Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

Differing molecular response of young and advanced maternal age human oocytes to IVM.

STUDY QUESTION: What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation? SUMMARY ANSWER: Although polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation. WHAT IS KNOWN ALREADY: Microarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes. STUDY DESIGN, SIZE, DURATION: The effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. LARGE SCALE DATA: Raw data from this study can be accessed through GSE95477. LIMITATIONS, REASONS FOR CAUTION: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility. STUDY FUNDING/COMPETING INTEREST(S): J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Air Force School of Health Care Science's quality of life study.

This was an empirically based assessment of non-prior service students' quality of life in the Air Force's School of Health Care Sciences. Analysis provided five results: (1) The overall quality of life at the school was good. (2) The variables accounting for student unhappiness were dormitory unsuitability and the students not being in their top-three career choices. (3) Structural changes were required at the dormitories. (4) The desire to succeed and how to achieve that success were the most important interests for students. (5) Loved ones and student independence were the greatest indicators of motivation. The findings resulted in three immediate corrections and two long-term recommendations to improve students' quality of life. The two long-term recommendations were to have an educational psychologist intervene when students are having significant learning problems, and to alter the selection process for recruiting. Both immediate corrections and long-term recommendations are useful for sister services.

The effect of anabolic steroids on the biomechanical and histological properties of rat tendon.

Twenty-four male rats were divided into four groups, with anabolic steroids and exercise as variables. Biomechanical tests and histological evaluations were performed. The results of the biomechanical tests suggested that anabolic steroids produce a stiffer tendon, which fails with less elongation. The energy at the time when the tendon failed, the toe-limit elongation, and the elongation at the time of the first failure were all affected significantly. Changes in the force at failure were not statistically significant. No alterations of structure were noted when the specimens were viewed with light microscopy. Alterations of the sizes of the collagen fibrils were noted on electron microscopy.

A high-strength Dacron augmentation for cruciate ligament reconstruction. A two-year canine study.

Augmentation and substitution grafting was performed in 48 adult canine stifles after anterior cruciate ligament resection. Four surgical reconstructions were performed: substitution in over-the-top fashion intra-articularly with just the Dacron implant; intra-articular substitution with tibial and femoral drill holes; over-the-top substitution intra-articularly and extra-articularly with the Dacron graft augmenting the iliotibial band wraparound transfer; and two-band extra-articular Dacron substitution with the Coker modification of the MacIntosh extra-articular reconstruction without iliotibial band. Sacrifice was performed at six, 12, 24, and 36 weeks postoperation with each specimen submitted for histologic or tensile strength testing. Four canines incurred infection and were omitted from follow-up study. Examination at six and 12 weeks revealed no graft breakage, stable knees, and no evidence of degenerative change radiographically in stifles. Fibrous ingrowth was present in the intra-articular substitution and augmentation stifles. No loss of ultimate failure strength was noted during failure mode testing. The direct substitution procedure revealed some evidence of cutting-edge disruption in 24-week canines. Beveling the drill holes could eliminate this problem. Follow-up evaluation of the augmentation techniques at 24 weeks revealed increased preservation of the iliotibial band-wrapped graft by a higher degree of fibrous ingrowth. No further loss in tensile strength of the graft was noted; concurrently, no increase in tensile strength was appreciated. The use of high-tensile-strength Dacron as an augmentation to biologic transfer appears to offer significant promise as a technique for human application. The study is continuing for long-term durability of the augmentation graft.

Acceleration of growth in two children treated with human growth hormone-releasing factor.

Two growth hormone-deficient children were treated with growth hormone-releasing factor for six months. The pattern of administration--1 to 3 micrograms per kilogram of body weight, given subcutaneously over one minute every three hours by infusion pump--was chosen to simulate growth hormone secretion in normal children. During the first week of therapy, both children had evidence of the metabolic effects of increased growth hormone secretion--i.e., nitrogen retention, demonstrated by decreased nitrogen excretion (P less than 0.05), and increased urinary calcium excretion (P less than 0.01). Growth hormone secretion was increased after pulses of growth hormone-releasing factor during the entire six-month period, and growth was accelerated. One child grew at a rate of 7.1 cm per year, as compared with 4.6 cm per year before therapy; the other grew at a rate of 13.7 cm per year, as compared with 2.1 cm per year before therapy, and had increased serum levels of somatomedin C. Growth hormone--releasing factor can restore growth hormone secretion and its biologic effects, including an increase in nitrogen retention, an increase in serum somatomedin C, and acceleration of linear growth in children with growth hormone deficiency. It is premature to speculate how useful this agent will prove to be in the treatment of children with growth hormone deficiency.

Extrahypothalamic growth-hormone-releasing factor (GRF) secretion is a rare cause of acromegaly: plasma GRF levels in 177 acromegalic patients.

To assess the frequency with which acromegaly is caused by ectopic secretion of GRF, we collected plasma samples from 177 unselected acromegalic patients. The samples together with those of three acromegalic patients with previously diagnosed tumors secreting GRF and of normal subjects were assayed in 3 independent GRF RIAs. Plasma immunoreactive GRF (IR-GRF) levels in normal subjects were either undetectable or detectable at levels up to 62.5 pg/ml. In none of the 177 specimens from acromegalic patients were IR-GRF values detectable in all assays, and in the most sensitive assay, the levels were similar to those in normal subjects, with the highest level measuring 82 pg/ml. In contrast, plasma IR-GRF found in the 3 patients with tumors that secreted GRF ranged from 2.0-24.4 ng/ml. These data suggest that extrahypothalamic GRF secretion is a rare cause of acromegaly. However, it is important that this rare cause of acromegaly be diagnosed before the patient has unnecessary surgery and/or irradiation directed at the pituitary. We recommend that plasma IR-GRF be measured in each new acromegalic patient.

Creatine kinase-MB activity: clinical and laboratory studies of specific immunochemical technique with optimized enzymatic assay.

A centrifugal analyzer method was developed for measuring the MB isoenzyme of creatine kinase (EC 2.7.3.2) in serum by use of a specific immunochemical technique that avoids interference from CK-BB and adenylate kinase. Enzymatic activity was measured kinetically at 30 degrees C with an optimized reagent containing creatine phosphate as substrate. The precision (CV) of the assay was 5 to 12 percent day-to-day (n = 39). The reference interval was 0 to 3.5 U per L (n = 45). Patient samples without detectable CK-MB in a widely-used electrophoretic assay contained up to 12 U per L of CK-MB by the new method. The new test was evaluated carefully in 99 patients consecutively admitted to the coronary care unit. Blood samples were obtained at frequent (four to eight hr) intervals. All patients with acute myocardial infarction (n = 27) had peak CK-MB greater than 7 U per L and greater than 3.5 percent of total CK. The predictive value of this result was 94 percent for the diagnosis of infarction. Abnormal results were documented at the same times (+/- four hr) following infarction by the electrophoretic and immunochemical techniques. Guidelines were evolved for interpreting the percent of MB (i.e., MB per total CK) at various time points. The test was reliable in documented early recurrent infarctions that occurred in three patients. The method appears to be an attractive alternative to electrophoretic techniques for use in diagnosis of acute myocardial infarction.

Kinematic analysis and simulation of vertebral motion under static load-part I: kinematic analysis.

The paper presents the kinematic analysis of motion segments of ten, humanlumbar spines. In order to achieve this objective, a spine fixture and a linkage transducer were designed. The spine fixture is capable of holding the motion segment in a prescribed plane of loading. With the designed fixture it becomes possible to apply three types of shear loads, and three types of bending loads. In addition, if desired, a compressive preload may be applied to a motion segment. The linkage transducer consists of six rotary potentiometers connected by seven rigid links. The transducer is capable of measuring all possible components of vertebral motion. The motion data in the motion segments of ten lumbar spine were collected under the influence of combined shear and bending loads applied in an incremental manner. Maximum shear load was 35.6 N and maximum bending load was 6780 N. mm. The motion segments did not show any appreciable difference in their motion beyond these loads. The motion segments were not subjected to any compressive preload. The range of motion data were collected in twelve loading planes 15 deg. apart. The characteristic vertebral motion may be described in terms of range of motion, its components, and the parameters associated with the screw motion. The paper presents data in the chart form to describe the kinematic characteristics of the lumbar motion segment.

Treatment program clients and emergency room patients: a comparison of two drug-using samples.

Research on institutionalized drug abusers has produced a profile of drug users which at times has been applied improperly to all individuals who manifest drug-related problems. This study compares a sample of drug treatment clients with a sample of emergency room patients who have acute drug reaction problems to ascertain the extent to which the traditional profile of drug abusers accurately describes both of these drug-using groups. The conclusion is that the treatment client sample does resemble the profile, but the emergency room patient sample does not. Treatment implications of this conclusion are discussed.