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.

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.

Metabolic clearance rate of dehydroepiandrosterone sulfate, its metabolism to testosterone, and its intrafollicular metabolism to dehydroepiandrosterone, androstenedione, testosterone, and dihydrotestosterone in vivo.

At the time of surgery, women were infused with [3H]dehydroepiandrosterone sulfate ([3H]DS)/[14C]testosterone ([14C]T) for 6 h; blood samples were obtained from an artery the ovarian veins, and a peripheral vein; and fluid was obtained from ovarian follicles. Both blood and follicular fluid samples were analyzed for radioactivity as DS, dehydroepiandrosterone (D), androstenedione (delta 4A). T, and dihydrotestosterone (DHT), and the blood was also analyzed for the concentration of nonisotopic DS by RIA. In other subjects the concentrations of D and DS were measured in paired samples of blood and follicular fluid. From these data, values of 13.6 +/- 0.69 L/day four (mean +/- SE; n = 4) for MCRDS, 607 +/- 90 L/day (n = 3) for MCRT, and 0.0190 +/- 0.0089 (n = 3) for [p]DS-T (fraction of plasma DS metabolized to plasma T) were obtained. The ratio of the concentration of the tracer-labeled steroid in the follicular fluid to the concentration in the arterial plasma sample was elevated significantly above 1 for three 3H-labeled and three [14C-labeled metabolites: [3H]D (21-fold; P less than 0.001), [3H]T (81-fold; P less than 0.001), [3H]DHT (19-fold; P less than 0.001), [14C]T (4-fold; P less than 0.025), [14C]DHT (21-fold; P less than 0.01), and [14C]delta 4A (50-fold; P less than 0.001). The estimated concentrations of steroids in follicular fluid derived from DS based on specific activity calculations were as follows: [geometric mean (95% confidence limits; n)]: DS, 5600 (4800-6500 nmol/L; 12); D, 370 (88-1500 nmol/L; 10); delta 4 A, 120 (67-220 nmol/L; 12); T, 130 (39-450 nmol/L; 10); and DHT, 64 (35-120 nmol/L; 8). Comparison of these data to known follicular fluid steroid concentrations shows that DS from the intravascular pool can be used as an ovarian prehormone.

Construction of GFP vectors for use in gram-negative bacteria other than Escherichia coli.

A set of vectors containing a mutated gfp gene was constructed for use with Gram-negative bacteria other than Escherichia coli. These constructs were: pTn3gfp for making random promoter probe gfp insertions into cloned DNA in E. coli for subsequent introduction into host strains; pUTmini-Tn5gfp for making random promoter probe gfp insertions directly into host strains; p519gfp and p519nfp, broad host range mob+ plasmids containing gfp expressed from a lac and an npt2 promoter, respectively.

A comparison of the morphology of testicular, epididymal, and ejaculated sperm from fertile men and men with obstructive azoospermia.

OBJECTIVE: To assess the morphology of testicular, epididymal, and ejaculated sperm. DESIGN: Morphology of the three types of sperm was assessed by using Tygerberg strict criteria. SETTING: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S): Thirty-two men with obstructive azoospermia and 10 fertile men. INTERVENTION(S): Trucut needle testicular biopsy and percutaneous epididymal sperm aspiration under local anesthetic. MAIN OUTCOME MEASURE(S): Percentages of normal sperm and sperm with head, midpiece, and tail defects for testicular, epididymal, and ejaculated sperm. Testicular sperm morphology in men with obstructive azoospermia was compared with that of fertile men. RESULT(S): The percentage of normal testicular sperm (4.3%) differed significantly from the percentages of normal epididymal (10. 8%) and ejaculated sperm (9.6%). Testicular sperm morphology in men with obstructive azoospermia did not differ from that in fertile men. CONCLUSION(S): Tygerberg strict criteria are not suitable for the assessment of testicular sperm morphology.

Comparison of the effects of two methods of cryopreservation on testicular sperm DNA.

OBJECTIVE: To assess the effects of two methods of freezing on testicular sperm DNA from subjects with obstructive azoospermia and to compare these with samples of fresh and freeze-thawed testicular sperm from fertile men. DESIGN: The Comet assay was used to determine the percentage of undamaged DNA in fresh testicular sperm, testicular sperm freeze-thawed in suspension and in a biopsy sample (men with obstructive azoospermia), and in fresh and freeze-thawed testicular sperm (fertile men). SETTING: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S): Twelve males with obstructive azoospermia (normal testicular volume and hormone profiles) and nine fertile control subjects. INTERVENTION(S): Trucut needle testicular biopsy under local anesthetic. MAIN OUTCOME MEASURE(S): Measurement of the percentage of undamaged DNA in fresh and freeze-thawed testicular sperm using the Comet assay. RESULT(S): No significant difference was found between the percentage of undamaged DNA of fresh testicular sperm and of both types of freeze-thawed testicular sperm. There was also no significant difference between the percentage of undamaged DNA in fresh or freeze-thawed testicular sperm from controls. Control ejaculated sperm DNA was significantly more damaged than testicular sperm DNA from control men. CONCLUSION(S): Cryopreservation of testicular sperm does not increase baseline levels of DNA damage.

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.

Antioxidant supplementation in vitro does not improve human sperm motility.

OBJECTIVE: To determine the effects of supplementation of preparation media with ascorbate and alpha-tocopherol on subsequent sperm motility and reactive oxygen species production. DESIGN: Prospective study to analyze postpreparation human sperm motility parameters and reactive oxygen species production following antioxidant supplementation. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. PATIENT(S): Sixty patients attending the Andrology Laboratory for semen analysis. INTERVENTION(S): Normozoospermic and asthenozoospermic semen samples (n = 10 for each control and antioxidant group) were prepared by Percoll density centrifugation (95%-47.5%) in media supplemented with ascorbate or alpha-tocopherol to different concentrations within physiologic levels. Controls were included that were not exposed to antioxidant. MAIN OUTCOME MEASURE(S): Sperm motility parameters were assessed using computer-assisted semen analysis. The generation of reactive oxygen species was determined using luminol-dependent chemiluminescence. RESULT(S): The production of reactive oxygen species by sperm was reduced by supplementation in vitro with ascorbate and alpha-tocopherol. However, progressive motility, average path velocity, curvilinear velocity, straight-line velocity, and linearity were decreased significantly, with the greatest inhibition observed with the highest concentrations of antioxidants. CONCLUSION(S): Supplementation of preparation media with ascorbate and alpha-tocopherol, either singly or in combination, is not beneficial to sperm motility.

Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle.

OBJECTIVE: To determine the presence and localization of leukocyte subsets in the human follicular wall and in the corpus luteum (CL) throughout the menstrual cycle. DESIGN: Tissue was obtained by biopsy at laparoscopy. Frozen sections were labeled with a panel of monoclonal antibodies against leukocyte subtype markers and detected by immunohistochemical methods. SETTING: Infertility clinic in large teaching hospital. PATIENTS: Eighteen women who underwent diagnostic laparoscopy. Women with abnormal endocrinology, endometriosis, or ovarian disease were excluded. RESULTS: Macrophages and neutrophilic granulocytes were present in high numbers in the collagen-rich tissues, including the thecal layer, of the follicle wall. At ovulation, there was a marked increase in the density of these cells in the follicle wall, especially in the thecal layer. In the CL, there was a higher density of macrophages (2- to 3-fold) and neutrophils (3- to 5-fold) in the theca lutein area compared with the granulosa lutein area. There were no significant changes in the density of these cell types when early and late luteal phase were compared, but the density of macrophages were markedly elevated in regressing CLs obtained during the subsequent follicular phase. T lymphocytes were present in low numbers in the follicle wall and in the CL, and no variation in the density was detected throughout the menstrual cycle. CONCLUSION: Macrophages and neutrophilic granulocytes are present in high numbers in the follicle wall and are further increased in the theca at ovulation, suggesting an active role for these leukocyte subtypes in tissue remodeling during the ovulatory process. Macrophages and neutrophils are also abundant in the CL, and an increase in the macrophage density at CL regression could imply a role for these cells in luteolysis.

Ultrasound, antisperm antibody, and hormone profiles after testicular Trucut biopsy.

OBJECTIVE: To assess the effect of Trucut needle biopsy on the ultrasound appearances of the testis in obstructive and nonobstructive azoospermia to test serum samples for antisperm antibodies and gonadotropin and testosterone levels. DESIGN: Prospective case analysis. SETTING: IVF unit. PATIENT(S): Sixteen subjects with obstructive azoospermia had postbiopsy ultrasound scans, 18 had assessment of hormone profiles, and 20 had evaluation of antisperm antibodies. INTERVENTION(S): Trucut needle testicular biopsies under local anesthetic. MAIN OUTCOME MEASURE(S): Postbiopsy testicular ultrasound, the presence of serum antisperm antibodies, and follicle stimulating and luteinizing hormone and testosterone levels. RESULT(S): There were no postbiopsy hematomas or scars, antisperm antibodies did not develop, and pituitary gonadotropins did not rise nor testosterone levels fall. CONCLUSION(S): Trucut needle testicular biopsy in men with obstructive azoospermia is not associated with defects of parenchymal structure or function.

Body weight, body mass index, and age: predictors of menotropin dose and cycle outcome in polycystic ovarian syndrome?

Whereas the dose of hMG is significantly correlated with both BMI and BW, commencing the more obese patient on higher hMG doses is not justified in view of the cancellation of cycles from over-responsiveness in the more obese patients started on higher hMG dosage regimens. If the more obese patient is slow to respond, however, a greater degree of clinical freedom may be exercised to increase the dose. The ultimate outcome of the cycle is not related to BMI, BW, or the dose of hMG used.

Testicular sperm extraction by Trucut needle and milking of seminiferous tubules: a technique with high yield and patient acceptability.

OBJECTIVE: To assess the sperm yield and patient acceptability of Trucut needle testicular biopsy followed by seminiferous tubule milking. DESIGN: Prospective case analysis. SETTING: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S): Forty-one males with obstructive azoospermia (normal testicular volume and FSH and LH levels). INTERVENTION(S): Trucut needle testicular biopsies under local anesthetic with milking of the seminiferous tubules. MAIN OUTCOME MEASURE(S): Quantitation of sperm retrieved per biopsy core and patient follow-up by questionnaire. RESULT(S): A mean of 105,634 sperm (range, 5,000-427,800) were retrieved, and the mean biopsy weight was 9.17 mg. Twenty-six subjects found the biopsy painless and 15 were pain-free after biopsy. CONCLUSION(S): The Trucut needle can be used in combination with seminiferous tubule milking to obtain large numbers of sperm in men with obstructive azoospermia.

Age and follicular phase estradiol are better predictors of pregnancy outcome than luteinizing hormone in menotropin ovulation induction for anovulatory polycystic ovarian syndrome.

OBJECTIVE: To examine the relationship of baseline and preovulatory serum E2, P, and LH levels and age with pregnancy outcome in polycystic ovarian syndrome (PCOS) patients undergoing hMG ovulation induction. DESIGN: Retrospective analysis of all available data over 2 years. SETTING: Tertiary referral ovulation induction clinic. SUBJECTS: Forty-four anovulatory PCOS patients with 25 ovulatory nonconception and 50 conception cycles after hMG ovulation induction. MAIN OUTCOME MEASURES: Ovulation (midluteal serum P > 25 nmol/L [7.86 ng/mL]); pregnancy (serum beta-hCG > 30 mIU/mL 16 days after ovulating injection); pregnancy outcome: pregnancy termination < 20 weeks' or > or = 20 weeks' amenorrhea. RESULTS: Of the endocrine parameters considered, none was significantly different in nonconceptive and conceptive ovulatory cycles. Miscarriage was associated with low basal serum E2: median value for pregnancies ending < 20 weeks, 105 pmol/L (28.6 pg/mL) and for > or = 20 weeks 150 pmol/L (40.9 pg/mL). It was also significantly associated with age. For patients > 29.5 years of age, (29.5 years, population mean age) a baseline E2 < or = 140 pmol/L (38.2 pg/mL) had sensitivity 92%, specificity 54%, positive predictive value 65%, and negative predictive value 87% for the prediction of miscarriage. The nature of the previous cycle, the day of the cycle on which therapy commenced, and a past history of miscarriage were not related either to pregnancy outcome or to basal serum E2.

Image analysis quantification of the Miles assay.

The Miles assay for vascular permeability has high intra- and interassay coefficients of variation (CVs). Quantification, usually by dye extraction and spectrophotometry, is time consuming. In this study, quantification by this means was compared with image analysis using the Olympus CUE-2 Image Analyzer (version 4). The test substance was recombinant human vascular permeability factor (rhVPF). The quantification process took approximately 10 min with image analysis. Formamide extraction and spectrophotometry required 1 hr of preparation, 4-6 days of incubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all < 10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectrophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 and 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R2 value for the correlation of image analysis with spectrophotometry was 81.4%. Test substances injected close to the spine evoked a greater permeability response than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng/mL p = 0.1 (unpaired t tests).

A comparison between randomly alternating imaging, normal laparoscopic imaging, and virtual reality training in laparoscopic psychomotor skill acquisition.

OBJECTIVES: To evaluate virtual reality as a laparoscopic training device in helping surgeons to automate to the "fulcrum effect" by comparing it to time-matched training programs using randomly alternating images (ie, y-axis inverted and normal laparoscopic) and normal laparoscopic viewing conditions. METHODS: Twenty-four participants (16 females and 8 males), were randomly assigned to minimally invasive surgery virtual reality (MIST VR), randomly alternating (between y-axis inverted and normal laparoscopic images), and normal laparoscopic imaging condition. Participants were requested to perform a 2-minute laparoscopic cutting task before and after training. RESULTS: In the test trial participants who trained on the MIST VR performed significantly better than those in the normal laparoscopic and randomly alternating imaging conditions. CONCLUSION: The results show that virtual reality training may provide faster skill acquisition with particular reference to automation of the fulcrum effect. MIST VR provides a new way of training laparoscopic psychomotor surgical skills.

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.