DNA fragmentation of sperm: a radical examination of the contribution of oxidative stress and age in 16 945 semen samples.

STUDY QUESTION: What is the relationship between sperm DNA fragmentation and oxidative stress (OS) with increasing male age? SUMMARY ANSWER: Sperm DNA fragmentation increases with age and is likely related to both defective spermatogenesis and increasing OS levels. WHAT IS KNOWN ALREADY: Sperm quality declines with age. The presence of DNA damage in a high fraction of spermatozoa from a raw semen sample is associated with lower male fertility in natural conception and intrauterine insemination. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of 16 945 semen samples analysed at a single reference laboratory between January 2010 and December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: All males were undergoing an infertility evaluation. The cohort was divided into seven age categories: <30, 30-34, 35-39, 40-44, 45-49, 50 to <54 and ≥55 years. The mean age was 37.6 years (SD 6.8). Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were calculated using flow cytometry. OS levels were measured using the oxidative stress adducts (OSA) test, by spectrophotometry. ANOVA with weighted polynomial contrast analysis was used to evaluate trends for DFI, OSA and HDS values across age categories. MAIN RESULTS AND THE ROLE OF CHANCE: Mean DFI significantly increased across all age groups (Ptrend < 0.001). OSA was lowest in patients <30 years old (mean 3.6, SD 1.0) and also increased as age increased (Ptrend < 0.001). There was a statistically significant difference between age groups for each of the three parameters (P < 0.001). There was a significant linear trend for DFI, OSA and HDS across the seven age categories (P < 0.001). Among patients with high DFI, there was a decreasing age-dependent trend in the patients observed with high OSA (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study. All males included in the study were undergoing a work-up for infertility and may not be representative of a fertile population. Additional patient demographics and clinical data were not available. WIDER IMPLICATIONS OF THE FINDINGS: DNA and/or oxidative damage in sperm may be just as important to understand as the chromosomal aberrations that are carried in the oocyte. Further studies are needed to evaluate the effect of advancing paternal age on the male genome and, ultimately, on the health of the offspring. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for this study. V.D. is an employee of Reprosource/Quest Diagnostics. D.S. reports he was a Scientific Advisor to Cooper Surgical. TRIAL REGISTRATION NUMBER: N/A.

Delivery outcomes for nulliparous women at the extremes of maternal age - a cohort study.

OBJECTIVE: To examine the associations between extremes of maternal age (≤17 years or ≥40 years) and delivery outcomes. DESIGN: Retrospective cohort study. SETTING: Urban maternity hospital in Ireland. POPULATION: A total of 36 916 nulliparous women with singleton pregnancies who delivered between 2000 and 2011. METHODS: The study population was subdivided into five maternal age groups based on age at first booking visit: ≤17 years, 18-19 years, 20-34 years, 35-39 years and women aged ≥40 years. Logistic regression analyses were performed to examine the associations between extremes of maternal age and delivery outcomes, adjusting for potential confounding factors. MAIN OUTCOME MEASURES: Preterm birth, admission to the neonatal unit, congenital anomaly, caesarean section. RESULTS: Compared with maternal age 20-34 years, age ≤17 years was a risk factor for preterm birth (adjusted odds ratio [adjOR] 1.83, 95% confidence interval [95% CI] 1.33-2.52). Babies born to mothers ≥40 years were more likely to require admission to the neonatal unit (adjOR 1.35, 95% CI 1.06-1.72) and to have a congenital anomaly (adjOR 1.71, 95% CI 1.07-2.76). The overall caesarean section rate in nulliparous women was 23.9% with marked differences at the extremes of maternal age; 10.7% at age ≤17 years (adjOR 0.46, 95% CI 0.34-0.62) and 54.4% at age ≥40 years (adjOR 3.24, 95% CI 2.67-3.94). CONCLUSIONS: Extremes of maternal age need to be recognised as risk factors for adverse delivery outcomes. Low caesarean section rates in younger women suggest that a reduction in overall caesarean section rates may be possible.

Genetic diversity of the rice bean (Vigna umbellata) genepool as assessed by SSR markers.

The genetic diversity of 472 rice bean accessions (388 cultivated and 84 wild) from 16 Asian countries was evaluated by 13 simple sequence repeat (SSR) markers. In total, 168 alleles were detected, and the numbers of alleles in cultivated and wild accessions were 129 and 132, respectively. The gene diversity in cultivated populations (0.565) was about 83% of that for wild (0.678) populations. Cultivated populations from Vietnam, Myanmar, Nepal, and India had the highest gene diversity (>0.5). East Asian accessions formed a distinct genepool. Indonesian cultivated accessions showed high genetic divergence from other cultivated populations and had the most similar genetic structure to wild accessions. In Nepalese cultivated accessions, many accessions from western regions were quite distinct from others and formed a specific group. These Nepalese accessions could be considered a unique gene source for rice bean breeding. In contrast, eastern Nepalese accessions showed an SSR profile similar to that of Southeast Asian rice beans. The present study represents the first comprehensive SSR analysis in cultivated and wild rice bean germplasm and clarifies geographical distribution of genetic profile that might be used to broaden the genetic base of currently grown rice bean cultivars.

Genetic diversity of the azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) gene pool as assessed by SSR markers.

To facilitate the wider use of genetic resources including newly collected cultivated and wild azuki bean germplasm, the genetic diversity of the azuki bean complex, based on 13 simple sequence repeat (SSR) primers, was evaluated and a core collection was developed using 616 accessions originating from 8 Asian countries. Wild germplasm from Japan was highly diverse and represented much of the allelic variation found in cultivated germplasm. The SSR results together with recent archaeobotanical evidence support the view that Japan is one center of domestication of azuki bean, at least for the northeast Asian azuki bean. Cultivated azuki beans from China, Korea, and Japan were the most diverse and were genetically distinct from each other, suggesting a long and relatively isolated history of cultivation in each country. Cultivated azuki beans from eastern Nepal and Bhutan were similar to each other and quite distinct from others. For two primers, most eastern Nepalese and Bhutanese cultivated accessions had null alleles. In addition, wild accessions from the Yangtze River region of China and the Himalayan region had a null allele for one or the other of these primers. Whether the distinct diversity of azuki bean in the Himalayan region is due to introgression or separate domestication events requires further study. In contrast, western Nepalese azuki beans showed an SSR profile similar to that of Chinese azuki beans. The genetic distinctness of cultivated azuki beans from Vietnam has been revealed for the first time. The specific alleles indicate that Vietnamese azuki beans have been cultivated in isolation from Chinese azuki beans for a long time. Wild germplasm from the Himalayan region showed the highest level of variation. Based on the results, Himalayan germplasm could be considered a novel gene source for azuki bean breeding. A comparison with mungbean SSR analysis revealed that the mean gene diversity of cultivated azuki bean (0.74) was much higher than that of cultivated mungbean (0.41). The reduction in gene diversity due to domestication, the domestication bottleneck, in azuki bean is not strong compared with that in mungbean.

From crop domestication to super-domestication.

Research related to crop domestication has been transformed by technologies and discoveries in the genome sciences as well as information-related sciences that are providing new tools for bioinformatics and systems' biology. Rapid progress in archaeobotany and ethnobotany are also contributing new knowledge to understanding crop domestication. This sense of rapid progress is encapsulated in this Special Issue, which contains 18 papers by scientists in botanical, crop sciences and related disciplines on the topic of crop domestication. One paper focuses on current themes in the genetics of crop domestication across crops, whereas other papers have a crop or geographic focus. One feature of progress in the sciences related to crop domestication is the availability of well-characterized germplasm resources in the global network of genetic resources centres (genebanks). Germplasm in genebanks is providing research materials for understanding domestication as well as for plant breeding. In this review, we highlight current genetic themes related to crop domestication. Impressive progress in this field in recent years is transforming plant breeding into crop engineering to meet the human need for increased crop yield with the minimum environmental impact - we consider this to be 'super-domestication'. While the time scale of domestication of 10 000 years or less is a very short evolutionary time span, the details emerging of what has happened and what is happening provide a window to see where domestication might - and can - advance in the future.

Development of a black gram [Vigna mungo (L.) Hepper] linkage map and its comparison with an azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] linkage map.

The Asian Vigna group of grain legumes consists of six domesticated species, among them black gram is widely grown in South Asia and to a lesser extent in Southeast Asia. We report the first genetic linkage map of black gram [Vigna mungo (L.) Hepper], constructed using a BC(1)F(1) population consisting of 180 individuals. The BC(1)F(1) population was analyzed in 61 SSR primer pairs, 56 RFLP probes, 27 AFLP loci and 1 morphological marker. About 148 marker loci could be assigned to the 11 linkage groups, which correspond to the haploid chromosome number of black gram. The linkage groups cover a total of 783 cM of the black gram genome. The number of markers per linkage group ranges from 6 to 23. The average distance between adjacent markers varied from 3.5 to 9.3 cM. The results of comparative genome mapping between black gram and azuki bean show that the linkage order of markers is highly conserved. However, inversions, insertions, deletions/duplications and a translocation were detected between the black gram and azuki bean linkage maps. The marker order on parts of linkage groups 1, 2 and 5 is reversed between the two species. One region on black gram linkage group 10 appears to correspond to part of azuki bean linkage group 1. The present study suggests that the azuki bean SSR markers can be widely used for Asian Vigna species and the black gram genetic linkage map will assist in improvement of this crop.

Population genetic structure of Japanese wild soybean (Glycine soja) based on microsatellite variation.

The research objectives were to determine aspects of the population dynamics relevant to effective monitoring of gene flow in the soybean crop complex in Japan. Using 20 microsatellite primers, 616 individuals from 77 wild soybean (Glycine soja) populations were analysed. All samples were of small seed size (< 0.03 g), were directly collected in the field and came from all parts of Japan where wild soybeans grow, except Hokkaido. Japanese wild soybean showed significant reduction in observed heterozygosity, low outcrossing rate (mean 3.4%) and strong genetic differentiation among populations. However, the individual assignment test revealed evidence of rare long-distance seed dispersal (> 10 km) events among populations, and spatial autocorrelation analysis revealed that populations within a radius of 100 km showed a close genetic relationship to one another. When analysis of graphical ordination was applied to compare the microsatellite variation of wild soybean with that of 53 widely grown Japanese varieties of cultivated soybean (Glycine max), the primary factor of genetic differentiation was based on differences between wild and cultivated soybeans and the secondary factor was geographical differentiation of wild soybean populations. Admixture analysis revealed that 6.8% of individuals appear to show introgression from cultivated soybeans. These results indicated that population genetic structure of Japanese wild soybean is (i) strongly affected by the founder effect due to seed dispersal and inbreeding strategy, (ii) generally well differentiated from cultivated soybean, but (iii) introgression from cultivated soybean occurs. The implications of the results for the release of transgenic soybeans where wild soybeans grow are discussed.

A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi].

To make progress in genome analysis of azuki bean (Vigna angularis) a genetic linkage map was constructed from a backcross population of (V. nepalensis x V. angularis) x V.angularis consisting of 187 individuals. A total of 486 markers-205 simple sequence repeats (SSRs), 187 amplified fragment length polymorphisms (AFLPs) and 94 restriction fragment length polymorphisms (RFLPs) -were mapped onto 11 linkage groups corresponding to the haploid chromosome number of azuki bean. This map spans a total length of 832.1 cM with an average marker distance of 1.85 cM and is the most saturated map for a Vigna species to date. In addition, RFLP markers from other legumes facilitated finding several orthologous linkage groups based on previously published RFLP linkage maps. Most SSR primers that have been developed from SSR-enriched libraries detected a single locus. The SSR loci identified are distributed throughout the azuki bean genome. This moderately dense linkage map equipped with many SSR markers will be useful for mapping a range of useful traits such as those related to domestication and stress resistance. The mapping population will be used to develop advanced backcross lines for high resolution QTL mapping of these traits.

The development of SSR markers by a new method in plants and their application to gene flow studies in azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi].

To gain a better understanding of wild and weedy azuki population structures in relation to the cultigens we have developed simple sequence repeat (SSR) markers based on a new methodology for plant material. In the azuki bean genome, the number of (AG)(n) and (AC)(n) motif loci per haploid genome has been estimated to be 3,500 and 2,100, respectively, indicating that (AG)(n) motifs are a rich source of markers. We constructed a (AG)(n)-SSR-enriched library in azuki bean in order to obtain a comprehensive range of SSR markers efficiently. The method applied in this study resulted in a 116-fold enrichment over the non-enriched genomic library, with a high percentage (98%) of successful single-locus amplification by the primer pairs designed. Consequently, this method can be applied to construct SSR-enriched libraries suitable for large-scale sequencing. We obtained 255 unique sequences from an (AG)(n)-enriched library for azuki bean. Fifty primer pairs were designed and screened against five populations of wild azuki bean. Among these five populations, one population from Bato town, Tochigi prefecture, Japan, showed greater polymorphism using these primers than the others and was therefore chosen for the in-depth study. The genotypes of 20 individuals were investigated using eight of the SSR primers developed. The genetic relationships among individuals revealed a complex spatial pattern of population structure. Although azuki bean is considered to be a predominantly self-pollinating species, 3 of the 20 individuals tested in the population showed heterozygous genotypes, indicating outcrossing. Allele size and DNA sequence in each of the 20 individuals were compared with those of landraces and released cultivars of azuki bean. Plants in part of the population had many alleles of the same size and with the same sequence as those in cultivated azuki bean, suggesting that gene flow from the cultigen to wild plants has occurred in this population. Unintentional transgene escape from azuki could therefore occur when transgenic azuki is grown in areas where its wild and weedy relatives occur. The approach used here could be applied to biosafety monitoring of transgenic azuki bean.

Characterization of resistance to three bruchid species (Callosobruchus spp., Coleoptera, Bruchidae) in cultivated rice bean (Vigna umbellata).

Resistance of wild and cultivated rice bean (Vigna umbellata [Thunberg] Ohwi and Ohashi) to three bruchid species, Callosobruchus chinensis L., Callosobruchus maculatus F., and Callosobruchus analis F., was evaluated. All but three accessions of cultivated, and all wild rice bean accessions tested, exhibited complete resistance to all three bruchid species. Rice bean seeds with seed coat removed also showed complete resistance to the three bruchid species. Results indicate that physical attributes and/or chemical(s) present in the seed coat of rice bean are not the main factors responsible for resistance. Feeding tests were performed by using artificial beans prepared with varying proportions of rice bean (resistant) and azuki bean (susceptible) flour. Number of bruchid adults that emerged decreased, and larval developmental period (days) was extended, when artificial beans with an increasing proportion of rice bean flour were used. These tests revealed that a chemical compound(s) contained in the cotyledon of rice bean has an inhibitory effect on the growth of these bruchid species. The results also indicate that the chemical(s) in rice bean cotyledon is most effective against C. maculatus.

Molecular phylogeny of genus Vigna subgenus Ceratotropis based on rDNA ITS and atpB-rbcL intergenic spacer of cpDNA sequences.

The genetic diversity and phylogenetic relationships among species in the genus Vigna subgenus Ceratotropis were investigated using sequence data from the ribosomal DNA ITS and atpB-rbcL intergenic spacer of chloroplast DNA regions. While both sets of sequences were of similar lengths about 700 bp the rDNA-ITS was more informative than atpB-rbcL having 170% more polymorphic sites and five times as many parsimony-informative sites. The atpB-rbcL spacer may be appropriate for analysis of taxa above the species level in the genus Vigna. Results of analyzing rDNA-ITS revealed, with low level of statistical bias, separation of the subgenus into three groups that correspond to the three sections Aconitifoliae, Angulares, and Ceratotropis. The ancestral section is Aconitifoliae based on comparison with the outgroup species cowpea, Vigna unguiculata. The V minima complex, V minima, V riukiuensis, and V nakashimae, has a distinct evolutionary path within section Angulares. Other species in section Angulares are very closely related except V trinervia. Vigna trinervia has an intermediate position between sections. Sequence data suggests one genome donor to V reflexo-pilosa came from a lineage within section Angulares close to V exilis, V hirtella, and V umbellata. Data presented supports the view that section Angulares is the most recently diversified section in the subgenus, as inferred by short terminal branch lengths among the species of this section.

Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis complex.

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 x 10(4) in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex.

Diversity in the integrase coding domain of a gypsy-like retrotransposon among wild relatives of rice in the Oryza officinalis complex.

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75-90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex.

Dental health issues in child-care centers.

In 1996, the privately owned licensed child day-care facilities in West Virginia were surveyed by mail to determine the level of compliance with the oral health regulations set by the State Board of Health. The number of children enrolled in the 119 responding facilities ranged from three to 218. Of these, 63 percent included tooth-brushing in the children's daily schedule. The majority of respondents (72%) reported inviting dental health care providers to discuss dental health with the enrollees. However, it was less commor (29%) for dental care providers to address the child-care staff regarding children's dental health. Forty percent of respondents reported that children had sustained dental injuries while at their facility. Nearly one-half (46%) maintained a record of each child's dentist. These findings support the need for establishing partnerships between dental health care providers and child-care centers to initiate continuous dental health programs for enrollees and to develop periodic staff educational workshops that focus on children's dental health. This study was funded by the West Virginia University Dental Corporation.

Need for intravenous hydrocortisone in addition to oral prednisolone in patients admitted to hospital with severe asthma without ventilatory failure.

52 severely ill asthmatic patients requiring acute admission to hospital entered a double-blind placebo-controlled trial to determine whether intravenous hydrocortisone given in addition to high-dose oral prednisolone and standard bronchodilator therapy accelerated recovery. Patients who had been given parenteral steroids before admission, by comparison with those who had not received such treatment, had been deteriorating for a shorter period before admission, had received more injected or nebulised bronchodilator therapy, and had higher admission peak flows. As judged by peak flow measurements 24 h after admission, parenteral steroids had no effect on the outcome, irrespective of whether they were given before or after (ie, intravenous hydrocortisone) admission. There is no evidence for the continued use of intravenous hydrocortisone in addition to oral prednisolone and bronchodilator therapy in patients admitted to hospital with severe asthma without ventilatory failure.