Preimplantation genetic testing using Karyomapping for a paternally inherited reciprocal translocation: a case study.

PURPOSE: Preimplantation genetic testing (PGT) using Karyomapping is used to screen embryos for single gene disorders prior to implantation. While Karyomapping is not designed to screen for abnormalities in chromosome copy number, this testing is based upon a genome-wide analysis of single nucleotide polymorphisms (SNP) and, as such, some chromosome abnormalities are detected. The aim of this study was to validate whether Karyomapping could provide reliable and accurate PGT for a paternal 46,XY,t(10;19)(p15;p13.3) reciprocal translocation. METHODS: Feasibility/validation for PGT was performed using DNA from the couple, as well as DNA from the paternal parents and from a previous unbalanced pregnancy. Karyomapping was performed using Illumina's HumanKaryomap-12 BeadChip microarray technology. SNP analysis was performed using BlueFuse Multi software (Illumina). Transmission of the translocation was assessed through the analysis of SNP markers on the chromosome regions of interest. RESULTS: PGT-SR was determined to be feasible as chromosomal SNP analysis could reliably distinguish normal/balanced outcomes from all unbalanced outcomes. The couple transferred a normal/balanced embryo in an elective single embryo transfer procedure following 2 IVF/PGT-SR cycles. A clinical pregnancy was achieved. CONCLUSION: This is the first report of PGT-SR test validation using Karyomapping for a 46,XY,t(10;19)(p15;p13.3) reciprocal translocation. Karyomapping may offer a means of detecting unbalanced forms of chromosome rearrangements when other PGT platforms fail.

Defining the limits of detection for chromosome rearrangements in the preimplantation embryo using next generation sequencing.

STUDY QUESTION: Is next generation sequencing (NGS) capable of detecting smaller sub-chromosomal rearrangements in human embryos than the manufacturer's quoted resolution suggests? SUMMARY ANSWER: NGS was able to detect unbalanced chromosome segments smaller than the manufacturer's resolution. WHAT IS KNOWN ALREADY: Array Comparative Genomic Hybridization (array-CGH) has been the gold standard platform used for PGD of chromosome rearrangements. NGS is a viable alternative to array-CGH for PGD of chromosome arrangements given that the manufacturer's guidelines quote a resolution of ≥20 Mb. However, as many patients carry a chromosome rearrangement <20 Mb, the detection limits of NGS warrant further investigation. STUDY DESIGN, SIZE, DURATION: This study involved a retrospective assessment of stored DNA samples from embryos that had previously been diagnosed as unbalanced by array-CGH as part of routine PGD in two separate IVF clinics between November 2013 and April 2017. SurePlex whole genome amplification (WGA) products derived from DNA extracted from an embryo biopsy sample known to carry an unbalanced form of a chromosome rearrangement were subjected to a specific NGS workflow (VeriSeq PGS). The results from the two technologies were compared for each sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: WGA products from 200 embryos known to carry unbalanced rearrangements were sequenced and analysed. These embryos had been created by 75 patients known to carry a chromosome rearrangement (68 reciprocal translocations, 3 pericentric inversions, 1 paracentric inversion, 2 insertions and 1 dual reciprocal and inversion). Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment and chromosome, segregation pattern, the number of bins in the analysis software used and concordance with array-CGH results. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 294 unbalanced chromosome segments were assessed. NGS was capable of detecting 285/294 (97%) unbalanced segments previously identified using array-CGH. The final PGD diagnosis was concordant for 200/200 (100%) embryos. In total, 44/75 (59%) patients contained an unbalanced chromosome segment below the quoted 20 Mb manufacturer's stated resolution. Of these, 35/44 (80%) patients had segments that were able to be detected using NGS, whilst maintaining clinical outcome concordance. LIMITATIONS, REASONS FOR CAUTION: Our study subset did not include any rearrangements involving the Y chromosome. NGS has less available bins per chromosome compared to the array-CGH platform used, thus it remains possible that chromosome rearrangements predicted to be small but still detectable by array-CGH may not be feasible for testing using NGS. This should be considered when undertaking a theoretical feasibility assessment for detecting the chromosome rearrangement in question. Only one specific workflow for WGA and NGS was investigated in this study. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that NGS is available for the detection of unbalanced chromosome rearrangements ≥10 Mb. STUDY FUNDING/COMPETING INTEREST(S): Part sponsorship of the VeriSeq PGS kits used was provided by Illumina. The remainder of the kits were provided by two commercial IVF clinics. None of the authors has any conflicting interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Preimplantation genetic diagnosis for chromosome rearrangements - one blastomere biopsy versus two blastomere biopsy.

PURPOSE: Preimplantation Genetic Diagnosis (PGD) has proven to be a useful reproductive option for carriers of some chromosome rearrangements. The data presented in this study compares the impact of one versus two blastomere biopsy on the likelihood of achieving a PGD result, as well as the effect on subsequent embryo development and clinical outcomes. METHODS: IVF-PGD couples had either one or two blastomeres biopsied from all embryos with ≥7 blastomeres on day 3 post oocyte collection. These blastomeres were assessed for the specific chromosome rearrangement using Fluorescent In-situ Hybridisation (FISH). Further embryo development was monitored on days 4 and 5. Clinical outcomes were assessed retrospectively. RESULTS: The data shows that statistically more embryos achieved a PGD result following two blastomere biopsy, compared with one blastomere biopsy (92 % versus 88 %, respectively). Furthermore it was found that embryo development and clinical outcomes were similar between the two biopsy groups. CONCLUSIONS: Based on this analysis it appears that the biopsy of two blastomeres from embryos with ≥7 blastomeres on day 3 is a valid and successful approach for couples presenting for IVF-PGD for a chromosome rearrangement.

WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression.

BACKGROUND AND PURPOSE: Antidepressants, which raise the CNS concentrations of 5-HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity. EXPERIMENTAL APPROACH: In this report, we present the in vitro pharmacological and in vivo pharmacokinetic and pharmacological properties of the novel, orally available and CNS penetrant inhibitor of the noradrenaline transporter (NET), WAY-318068 (1-[(1S,2R)-1-(3,5-difluorophenyl)-2-hydroxy-3-(methylamino)propyl]-7-fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one). KEY RESULTS: WAY-318068 is a potent and effective inhibitor of the NET with a K(i) of 8.7 nM in a binding assay, and an IC(50) of 6.8 nM in an assay of transporter function, without significant binding to the dopamine transporter. Furthermore, the compound has only weak activity at the 5-HT transporter, leading to a functional selectivity of greater than 2500-fold. It is orally bioavailable with substantial quantities of the compound found in the CNS after oral dosing. As measured by microdialysis in rats, the compound causes a robust and significant increase in cortical noradrenaline levels without affecting 5-HT. WAY-318068 was effective in models of acute, visceral, inflammatory, osteoarthritic, neuropathic, diabetic and bone cancer pain, as well as in traditional models of depression at doses that do not cause motor deficits. CONCLUSIONS AND IMPLICATIONS: Collectively, the present results support the conclusion that selectively increasing CNS levels of noradrenaline is sufficient for efficacy in models of depression and pain.

Preclinical characterization of WAY-211612: a dual 5-HT uptake inhibitor and 5-HT (1A) receptor antagonist and potential novel antidepressant.

BACKGROUND AND PURPOSE: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models. EXPERIMENTAL APPROACH: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models. KEY RESULTS: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model. CONCLUSIONS AND IMPLICATIONS: These findings suggest that WAY-211612 may represent a novel antidepressant.

Preimplantation genetic screening outcomes are associated with culture conditions.

BACKGROUND: Although preimplantation genetic screening (PGS) is widely offered, there are contradictory reports on the clinical merit of this procedure. Any gain from embryo selection following aneuploidy screening must significantly outweigh the impact of the procedure. Variability of technical expertise in embryo biopsy, blastomere fixation, fluorescence in situ hybridization analysis, along with suboptimal laboratory quality control and inappropriate patient selection may impact PGS outcomes. METHODS: To investigate such effects, a total of 1508 stimulated in vitro fertilization (IVF) cycles were retrospectively analysed. During 2004, a significant change was made to the embryo culture media used. Clinical outcomes from cycles with PGS were compared prior to and after the change in media and compared with matched controls not utilizing PGS during the same period. RESULTS: Clinical PGS success rates were found to improve following the media change. For patients aged less than 40, clinical outcomes following PGS were significantly lower than those without PGS prior to the change, but became equivalent after the change. For patients >or=40 years and

Characterization of the role of medial prefrontal cortex dopamine receptors in cocaine-induced locomotor activity.

Medial prefrontal cortex (mPFC) dopamine (DA) modulates the motor-stimulant response to cocaine. The present study examined the specific mPFC DA receptor subtypes that mediate this behavioral response. Intra-mPFC injection of the DA D2-like receptor agonist quinpirole blocked cocaine-induced motor activity, an effect that was prevented by coadministration of the D2 receptor antagonist sulpiride. Intra-mPFC injection of the selective D4 receptor agonist PD 168,077 or the selective D1 receptor agonist SKF 81297 did not alter the motor-stimulant response to cocaine. Finally, it was found that an intermediate dose of quinpirole, which only attenuated cocaine-induced motor activity, was not altered by SKF 81297 coadministration, suggesting a lack of synergy between mPFC D1 and D2 receptors. These results suggest that D2 receptor mechanisms in the mPFC are at least partly responsible for mediating the acute motor-stimulant effects of cocaine.

Repeated exposure to inhaled toluene induces behavioral and neurochemical cross-sensitization to cocaine in rats.

RATIONALE: Toluene is a solvent found in many commercial products and is frequently abused by inhalation. Whether previous exposure to toluene alters subsequent responses to other drugs of abuse is not known. OBJECTIVES: This study determined the effects of repeated toluene exposure on the acute motor-stimulant response to cocaine and on cocaine-induced dopamine (DA) concentrations in the nucleus accumbens (NAc). METHODS: One week following bilateral cannulae implantation over the NAc, 27 adult, male Wistar rats began a daily 30-min exposure regimen to either toluene (8,000 ppm) or air for ten sessions. Approximately 24 h or 96 h after their last exposure, animals were injected with either saline or cocaine (15 mg/kg, i.p.) and locomotor activity and DA concentrations in the NAc were measured. RESULTS: Exposure to toluene rendered the rats immobile, and the time required for recovery of normal posture decreased across the ten sessions. In all animals tested, systemic cocaine administration enhanced both locomotor activity and DA concentrations in the NAc. These increases, however, were significantly greater in rats previously exposed to toluene. CONCLUSIONS: Overall, these findings show that repeated toluene exposure enhances behavioral and neurochemical responses to subsequent cocaine administration.

Intra-medial prefrontal cortex injection of quinpirole, but not SKF 38393, blocks the acute motor-stimulant response to cocaine in the rat.

RATIONALE: Considerable evidence suggests that the medial prefrontal cortex (mPFC) is an important region in mediating certain behavioral and neurochemical responses to cocaine. However, a role for cortical dopamine (DA) receptor subtypes in modulating these responses has yet to be elucidated. OBJECTIVES: This study investigated the effects of intra-mPFC administration of DA agonists on the acute motor-stimulant response to cocaine. In addition, in vivo microdialysis techniques were employed to determine the effects of intracortical injection on cocaine-induced extracellular DA concentrations in the nucleus accumbens (NAC). METHODS: One week following bilateral cannulae implantation over the mPFC and the NAC (for dialysis experiments), male Sprague-Dawley rats received an intra-mPFC injection of saline, the DA D2-like agonist quinpirole (0.015, 0.05, 0.15, 0.5, 1.5, or 5.0 nmol per side) or the partial DA D1-like agonist SKF 38393 (0.5, 1.5, or 5.0 nmol per side) approximately 5 min before peripheral administration of saline or cocaine (15 mg/kg, i.p.). For dialysis experiments, only the highest dose of quinpirole was examined. RESULTS: Pretreatment with quinpirole produced a dose-dependent decrease in cocaine-induced motor activity, with the highest doses resulting in a complete abolition of the acute motor-stimulant response to cocaine. In contrast, intra-mPFC administration of SKF 38393 was not shown, at the doses tested, to alter cocaine-induced motor activity. In agreement with the behavioral effects, intra-mPFC quinpirole injection (5 nmol per side) significantly blocked cocaine-induced DA overflow in the NAC. CONCLUSIONS: The results of the present study provide additional support that the mPFC is a neural substrate through which cocaine, in part, produces its motor-stimulant effects. In addition, these data suggest that modulation of cortical DA D2 receptors can block acute cocaine-induced behavioral (locomotor activity) and neurochemical (DA concentrations in the NAC) responses in the rat.

Dopamine depletion in the medial prefrontal cortex induces sensitized-like behavioral and neurochemical responses to cocaine.

It has been postulated that behavioral sensitization to cocaine is associated with an attenuation of cocaine-induced dopamine (DA) transmission in the medial prefrontal cortex (mPFC). Hence, experiments were designed to examine the effects of chemically-induced cortical DA depletion on the acute behavioral and neurochemical responses to cocaine. One week following two bilateral 6-hydroxydopamine (6-OHDA) injections into the mPFC, animals received injections of cocaine (7.5, 15 or 30 mg/kg, i.p.) or saline (1 ml/kg, i.p.) in a randomized fashion with a minimum 3 day intertrial interval. Cocaine produced a dose-dependent increase in motor activity which was significantly enhanced in animals depleted (mean of 76%) of dopamine in the mPFC. Likewise, 6-OHDA lesions of the mPFC produced a significant enhancement of cocaine-induced DA transmission in the nucleus accumbens (NAC) as estimated by in vivo microdialysis. These data indicate a permissive involvement of cortical DA in mediating behavioral and neurochemical responses to cocaine, as well as confirm the ability of the mPFC to influence subcortical structures in response to an acute injection of cocaine. Collectively, the present findings suggest that alterations in cortical DA transmission may be a neural substrate mediating the development of sensitization to cocaine, and thus, may contribute to the addictive properties of cocaine.

Sexual coercion content in 21 sexuality education curricula.

Sexual coercion, a topic of relevance to school health personnel, may be as common in high school populations as in university populations. Twenty-one sexuality education curricula were examined for information on the topics of date rape, stranger rape, pressure, incest, sexual harassment, unwanted/inappropriate touch, and exploitation/victimization. Curricula scoring highest in total coverage also were the most comprehensive with six of the seven sexual coercion topics covered. Overall, pressure and exploitation/victimization received the greatest attention, while sexual harassment was not covered in any of the curricula. Common themes occurring within the coercion topic areas included guilt, communication/assertiveness skills, blame, drug use, premeditation, fear, sources of help. Results suggest sexuality education curricula have not responded to the increased concern regarding sexual harassment in schools.

PIP: This study is an analysis of sexual coercion content in sexuality education curricula. Copies of 21 adolescent sexuality education curricula materials published from 1988 or later were obtained and the information therein reviewed for sexual coercion topics such as date/stranger rape, incest, sexual harassment or pressure, inappropriate touch, and exploitation/victimization. The results revealed that 3 of 21 curricula (Streetwise to Sex-wise, Values and Choices, and Life Planning Education-DC) addressed all the sexual coercion topics with the exception of sexual harassment. The topic covered by the greatest number of curricula was pressure. Date rape was addressed in 14 curricula, 12 covered the exploitation topic, inappropriate/unwanted touch was addressed by 11, stranger rape was addressed by 7, and incest was covered by 4 curricula. The curricula that covered the fewest number of topics also offered the least amount of depth as measured by the number of paragraphs. The most significant finding from the analysis was that none of the 21 curricula addressed sexual harassment. This article recommends that all facets of sexual coercion should be equally addressed in junior high/middle school and high school curricula. Even though the topic may seem sensitive or controversial, it should be covered because these adolescents deserve to have information and skills to prevent or effectively deal with sexual coercion.

The GABAB agonist baclofen modifies cocaine self-administration in rats.

The present experiments were conducted to examine further the ability of GABAergic compounds to modify the reinforcing effects of cocaine. In male Sprague-Dawley rats, behaviour was maintained under a fixed-ratio (FR)-5 with a 240 s timeout (TO) multiple schedule of cocaine (0.66 mg/kg/infusion) and food (45 mg) in 180 min sessions. Once rats could reliably nose-poke for comparable number of reinforcers over sessions, and demonstrate extinction selectively for each reinforcer, pretreatments were examined. The GABAB agonist baclofen (2.5-10.0 mg/kg i.p.) administered 30 min before the start of the session, dose-dependently attenuated behaviour maintained by cocaine, whereas responding maintained by food was marginally decreased. 4,5,6,7-Tetrahydroisoxazolo [5,4-c]pyridin-3-ol hydrochloride (THIP) (2-8 mg/kg i.p.) a GABAA agonist failed to modify cocaine-maintained or food-maintained responding. In another experiment, behaviour maintained by cocaine (0.66 mg/kg/infusion) under an FR-5 TO 20 s schedule of reinforcement was attenuated by intra-nucleus accumbens (100-300 ng) or intra-ventral tegmental area (300 ng) administration of baclofen. Similar doses of baclofen administered into the striatum had no effect. Repeated systemic pretreatment for 3 days with baclofen (2.5 mg/kg i.p.) produced gradual decreases in cocaine-maintained responding. A larger dose (5.0 mg/kg i.p.) tested repeatedly for 5 days decreased the number of cocaine injections self-administered. The present findings demonstrate that modulation of GABA systems may have therapeutic potential for the treatment of psychomotor stimulant abuse.

Antioxidant properties of melatonin--an emerging mystery.

Over three centuries ago, the French philosopher René Descartes described the pineal gland as "the seat of the soul." However, it was not until the late 1950s that the chemical identity and biosynthesis of melatonin, the principal hormone secreted by the pineal body, were revealed. Melatonin, named from the Greek melanos, meaning black, and tonos, meaning color, is a biogenic amine with structural similarities to serotonin. The mechanisms mediating the synthesis of melatonin are transcriptionally regulated by the photoperiodic environment. Once synthesized, the neurohormone is a biologic modulator of mood, sleep, sexual behavior, reproductive alterations, immunologic function, and circadian rhythms. Moreover, melatonin exerts its regulatory roles through high-affinity, pertussis toxin-sensitive, G-protein (or guanine nucleotide binding protein) coupled receptors that reside primarily in the eye, kidney, gastrointestinal tract, blood vessels, and brain. Additional evidence also indicates a role for melatonin in aging and age-related diseases, probably related to its efficient free radical scavenger (or antioxidant) activity. The potential clinical benefit of melatonin as an antioxidant is remarkable, suggesting that it may be of use in the treatment of many pathophysiological disease states including various cancers, hypertension, pulmonary diseases, and a variety of neurodegenerative diseases such as Alzheimer's disease. This review summarizes the biosynthesis of melatonin and its many endocrine and physiological functions, including its therapeutic potential in human disease states. Emphasis is placed on the recent speculations indicating that this pineal hormone serves as an endogenous antioxidant agent with proficient free radical scavenging activity.

Gender representation in illustrations, text, and topic areas in sexuality education curricula.

A content analysis instrument was developed to examine differences in gender representation in sexuality education curricula. Fourteen middle school and high school curricula, published between 1985 and 1995, were examined. Variables included illustrations, noun/pronoun usage, and specific topics within the two categories of biological function and mutual interest. The study examined differences in gender representation in illustrations, photographs, cartoons, drawings, and text. Omission of sexuality topics related to gender also was examined. Little difference occurred within the text of the curricula in noun and pronoun usage. Illustrations were few in number, but showed greater female representation. Greater male representation occurred in topics related to drug use, sexual exploitation, sexual desire, and homosexuality. Topics with greater female representation included body image, diseases of the reproductive organs, and hygiene.