression profiling of human healthy tissues. MATERIALS, SETTING, METHODS: Non-liquefied seminal plasma proteins from a healthy donor were prefractionated using two sequential Proteominer™ libraries. Eight subproteome fractions were collected, trypsin digested and subjected to three successive mass spectrometry analyses for peptide characterization. The list of identified proteins was compared with and merged with other available data sets of the human seminal plasma proteome. The expression of corresponding genes was then investigated using tissue transcriptome profiles to determine where, along the male reproductive tract, these proteins were produced. Finally, tissue specificity of a selected subset of biomarker candidates was validated on human tissues. MAIN RESULTS AND THE ROLE OF CHANCE: We first performed a proteomic analysis of the human seminal plasma and identified 699 proteins. By comparing our protein list with other previous proteomic data sets, we found that 2545 unique proteins have been described so far in the human seminal plasma. We then profiled their expression at the gene level and identified 83 testis, 42 epididymis, 7 seminal vesicle and 17 prostate candidate protein markers. For a subset of testis-specific candidates, i.e. TKTL1, LDHC and PGK2, we further validated their germ cell expression and demonstrated that such markers could distinguish between semen from fertile and infertile men. LIMITATIONS, REASONS FOR CAUTION: While some of the markers we identified are well-known tissue-specific products, further dedicated studies to validate the biomarker status of new candidates will be required. Additionally, whether or not the abundance of these proteins is indeed decreased in some specific pathological situations remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Using an integrative genomics approach, we identified biomarker candidates for each of the organs participating in the seminal plasma production. In this study, we essentially focused on germ cell markers and their potential application for the diagnosis of male infertility. Other types of markers also deserve a focused attention given their potential predictive value for various reproductive disorders, notably for prostate cancers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Proteomics Core Facility at Biogenouest and was funded by Conseil Régional de Bretagne, IBiSA and Agence de la Biomédecine grants. The authors declare that there exists a competing financial interest in this work that is related to a patent application on the use of identified germ cell-specific proteins in an antibody-based assay (Fertichip™) to predict the successful testicular biopsy outcomes in human non-obstructive azoospermia.

Perinatal xenohormone exposure impacts sweet preference and submandibular development in male rats.

OBJECTIVE: To determine the effect of perinatal exposure to low doses of genistein and/or vinclozolin on submandibular salivary gland (SSG) development in juvenile and adult male rats and to establish a link with sweet preference. MATERIAL AND METHODS: Female rats received orally (1 mg kg(-1) body weight/day) genistein and vinclozolin, alone or in combination, from the first gestational day up to weaning. Sweet preference was assessed at weaning and in adulthood in male offspring; submandibular glands were then collected to study the morphogenesis and mRNA expression of steroid receptors, growth factors and taste related proteins. RESULTS: Exposure to genistein and/or vinclozolin resulted in a higher saccharin intake on postnatal day 25 (P < 0.05) linked to a higher number of pro-acinar cells (P < 0.01) and mRNA expression of progesterone receptor, growth factors and gustine (P < 0.01). These increases disappeared in adulthood, but mRNA expressions of sex hormone receptors and growth factors were strongly repressed in all treated groups (P < 0.01). CONCLUSION: Our findings confirm that the SSG are target for xenohormones and provide evidence that perinatal exposure to low doses of genistein and/or vinclozolin could simultaneously disrupt not only the salivary gland prepubertal development and sweet intake but also endocrine gene mRNA expression later in life.

Diaporthe ambigua Associated with Post-Harvest Fruit Rot of Kiwifruit in Chile.

Kiwifruit is an important, expanding commercial crop in Chile. Several fungi have been reported to be associated with post-harvest rots of kiwifruit worldwide (2). During 2011 and 2012, kiwifruit produced in the VI and VII regions of Chile, showing symptoms of inner rot, were investigated with the aim of identifying the disease agent. The affected fruits had brown pubescent skin at the stem end that became soft and lighter in color than the adjacent firm healthy tissues. A watery exudate and white to pale grayish mycelial mats frequently developed at the stem end of the fruit, causing a water-drop stain down the sides on the dry brown healthy skin. The underlying tissue accessed by peeling off the skin was usually water soaked, soft, and lighter green than the healthy tissue. A fermented sour odor occurred frequently on severely decayed fruits. After incubation at 25°C over 7 days on potato dextrose agar (PDA), white to grayish, pale aerial mycelial mats were recovered from fragments of symptomatic kiwifruit superficially disinfected with 95% ethanol. After 2 weeks, black, spherical pycnidia developed, bearing hyaline, ellipsoidal, biguttulate conidia (5.4 to 12.6 × 2.1 to 4.7 µm). After 3 weeks, abundant perithecia embedded in a distinct, black, elevated stroma developed. Perithecia were black, globose, 200 to 500 µm in diameter with necks sinuous, filiform, 550 to 840 × 50 to 95 µm. Clavate, sessile asci, 30.9 to 50.2 × 6.6 to 12.5 µm contained ascospores biseriate, hyaline, smooth, fusoid-ellipsoid, widest just above the septum, tapering towards both ends, medianly septate, constricted at the septum at maturity, with 1 to 2 guttules per cell ascospores, 5.9 to 9.7 × 2.5 to 4.3 µm. All colonies obtained from kiwifruit displayed the same morphological traits and were consistent with those of a Diaporthe sp. (1). The internal transcribed spacer (ITS) region was sequenced using ITS1/ITS4 primers (4). Analysis of ITS region of kiwifruit isolates Damb_12 and Damb_16 (GenBank Accession Nos. KC294545 and KC294544, respectively) revealed 100% nucleotide identity to Diaporthe ambigua (HM575420, HM575419, DQ286274, DQ286270, and DQ286271). Six millimeter plugs from fungal colonies growing on PDA at 25°C for 7 days were used to inoculate 15 healthy untreated, ripe 'Hayward' kiwifruits. Control fruits were inoculated with agar plugs. Inoculated fruits were incubated at 25°C and 80% relative humidity. After 7 days, white to pale grayish mycelial mats developed only on the inoculated fruits, releasing a water drop stain. The underlying tissue was lighter green and water soaked. D. ambigua was reisolated only from the inoculated fruits. D. actinidiae has been previously reported on kiwifruit canes in Chile (3); however, to our knowledge, this is the first report on the occurrence of D. ambigua (Phomopsis ambigua) on kiwifruit in Chile. The fungal isolates (no. 1 to 21) have been deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) J. C. Jansen van Rensburg et al. Studies in Mycol. 55:65, 2006. (2) L. Luongo et al. J. Plant Pathol. 93:205, 2011. (3) A. Palma and E. Piontelli. E. Bol. Micol. 15:79, 2000. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Neofusicoccum australe (Botryosphaeria australis), as a Branch Dieback Pathogen of Avocado Trees in Chile.

Since 2007, Chilean avocado (Persea americana Mill.) orchards have been exposed to several abiotic stress conditions, namely frost damage and drought, due to three consecutive seasons of cold winters and shortage of irrigation water. At the same time, a severe disease resulting in tree dieback of cv. Hass, specifically, was observed in north-central Chile. Symptomatic trees exhibited abundant dead twigs in the tree canopy, and wilted leaves remained attached to the twigs in autumn. Closer inspection revealed reddish-brown necrotic lesions on the bark of the dead twigs, which girdled these symptomatic branches. When the bark was removed, the wood below appeared dark brown, in contrast to the yellowish-green coloring of healthy. The fungus was also consistently isolated from rotted fruit. A Neofusicoccum sp. with a yellow colony was consistently isolated from the necrotic lesions on PDA and incubated at room temperature for 3 days. Conidia produced in black pycnidia growing on 2% water agar with sterilized pine needles were smooth, unicellular, hyaline, and with granular contents. One or two septa developed at germination, but rarely before. The average length of the conidia was 27.0 ± 0.9 µm, with a length/width ratio of 3.9 ± 0.2 µm. Based on culture and conidial morphology, the isolates were putatively identified as Neofusicoccum luteum (1). DNA sequence analysis of the rDNA internal transcribed spacer (ITS) region was conducted for four representative isolates using primers ITS1 and ITS4 (4). The sequence analysis of ITS region of kiwifruit isolate H1M4 (Accession No. KC330230) reveled 100% nucleotide identity to N. australe (FJ157187 to FJ157192) (3). Pathogenicity tests were conducted with stem inoculations of 2-year-old cv. Hass plants grow in plastic containers in a sand/lime/peat mixture. For each inoculated plant (n = 8), a 7-mm-diameter agar plug from the margin of a 3-day culture was used as inoculum after wounding the stem to the depth to 7 mm with a cork borer. Negative control (n = 8) were wounded and then 'mock-inoculated' with sterile agar plugs. The inoculation sites were wrapped with Parafilm. All plants were kept in a greenhouse. After 5 months, all inoculated plants showed bark cankers and necrotic lesions beneath the bark, which were 5.2 cm long (n = 8). No symptoms developed on the control plants. N. australe was recovered from the margin of the necrosis lesion of every inoculated plant, thus fulfilling Koch's postulates and confirming its pathogenicity. Botryosphaeraceae spp. are the commonly reported to have ability to survive endophytically in their host, causing disease only when the host is exposed to a stress condition (2). To our knowledge, this is the first report of N. australe as a pathogen of avocado in Chile. The fungal isolates (PaHass No. 1 to 4) were deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) A. J. L. Phillips. http://www.crem.fct.unl.pt/botryosphaeria_site/ Accessed November 20, 2011. (2) B. Slippers and M. J. Wingfield. Fungal Biol. Rev. 21:90, 2007. (3) B. Slippers et al. Mycologia 96:1030, 2004. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Second to fourth digit ratios, male genital development and reproductive health: a clinical study among fertile men and testis cancer patients.

Second- to fourth-digit length ratio, 2D:4D, is a marker of testosterone level during foetal life that was found associated with sperm concentration or testosterone levels in some studies, but not in others, a difference possibly related to the way the ratio is assessed. In this study, 2D:4D was assessed in 122 men partners of pregnant women and in 71 testicular cancer patients using a new method based on direct measurements of finger lengths. In addition, we investigated the association between 2D:4D, birth weight, testicular volume, semen quality and time to pregnancy. A validation study of the method demonstrated high reliability and reproducibility. Neither digit lengths nor 2D:4D significantly differed in both groups of men. We found a significant negative association between 2D:4D and birth weight in testicular cancer patients. In fertile men, 2D:4D was associated with testicular volume (r=-0.36, p<0.001), total sperm number (r=-0.18, p=0.04) and time to pregnancy (r=0.24, p<0.02). In addition, participants with a history of epididymal cyst had a significantly higher 2D:4D than those without cysts. In conclusion, all significant findings indicate that the human male reproductive function is negatively related to 2D:4D. However, 2D:4D for testicular cancer patients does not point to a hormonal imbalance during foetal life as the common cause for developing germ-cell cancer. Such results obtained, thanks to an easy, direct and reliable method for measuring finger lengths, suggest the usefulness of this new tool in fertility studies as well as for studying men with developmental disorders of the reproductive tract.

[The French linguistic validation of the Ureteric Stent Symptom Questionnaire (USSQ)]. / Validation en langue française du questionnaire << Ureteric Stent Symptom Questionnaire >> (USSQ).

PURPOSE: Translation and linguistic validation of the French version of the Ureteral Stent Symptom Questionnaire (USSQ). MATERIALS AND METHODS: A double-back translation of the original Ureteral Stent Symptom Questionnaire was performed. First, two urologists translated the English version in French. Then a first consensus meeting between the translators and a group composed with three urologists, one general practitioner and two nurses was achieved. Back-translation of this version was then done by professional translators (Nagpal, Paris) to ensure that no distortion was detected between the two questionnaires. Finally, a pilot test followed by an interview was carried out among two men and two women who had an indwelling ureteral stent. RESULTS: The consensus version is attached to the article. No difficulties were reported by the pilot population to comprehend or to complete this USSQ French version. CONCLUSION: This USSQ version - attached to the article - makes it possible for researchers among a French population to use this validated and internationally recognized tool that provides reproducible and measurable endpoints on tolerance of ureteral stents.

Pierce's disease of grapevines: evidence for a bacterial etiology.

A Gram-positive, rod-shaped bacterium was isolated and grown in pure culture on artificial mediums from the leafhopper Draeculacephala minerva Ball which had fed on plants infected with Pierce's disease. The bacterial culture was injected into uncontaminated leafhoppers which were than allowed to feed on healthy grapevines. After exposure to these leafhoppers, typical symptoms of Pierce's disease developed; however, no symptoms developed on plants exposed to leafhoppers injected with sterile culture medium. The same organism was reisolated from the experimentally inoculated plants. Electron microscopic examination of these infected plants revealed bacterial cells localized in the xylem tissues. No such cells were seen in healthy or control plants.

Xiphinema rivesi from Chile Transmits Tomato ringspot virus to Cucumber.

The dagger nematode, (Xiphinema rivesi Dalmasso), a member of the X. americanum group, was first reported in 2002 in Chile (3). X. rivesi is a vector of at least four North American nepoviruses including Cherry rasp leaf virus (CRLV), Tobacco ringspot virus (TobRSV), Tomato ringspot virus (TomRSV), and Peach rosette mosaic virus (PRMV) (2). TomRSV, first reported in Chile in 1984, was associated with raspberry decline and lately with brownline disease in D'Agen prune trees (1), however none of the Xiphinema spp. found in Chile have been reported to transmit this nepovirus. Two virus isolates, TomRSV (prune brownline isolate PBL-08) and Grapevine fanleaf virus (GFLV) (Yellow mosaic isolate GFLV-012), from the virus collection of the Departamento de Sanidad Vegetal, Universidad de Chile were used in transmission tests with a population of X. rivesi found in Chile. X. rivesi is not known to transmit GFLV and this virus was included as a check. The nematodes were extracted from soil from a D'Agen prune orchard, and transmission tests were done in compliance with the criteria proposed by Trudgill et al. (4). Cucumis sativus cv. National Pickling were grown in a growth chamber at 25°C and used as acquisition hosts and transmission bait plants. Acquisition hosts were mechanically inoculated with GFLV or TomRSV, displaying systemic symptoms in 15 to 20 days. Noninoculated cucumber plants were included as controls. Virus infection was confirmed by double-antibody sandwich (DAS)-ELISA before the introduction of nematodes to the soil. After a 20-day acquisition feeding period, the nematodes were wet screened from the soil and added to the healthy bait plants and allowed a 20-day inoculation feeding period. X. rivesi transmitted TomRSV but not GFLV. TomRSV bait plants developed systemic symptoms 5 weeks after the nematodes were transferred. Transmission of TomRSV was confirmed by testing leaf and root sap of bait plants in a DAS-ELISA. High virus concentrations were detected in the roots and leaves of TomRSV symptomatic plants. Bait plants on which nematodes had been allowed to feed following virus acquisition from GFLV-infected or from virus-free control plants tested negative by ELISA. No symptoms appeared on bait plants used for GFLV transmission or the control bait plants. To our knowledge, this is the first report of transmission of TomRSV with a Xiphinema population from Chile and South America. References: (1) J. Auger. Acta Hortic. 235:197, 1988. (2) D. J. F. Brown et al. Phytopathology 84:646, 1994. (3) G. Leal et al. Fitopatología 37:75, 2002. (4) D. L. Trudgill et al. Rev. Nematol. 6:133, 1983.

First Report of Verticillium Wilt of Gold Kiwifruit, Actinidia chinensis Cv. Hort 16A, Caused by Verticillium albo-atrum in Chile.

Gold kiwifruit, Actinidia chinensis Planch cv. Hort 16A, was first planted in Chile in 2003 and vines started dying within 2 years. By the end of the 2007-2008 growing season, as much as 80% of the plants in several orchards had died. The disease was characterized by a conspicuous reddish brown discoloration of the xylem and the sudden wilting and dieback of plants any time during the growing season. In the spring, entire plants or parts of plants failed to break buds. In others, the buds broke, but juvenile leaf clusters then wilted and died. On severely affected plants, scion watershoots wilted and died. The disease was often accompanied by shallow cracking of the bark and slight sponginess of the underlying cortex. The disease was apparently most severe in sites that had been planted to Gold kiwifruit immediately after removal of apple, pear, citrus, or grape. Orchards planted following long-term maize, wheat, or grass culture were almost disease free. A fungus was consistently isolated from symptomatic vascular tissue disinfected in 1% sodium hypochlorite and plated on potato dextrose agar. Conidiogenous cells were arranged in verticels; conidia were hyaline, elliptical, single celled, and measured 3.5 to 8.5 × 1.8 to 4.3 µm (average 5.5 × 2.5 µm). Dark, resting mycelium developed after 1 to 2 weeks of incubation. On the basis of these morphological characteristics, the fungus was identified as Verticillium albo-atrum Reinke & Berthier. Identification was confirmed by sequencing part of the internal transcribed spacer (ITS) region with primers ITS1 and ITS4. The sequence of a representative isolate showed high homology (98% identity over a length of 494 bp) with a DNA fragment (NCBI Accession No. 108476) of V. albo-atrum from alfalfa. To complete pathogenicity tests, 20 healthy, 1-year-old Hort 16A kiwi vines grafted on Hayward kiwifruit (A. deliciosa Chevalier) seedlings were inoculated by injection of 20 µl of 106 conidia/ml into stems of the scion. Twenty control plants were injected with an equal volume of sterile distilled water. Plants were held in a controlled environment facility at 24°C with 16 h of light per day. Eight weeks after inoculation, typical wilting and dieback symptoms developed on 90% of the plants. Control plants injected with water remained healthy. Verticillium wilt has never been reported on kiwifruit (A. deliciosa) in Chile. V. albo-atrum has a rather narrow host range and is mainly reported as a pathogen on alfalfa, hop, soybean, tomato, and potato (1). To our knowledge, this is the first report of V. albo-atrum causing wilt and dieback on Gold kiwifruit (A. chinensis) cv. Hort 16A. The fungal isolates have been deposited in the Plant Pathology Laboratory of the Sanidad Vegetal Department of Agricultural Sciences Faculty of University of Chile under the name Actinidia chinensis/V. albo-atrum No. 1 to 8. Reference: (1) E. K. Ligoxigakis et al. Phytoparasitica 30:511, 2002.

A new LH receptor splice mutation responsible for male hypogonadism with subnormal sperm production in the propositus, and infertility with regular cycles in an affected sister.

BACKGROUND: Inactivating LH receptor (LHR) mutations have been described so far in men as well as in women. Phenotypes in men have been variable with in nearly all cases impairment of sex differentiation or azoospermia. We report a milder reproductive phenotype both in a male patient and his sister. METHODS AND RESULTS: We describe a family that carries a homozygous mutation G-->A at position -1 at the intron 10-exon 11 boundary of the LHR gene. The male patient presented with delayed puberty, micropenis and oligospermia. Two of his sisters were homozygous for the same mutation and were infertile. Surprisingly, one of them was found to have had regular ovarian cycles for years and showed normal LH values (6.5 and 10.6 mIU/ml for LH and FSH, respectively). In vitro analysis showed that this altered splicing resulted in an LHR from which eight amino acids are deleted from the extracellular domain (Delta Tyr(317)-Ser(324)). In vitro expression has shown that the receptor was expressed and capable of LH-induced signaling, albeit with reduced potency (P < 0.001). CONCLUSIONS: LHR mutations may represent an underestimated cause of infertility in women, in addition to being responsible for male hypogonadism with reduced spermatogenesis.

Psychological factors in male partners of infertile couples: relationship with semen quality and early miscarriage.

In this study we sought to evaluate whether psychological factors in males affect semen quality and pregnancy. In 1076 men of infertile couples, psychological factors, i.e. exposure to acute stress, coping with stress, the WHO (five) Well-Being Index and the Zung's Anxiety Scale Inventory scores were assessed by a questionnaire at the time of semen analysis. Relationships between psychological factors and semen quality (sperm concentration, rapid and progressive motility and normal morphology) were assessed. In 353 men with infertility duration of < or =1.5 years, sperm concentration > or =5 x 10(6) sperm/mL and a female partner with a laparoscopically confirmed tubal patency, we looked prospectively for relations between psychological factors and the occurrence of a natural pregnancy at a 6-month follow-up (n = 124), and first-trimester loss (n = 18). Anxiety trait, found in 19% of men, was related to previous in vitro fertilization/intracytoplasmic sperm injection attempts (p = 0.014), cigarette intake (p = 0.006), alcohol intake (p = 0.026) and sexual difficulties (p < 0.001). Regression analyses indicated a significant positive relationship between the level of sperm concentration and the WHO (five) Well-Being Index score, each successive score number accounting for a 7.3% increase in sperm concentration (p = 0.039), whereas no correlation was found between psychological factors and sperm rapid progressive motility and normal morphology. Poorer coping with stress was related to the occurrence of a first-trimester miscarriage (p = 0.016) in the female partner. Possible depression in males is related to decreased sperm concentration, and poor coping with stress is associated with increased occurrence of early miscarriage.

Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines.

BACKGROUND: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRgamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). OBJECTIVE: To determine why GPVI-FcRgamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. METHODS AND RESULTS: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcRgamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. CONCLUSION: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRgamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

BACKGROUND: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation. OBJECTIVE: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin). RESULTS: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model. CONCLUSIONS: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

A major role for Scar/WAVE-1 downstream of GPVI in platelets.

BACKGROUND: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex. OBJECTIVE: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization. METHODS AND RESULTS: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1. CONCLUSION: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.

Glycol ethers and semen quality: a cross-sectional study among male workers in the Paris Municipality.

OBJECTIVES: Apparent increases in human male reproductive disorders, including low sperm production, may have occurred because of increased chemical exposure. Various glycol ether-based solvents have pronounced adverse effects on sperm production and male fertility in laboratory animals. The authors investigated the effects of past and current exposure to glycol ether-containing products on semen quality and reproductive hormones among men employed by the Paris Municipality. METHODS: Between 2000 and 2001 the authors recruited 109 men who gave semen, blood and urine samples and underwent an andrological examination. Information on lifestyle, occupation, exposure and medical history was obtained by interview. According to their job and chemical products used during the period 1990-2000, men were classified as either occupationally exposed or non-exposed. Current exposure levels to glycol ethers at the time of the study were evaluated by biological monitoring of six urinary metabolites. RESULTS: Previous exposure to glycol ethers was associated with an increased risk for sperm concentration, for rapid progressive motility and for morphologically normal sperm below the World Health Organization semen reference values. No effect of previous glycol ether exposure on hormones levels was observed. By contrast, current glycol ether exposure levels were low and not correlated with either seminal quality or hormone levels. CONCLUSIONS: This study suggests that most glycol ethers currently used do not impact on human semen characteristics. Those that were more prevalent from the 1960s until recently may have long lasting negative effects on human semen quality.

First Report of Black Foot Disease of Grapevine Caused by Cylindrocarpon macrodidymum in Chile.

Black foot disease, caused by Cylindrocarpon macrodidymum Halleen, Schroers & Crous, is reported damaging table and wine grapes (Vitis vinifera L.) for the first time in Chile. During the summer of 2006, 2- to 5-year-old grapevines showed reduced vigor, shortened internodes, and drying and dying shoots along with abnormal development of roots with growth parallel to the soil surface, necrotic root crowns, and development of secondary roots. Internal necrosis extended from the bark to the pith in diseased parts of the plants. Other symptoms included black discoloration of the wood, gum inclusions in xylem vessels, black streaks in the vascular tissue, and reduction in root biomass, with sunken, necrotic root lesions. Eighteen Cylindrocarpon isolates were collected from roots, vascular elements, and pith tissue of grapevines cultivars (Flame Seedless, Red Globe, Thompson Seedless, Merlot, Carmenere, and Cabernet Sauvignon) from 12 locations in Chile. The isolates were identified on the basis of morphological features. All isolates produced micro- and macroconidia (one to three septa) and chlamydospores in short and intercalary chains (1,4), and by internal transcribed spacer (ITS1-5,8S-ITS4) rDNA and ß -tubulin (BT1, and BT2) partial sequences, identical to those of C. macrodidymum (isolate USS074, GenBank Accession No. AY 997558 and isolate USSO150, GenBank Accession No. AY 997598) (2). Phylogenetic analyses placed these isolates in a clade closely related, but clearly distinct from other clades, to C. destructans and C. liriodendri (2,3). Pathogenicity tests were completed by drench inoculation onto 50 6-month-old rooted cuttings of 'Red Globe' with 25 ml of conidia suspension (106 conidia ml-1) obtained from four isolates. Ten control cuttings of 'Red Globe' were inoculated with an equal volume of sterile distilled water. The plants were incubated for 4 months in a controlled environment facility at 24°C. All isolates tested were pathogenic. In addition, they caused significant root rot (t-test of disease incidence, P = 0.0048) and no significant level of variation was detected between different isolates. C. macrodidymum was reisolated from the region of brown streaking in all the inoculated cuttings and was not isolated from the water-treated controls. To our knowledge, this is the first report of C. macrodidymum causing black foot disease on grapevine in Chile. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1966. (2) F. Halleen et al. Stud. Mycol. 50:431, 2004. (3) F. R. Mantiri et al. Can. J. Bot. 79:334, 2001. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.

Effect of short-term exposure to two hydrophilic-coated and one gel pre-lubricated urinary catheters on sperm vitality, motility and kinematics in vitro.

AIM: This study aimed to determine the in vitro effect of a short-term exposure to two hydrophilic-coated and one gel pre-lubricated urinary catheters on human sperm quality. METHODS: Semen samples of various qualities were coincubated with each catheter for 5 min at 37 degrees C. The percentages of live and motile sperm with their kinematic characteristics were blindly assessed in control and treated samples at the end of the coincubation and 10 and 55 min later. RESULTS: The three catheters had no effect on sperm vitality. Similarly, the lubricated catheter and one hydrophilic-coated catheter negligibly modulated sperm motility. In contrast, the other hydrophilic-coated catheter tested had a significant negative effect on sperm movement. CONCLUSION: Further studies are warranted, the issue being especially relevant to the collection of spermatozoa in spinal cord diseased patients catheterizing themselves several times a day. In this population, compounds releasing from the catheter and accumulating in the urethra could be an additional factor contributing to the poor sperm quality.

First Report of Fenhexamid Resistant Isolates of Botrytis cinerea on Grapevine in Chile.

Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a haploid, filamentous ascomycete that causes gray mold on many economically important crops in temperate regions, especially grapevine. The management of gray mold on table grape in Chile involves cultural and chemical methods. Currently, protection programs are based on several fungicide families (dicarboximides, anilinopyrimidines, mixture of anilinopyrimidines and phenylpyrroles, and hydroxyanilides [fenhexamid]). During the last 25 years, B. cinerea developed resistance to virtually all specific fungicides used to control gray mold. Field resistance to benzimidazoles, phenylcarbamates, and dicarboximides was detected soon after their introduction. Recent studies using PCR-duplex and specific primers for the detection of transposable elements on Chilean B. cinerea isolates recovered from different table grape cultivars corroborated the presence of two sibling cryptic populations, transposa and vacuma (3). Some vacuma isolates have shown natural resistance to fenhexamide (HydR1) and it has been separated into two groups on a molecular basis using a marker gene (Bc-hch): Group I, fenhexamid-resistant vacuma isolates; Group II, vacuma and transposa isolates sensitive to this fungicide (HydS) (2). Group I and II isolates can not interbred (1,2). Other B. cinerea resistant phenotypes, HydR2 and HydR3, have been reported as belonging to Group II (1,4). Single-spore isolates of B. cinerea (472) were collected from different table grape cultivars from 13 locations in the Chilean Central Valley. The isolation was done during harvest time from rotting berries. Fenhexamid (Teldor; Bayer CropScience, Monheim, Germany) was diluted to 10 µg a.i./ml and added to the solid medium (10 g of glucose, 1.5 g of K2HPO4, 2 g of KH2PO4, 1 g of (NH4)2SO4, 0.5 g of MgSO4·H2O, 2 g of yeast extract, and 12.5 g of agar in 1 liter) to reach concentrations of 0, 0.025, 0.05, and 0.1 µg a.i./ml. A 5-mm mycelial plug from each isolate of B. cinerea was cut from the edge of 4-day-old colonies placed in the center of petri dishes with the described fungicide-amended medium and incubated at 20°C for 5 days. Two measurements, octogonal diameters, were taken from each of three replicates per treatment. Means were calculated and the diameter of the inoculated plug was subtracted from each mean. For each isolate, a linear regression of the percent inhibition of mycelial growth versus the Log10 transformation for each of the four concentrations of fenhexamid was obtained. The 50% effective concentration of fenhexamid (EC50) was calculated with the regression equation for each isolate. So, 95.3% of B. cinerea isolates were sensitive (EC50 under 0.083 µg/ml), 1.9% were less sensitive (EC50 between 0.084 and 0.1 µg/ml), and 2.8% (13 isolates) were resistant EC50 values ranging from 0.1 to 8.4 µg/ml. Through PCR-restriction fragment length polymorphism, according to the Bc-hch gene restriction pattern, all resistant isolates analyzed belong to Group II of B. cinerea (Bc-hch2) (2). To our knowledge, this is the first report of fenhexamid resistant isolates of B. cinerea on grapevine in Chile and South America. It would be necessary to study the population dynamics of these isolates, although failure of botrytis control in the field with this compound has not been reported. References: (1) C. Albertini et al. Mycol. Res. 106:1171, 2002. (2) E. Fournier et al. Mycologia 97:1251, 2005. (3) T. Giraud et al. Mol. Biol. Evol. 14:1177, 1997. (4) P. Leroux et al. Phytoma 599:31, 2006.