Evolutionary dynamics of West Nile virus in Georgia, 2001-2011.

From 1999-2001, West Nile virus (WNV) spread throughout the eastern United States (US) and was first detected in Georgia in 2001. To date, the virus has been detected in over 2,500 dead wild bird and mosquito samples from across Georgia. We sequenced the premembrane (preM) and envelope gene (E) (2004 bp) from 111 isolates collected from 2001 to 2011. To assess viral gene flow from other geographic regions in the US, we combined our data with WNV sequences available at the National Center for Biotechnology Information (NCBI) and performed phylogenetic analysis. We found evidence that WNV isolates detected in Chatham County Georgia most likely originated from the Northeastern United States. These results highlight the growing importance of adequate genetic surveillance for monitoring and controlling viruses of public health concern.

Multilocus estimation of selfing and its heritability.

We describe a new method of estimating the selfing rate (S) in a mixed mating population based on a population structure approach that accounts for possible intergenerational correlation in selfing rate, giving rise to an estimate of the upper limit for heritability of selfing rate (h2). A correlation between generations in selfing rate is shown to affect one- and two-locus probabilities of identity by descent. Conventional estimates of selfing rate based on a population structure approach are positively biased by intergenerational correlation in selfing. Multilocus genotypes of individuals are used to give maximum-likelihood estimates of S and h2 in the presence of scoring artifacts. Our multilocus estimation of selfing rate and its heritability (MESH) method was tested with simulated data for a range of conditions. Selfing rate estimates from MESH have low bias and root mean squared error, whereas estimates of the heritability of selfing rate have more uncertainty. Increasing the number of individuals in a sample helps to reduce bias and root mean squared error more than increasing the number of loci of sampled individuals. Improved estimates of selfing rate, as well as estimates of its heritability, can be obtained with this method, although a large number of loci and individuals are needed to achieve best results.

[The influence of diabetes mellitus on male reproductive function: a poorly investigated aspect of male infertility]. / Einfluss des Diabetes mellitus auf die Fertilität des Mannes : Ein wenig untersuchter Faktor der männlichen Infertilität.

Whilst diabetes mellitus is known to have many systemic complications, male infertility, beyond impotence, retrograde ejaculation and hypogonadism, has not been widely recognised to be one of them. Due to the paucity of studies and inconsistencies regarding the condition's impact on semen quality, few fertility specialists consider the condition noteworthy. As a consequence little information exists as to its prevalence amongst infertile men. Recently the prevailing view has been challenged by findings showing that diabetes induces subtle molecular changes that are important for sperm quality and function. Diabetic men have been found to have a significantly higher percentage of sperm with nuclear DNA damage, a factor known to be associated with compromised fertility and increased miscarriage rates. The mechanism by which this diabetes-related sperm nDNA damage occurs remains unknown. The identification of high levels of advanced glycation end products (AGEs) and their receptor (RAGE) throughout the male reproductive tract coupled to changes in testicular metabolite levels and spermatogenic gene expression suggest that glycation may play an integral role in oxidative stress which in turn causes sperm nDNA damage. As glycation is a normal consequence of life and has been implicated in DNA fragmentation in a variety of seemingly unconnected conditions, it may constitute a common mechanism for the damage seen in sperm DNA.

Differences in mouse models of diabetes mellitus in studies of male reproduction.

Diabetes Mellitus (DM) has been found to have subtle yet profound effects on the metabolic status of the testis, the expression of numerous spermatogenic genes and is associated with increased numbers of sperm with nuclear DNA damage. The precise mechanism causing these detrimental effects remains unknown. The presence of increased levels of the most prominent member (carboxymethyllysine - CML) of the advanced glycation end product adducts and their receptor (RAGE) in the reproductive tract of DM men has provided a new avenue for research. As there are suspicions that the antibiotic (streptozotocin - STZ) employed to induce DM is also capable of causing oxidative stress and DNA damage, we compared CML and RAGE levels in the reproductive tract and sperm nDNA status of STZ mice with the levels in the Ins(2Akita) mouse to determine which more closely mimics the situation described in the human diabetic. CML was observed in the testes, epididymes and sperm of all animals. Sperm from DM mice showed particularly strong CML immunolocalization in the acrosomal cap, the equatorial region and whenever present, cytoplasmic droplets. Although increased, the level of CML on the sperm of the STZ and Ins(2Akita) DM mice did not reach statistical significance. RAGE was present on the developing acrosome and epididymal sperm of all animals and in discrete regions of the epididymes of the DM models. Only the epididymal sperm of the Ins(2Akita) mice were found to have significantly increased (p < 0.0001) nDNA damage. The Ins(2Akita) mouse therefore appears to more accurately reflect the conditions found in the human and, as such, is a more representative model for the study of diabetes and glycation's influence on male fertility.

Metabolic profile changes in the testes of mice with streptozotocin-induced type 1 diabetes mellitus.

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the effect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin-induced diabetic C57BL/6 mice, 2, 4 and 8 weeks post-treatment. Diabetic status was confirmed by glycated haemoglobin, non-fasting blood glucose, physiological condition and body weight. A novel extraction procedure was utilized to obtain protein free, low-molecular weight, water soluble extracts which were then assessed using (1)H nuclear magnetic resonance spectroscopy. Principal component analysis of the derived profiles was used to classify any variations, and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and type 1 diabetic animals with the most distinctive being from mice with the largest physical deterioration and loss of body weight. Eight streptozotocin-treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in a few animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognized manner.

Advanced glycation end products accumulate in the reproductive tract of men with diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

A comparison of spontaneously conceived twins and twins conceived by artificial reproductive technologies.

Evidence on pregnancy outcomes of twins conceived by artificial reproductive technology (ART) compared with those naturally conceived (NC) is conflicting. We retrospectively audited outcomes of ART and NC twin births at Royal Maternity Hospital, Belfast from 01.01.2002-31.12.2003. Of 202 twins, 53 (26%) were ART and 149 (74%) were NC. ART group had increased maternal age (p < 0.001), more primiparous mothers (p < 0.001), used more peri-conceptual folic acid (p = 0.01), booked earlier for antenatal care (p < 0.05), delivered by caesarean section (p = 0.035) more often at a later gestation (p = 0.048) with a higher birth weight (p < 0.05). The NC group had increased congenital anomalies (p = 0.005) and babies requiring neonatal intensive care (p = 0.003). Of confirmed cases, ART twins were all dichorionic compared with 81.7% of NC (p = 0.002). Most neonatal outcome differences disappeared when NC dichorionic twins were compared with ART twins. ART twins have fewer complications than NC twins. Chorionicity accounts for most of these differences.

Increased concentrations of the oxidative DNA adduct 7,8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes.

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 10(5) molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 10(5) molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Insulin dependant diabetes mellitus: implications for male reproductive function.

BACKGROUND: Diabetes mellitus (DM) is increasing in men of reproductive age. Despite this, the prevalence of diabetes in men attending fertility clinics is largely unknown. Furthermore, studies examining the effects of DM on sperm fertility potential have been limited to conventional semen analysis. METHODS: Conventional semen analysis (semen volume, sperm count, motility and morphology) was performed for 27 diabetic (mean age 34+/-2 years) and 29 non-diabetic subjects (control group, men undergoing routine infertility investigations, mean age 33+/-1 years). Nuclear DNA (nDNA) fragmentation was assessed using the alkaline Comet assay and mitochondrial DNA (mtDNA) deletions by Long-PCR. RESULTS: Other than a small, but significant, reduction in semen volume in diabetic men (2.6 versus 3.3 ml; P<0.05), conventional semen parameters did not differ significantly from control subjects. Diabetic subjects had significantly higher mean nDNA fragmentation (53 versus 32%; P<0.0001) and median number of mtDNA deletions (4 versus 3; P<0.05) compared with control subjects. CONCLUSIONS: Diabetes is associated with increased sperm nuclear and mtDNA damage that may impair the reproductive capability of these men.

Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI.

BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with ICSI. The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with yields of fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5 years previously or had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells and spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS AND CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomized men compared with fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons.

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.

An algorithm to predict pregnancy in assisted reproduction.

BACKGROUND: Male fertility potential cannot be measured by conventional parameters for the assisted reproduction technique; ICSI. This study determines the relationship between testicular and ejaculated sperm mitochondrial (mt) DNA deletions, nuclear (n) DNA fragmentation, and fertilization and pregnancy rates in ICSI. METHODS: Ejaculated sperm were obtained from 77 men and testicular sperm from 28 men with obstructive azoospermia undergoing ICSI. Testicular sperm were retrieved using a Trucut needle. mtDNA was analysed using a long PCR. The alkaline Comet assay determined nDNA fragmentation. RESULTS: Of subjects who achieved a pregnancy (50%) using testicular sperm, only 26% had partners' sperm with wild-type (WT) mtDNA. Of pregnant subjects (38%) using ejaculated sperm, only 8% had partner sperm with WT mtDNA. In each, the successful group had less mtDNA deletions and less nDNA fragmentation. There were inverse relationships between pregnancy and mtDNA deletion numbers, size and nDNA fragmentation for both testicular and ejaculated sperm. No relationships were observed with fertilization rates. An algorithm for the prediction of pregnancy is presented based on the quality of sperm nDNA and mtDNA. CONCLUSION: In both testicular and ejaculated sperm, mtDNA deletions and nDNA fragmentation are closely associated with pregnancy in ICSI.

The influence of the chemical composition of drinking water on cuprosolvency by biofilm bacteria.

AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.

Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm.

BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia.

BACKGROUND: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. METHODS: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men undergoing vasectomy (n = 11) and testicular sperm from men with obstructive azoospermia (n = 25). Nuclear DNA (nDNA) fragmentation was measured by an alkaline gel electrophoresis (comet) assay. RESULTS: Wild-type mtDNA was detected in only 60% of fertile men's testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different, but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (P < 0.02). NDNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. CONCLUSION: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia, the mtDNA deletions in testicular sperm are of a larger scale.

The effects of cryopreservation on sperm morphology, motility and mitochondrial function.

BACKGROUND: The effects of cryoinjury were determined simultaneously on the mitochondrial function, motility, morphology and viability of ejaculated human sperm. METHOD: Rhodamine 123 (R123) uptake (% of sperm) and stain intensity were used to determine sperm mitochondrial activity before and after cryopreservation from the semen of 50 men attending for infertility investigation. Morphology was assessed using Tygerberg's strict criteria and viability was assessed by eosin Y. Sperm motility was measured using computer-assisted semen analysis (CASA). RESULTS: Freeze-thawing caused a 37% (P = 0.001) reduction in normal morphological forms of sperm. All CASA sperm motility parameters except amplitude of lateral head displacement were similarly reduced. R123 uptake and intensity within sperm mitochondria decreased by 36 and 47% respectively (both P = 0.001). In addition, there was a similar significant decrease (31%, P = 0.001) in the viability of the sperm. CONCLUSIONS: Sperm morphology, motility, mitochondrial activities and viability are equally susceptible to cryopreservation-induced damage. R123 intensity is a novel and robust indicator of mitochondrial function before and after such trauma.

Virtual reality training leads to faster adaptation to the novel psychomotor restrictions encountered by laparoscopic surgeons.

BACKGROUND: The fulcrum effect of the body wall on instrument handling poses a major obstacle to the mastery of instrument coordination for junior laparoscopic surgeons. This study evaluated three types of laparoscopic simulator training to assess their ability to promote the user's adaptation to the fulcrum effect. METHODS: Thirty-two participants with no previous experience in laparoscopic surgery were randomly assigned to one of four groups representing different training conditions. One group was assigned to use a virtual reality simulator (MIST VR); two others were given a laparoscopic Z or U maze-tracking task. The control group received no training. Subjects were asked to perform a 2-min laparoscopic cutting task under normal laparoscopic imaging conditions first before and then after training. RESULTS: In the test trial, subjects who trained on MIST VR made significantly more correct incisions (p < 0.0001) and fewer incorrect incisions (p < 0.0001). CONCLUSION: Training on a virtual reality simulator such as MIST VR helps laparoscopic novices adapt to the fulcrum effect faster.

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity.

OBJECTIVE: To investigate effects of cryopreservation on sperm motility and DNA integrity. DESIGN: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. SETTING: A hospital andrology laboratory. PATIENT(S): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. INTERVENTION(S): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. MAIN OUTCOME MEASURE(S): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. RESULT(S): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. CONCLUSION(S): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Biofilms and microbially influenced cuprosolvency in domestic copper plumbing systems.

AIMS: To survey biofilm accumulation within domestic copper plumbing pipes in South Australian drinking water distribution systems and examine its role in copper solvation (cuprosolvency). METHODS AND RESULTS: Cold water copper pipes were sampled from two different plumbing systems receiving filtered and unfiltered potable water respectively. Biomass was quantified by total organic carbon measurements and viable cell counts and microbial activity by respirometry. Biofilm accumulation was related to water chemistry within the systems, particularly nutrients, alkalinity and conductivity, as well as water turbulence. Laboratory coupon experiments were used to determine the effect of extracted biofilm on copper solvation. Biofilms were shown to be capable of both increasing and decreasing aqueous copper concentrations in comparison to sterile controls. CONCLUSIONS: The results suggest that water quality may influence the accumulation of biofilms in copper plumbing systems, as well as potential cuprosolvency activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of biofilms in copper plumbing systems and their ability to influence aqueous copper concentrations has implications for both public health and the management of distribution systems.