The Use of Expanded Carrier Screening in Reproductive Medicine: Scientific Impact Paper No. 74.

Expanded carrier screening (ECS) is a genetic screening test carried out by analysing a blood sample. This screen can be used to detect whether the individual unknowingly carries gene variants associated with common genetic conditions, such as cystic fibrosis, that may be passed on to their children. It is typically performed in reproductive medicine for those who are considering having a family either naturally or via fertility treatment. Many donor sperm and egg banks, particularly in the USA and Europe, also perform blanket ECS testing on all their prospective sperm and egg donors. ECS is not currently routine practice in the UK, but a growing number of patients are requesting it before treatment. All of us carry gene variants of some sort that may cause autosomal recessive disease in their children if their partner or donor also carry a variant in the same gene. An autosomal recessive disease means two copies of an abnormal gene must be present in order for the disease or trait (such as cystic fibrosis or sickle cell disease) to develop. One copy of the variant means the person is a carrier but does not have the condition. Two copies, i.e. from the mother and father, means the child has a 25% chance of having the genetic disease. Carrying a gene variant does not mean that the individual would necessarily have any symptoms of the disease or any features of the condition. Genetic tests for specific conditions are currently available either before or during pregnancy for prospective parents who have a family or personal history of a genetic condition, or for those from ethnic backgrounds where certain conditions - such as haemoglobinopathies (blood disorders) - are common, prompting referral to a clinical genetics department. Expanded carrier screens may test for more than 100 genetic conditions. The list of conditions screened for is called a panel. Common panels are 250 or 600 genes. Not all expanded carrier screens that are available analyse the same genes. Some may test for genes that do not cause serious disease, or cause diseases that occur in later life; others test for genes that cause severe conditions in childhood. There is no agreement as to which panel of genes should be tested for in an ECS. Understanding the screening that is being offered, and the meaning of any results, is complicated and requires support from appropriately trained professionals to best inform the prospective parent or parents.

Strong Evidence for

The boundaries of the chart of nuclides contain exotic isotopes that possess extreme proton-to-neutron asymmetries. Here we report on strong evidence of ^{9}N, one of the most exotic proton-rich isotopes where more than one half of its constitute nucleons are unbound. With seven protons and two neutrons, this extremely proton-rich system would represent the first-known example of a ground-state five-proton emitter. The invariant-mass spectrum of its decay products can be fit with two peaks whose energies are consistent with the theoretical predictions of an open-quantum-system approach; however, we cannot rule out the possibility that only a single resonancelike peak is present in the spectrum.

Observation of the Exotic Isotope

A ^{13}F resonance was observed following a charge-exchange reaction between a fast ^{13}O beam and a ^{9}Be target. The resonance was found in the invariant-mass distribution of 3p+^{10}C events and probably corresponds to a 5/2^{+} excited state. The ground state was also expected to be populated, but was not resolved from the background. The observed level decays via initial proton emissions to both the ground and first 2^{+} state of ^{12}O, which subsequently undergo 2p decay. In addition, there may also be a significant proton decay branch to the second 2^{+} level in ^{12}O. The wave function associated with the observed level may be collectivized due to coupling to the continuum as is it located just above the threshold for proton decay to the 2_{2}^{+} state of ^{12}O.

First Observation of the Four-Proton Unbound Nucleus

^{18}Mg was observed, for the first time, by the invariant-mass reconstruction of ^{14}O+4p events. The ground-state decay energy and width are E_{T}=4.865(34) MeV and Γ=115(100) keV, respectively. The observed momentum correlations between the five particles are consistent with two sequential steps of prompt 2p decay passing through the ground state of ^{16}Ne. The invariant-mass spectrum also provides evidence for an excited state at an excitation energy of 1.84(14) MeV, which is likely the first excited 2^{+} state. As this energy exceeds that for the 2^{+} state in ^{20}Mg, this observation provides an argument for the demise of the N=8 shell closure in nuclei far from stability. However, in open systems this classical argument for shell strength is compromised by Thomas-Ehrman shifts.

First Observation of Unbound

The structure of the extremely proton-rich nucleus _{8}^{11}O_{3}, the mirror of the two-neutron halo nucleus _{3}^{11}Li_{8}, has been studied experimentally for the first time. Following two-neutron knockout reactions with a ^{13}O beam, the ^{11}O decay products were detected after two-proton emission and used to construct an invariant-mass spectrum. A broad peak of width ∼3.4 MeV was observed. Within the Gamow coupled-channel approach, it was concluded that this peak is a multiplet with contributions from the four lowest ^{11}O resonant states: J^{π}=3/2_{1}^{-}, 3/2_{2}^{-}, 5/2_{1}^{+}, and 5/2_{2}^{+}. The widths and configurations of these states show strong, nonmonotonic dependencies on the depth of the p-^{9}C potential. This unusual behavior is due to the presence of a broad threshold resonant state in ^{10}N, which is an analog of the virtual state in ^{10}Li in the presence of the Coulomb potential. After optimizing the model to the data, only a moderate isospin asymmetry between ground states of ^{11}O and ^{11}Li was found.

Near-perfect absorption in epsilon-near-zero structures with hyperbolic dispersion.

We investigate the interaction of polarized electromagnetic waves with hyperbolic metamaterial structures, whereby the in-plane permittivity component εx is opposite in sign to the normal component εz. We find that when the thickness of the metamaterial is smaller than the wavelength of the incident wave, hyperbolic metamaterials can absorb significantly higher amounts of electromagnetic energy compared to their conventional counterparts. We also demonstrate that for wavelengths leading to ℜ(εz) ≈ 0, near-perfect absorption arises and persists over a range of frequencies and subwavelength structure thicknesses.

Observation of long-range three-body coulomb effects in the decay of (16)Ne.

The interaction of an E/A=57.6-MeV ^{17}Ne beam with a Be target is used to populate levels in ^{16}Ne following neutron knockout reactions. The decay of ^{16}Ne states into the three-body ^{14}O+p+p continuum is observed in the High Resolution Array (HiRA). For the first time for a 2p emitter, correlations between the momenta of the three decay products are measured with sufficient resolution and statistics to allow for an unambiguous demonstration of their dependence on the long-range nature of the Coulomb interaction. Contrary to previous measurements, our measured limit Γ<80 keV for the intrinsic decay width of the ground state is not in contradiction to the small values (of the order of keV) predicted theoretically.

Anandamide modulates human sperm motility: implications for men with asthenozoospermia and oligoasthenoteratozoospermia.

STUDY QUESTION: What are the levels of anandamide (N-arachidonoylethanolamide, AEA) in human seminal plasma and how are these related to abnormal spermatozoa? SUMMARY ANSWER: Seminal plasma AEA levels were lower in men with asthenozoospermia and oligoasthenoteratozoospermia compared with normozoospermic men. WHAT IS KNOWN ALREADY: AEA, a bioactive lipid, synthesized from membrane phospholipids may signal through cannabinoid receptors (CB1 and CB2) to regulate human sperm functions and male reproduction by modulating sperm motility, capacitation and the acrosome reaction in vitro. Local AEA levels are regulated by the synthetic and degradative enzymes, NAPE-PLD and FAAH, respectively. How the deregulation of this endogenous signalling pathway affects human sperm function(s) is not clear. STUDY DESIGN, SIZE AND DURATION: This was a cross-sectional study of 86 men presenting at an infertility clinic for semen analysis over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: AEA was quantified, by ultra-high performance liquid chromatography-tandem mass spectrometry, in seminal plasma from 86 volunteers. Using qRT-PCR, CB1, CB2, NAPE-PLD and FAAH transcript levels were determined in spermatozoa from men with normozoospermia, asthenozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Normal spermatozoa were exposed in vitro to methanadamide (meth-AEA) to determine its effect on sperm motility, viability and mitochondrial activity. MAIN RESULTS AND THE ROLE OF CHANCE: Seminal plasma AEA levels (mean ± SEM) were significantly lower in men with asthenozoospermia (0.080 ± 0.01 nM; P < 0.05) or oligoasthenoteratozoospermia (0.083 ± 0.01 nM; P < 0.05) compared with normozoospermic men (0.198 ± 0.03 nM). In addition, the levels of spermatozoal CB1 mRNA were significantly decreased in men with asthenozoospermia (P < 0.001) or oligoasthenoteratozoospermia (P < 0.001) compared with normozoospermic controls. Supra-physiological levels of meth-AEA decreased sperm motility and viability, probably through CB1-mediated inhibition of mitochondrial activity. LIMITATIONS, REASONS FOR CAUTION: The inhibitory effect of meth-AEA was only shown in vitro and may not reflect what happens in vivo. WIDER IMPLICATIONS OF THE FINDINGS: As the regulation of the endocannabinoid system appears to be necessary for the preservation of normal sperm function and male fertility, there may be implications for the adverse reproductive consequences of marijuana use. Exocannabinoids, such as Δ(9)-THC, are likely to compete with endocannabinoids at the cannabinoid receptors, upsetting the finely balanced endocannabinoid signalling system. The importance of the endocannabinoid system makes it an attractive target for pharmacological interventions to control male fertility. STUDY FUNDING/COMPETING INTEREST(S): This work was funded in part by miscellaneous educational funds from the University Hospitals of Leicester National Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. The authors declare no competing interests.

Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa.

Quantitative real-time polymerase chain reaction (qRT-PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. ß-Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2 M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT-PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2 M and ACTB.

Applying an integrated refuge to manage western corn rootworm (Coleoptera: Chrysomelidae): effects on survival, fitness, and selection pressure.

The refuge strategy can delay resistance of insect pests to transgenic maize producing toxins from Bacillus thuringiensis (Bt). This is important for the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), because of its history of adaptation to several management practices. A 2-yr study across four locations was conducted to measure the effects of integrated refuge (i.e., blended refuge) on western corn rootworm survival to adulthood, fitness characteristics, and susceptibility to Bt maize in the subsequent generation. The treatments tested in this study were as follows: a pure stand of Bt maize (event DAS-59122-7, which produces Bt toxins Cry34Ab1/Cry35Ab1), a pure stand of refuge (non-Bt maize), and two variations on an integrated refuge consisting of 94.4% Bt maize and 5.6% non-Bt maize. Within the two integrated refuge treatments, refuge seeds received a neonicotinoid insecticidal seed treatment of either 1.25 mg clothianidin per kernel or 0.25 mg thiamethoxam per kernel. Insects in the pure stand refuge treatment had greater survival to adulthood and earlier emergence than in all other treatments. Although fecundity, longevity, and head capsule width were reduced in treatments containing Bt maize for some site by year combinations, Bt maize did not have a significant effect on these factors when testing data across all sites and years. We found no differences in susceptibility of larval progeny to Bt maize in bioassays using progeny of adults collected from the four treatments.

Entomopathogenic Nematodes for Field Control of Onion Maggot (Delia antiqua) and Compatibility with Seed Treatments.

Onion maggot (Delia antiqua) is a prominent pest of allium crops in temperate zones worldwide. Management of this pest relies on prophylactic insecticide applications at planting that target the first generation. Because effective options are limited, growers are interested in novel tactics such as deployment of entomopathogenic nematodes. We surveyed muck soils where onions are typically grown to determine if entomopathogenic nematode species were present, and then evaluated the compatibility of entomopathogenic nematode species with the insecticides commonly used to manage D. antiqua. We also evaluated the efficacy of these entomopathogenic nematodes for reducing D. antiqua infestations in the field. No endemic entomopathogenic nematodes were detected in surveys of muck fields in New York. Compatibility assays indicated that, although insecticides such as spinosad and, to some extent, cyromazine did cause mortality of entomopathogenic nematodes, these insecticides did not affect infectivity of the entomopathogenic nematodes. Field trials indicated that applications of entomopathogenic nematodes can reduce the percentage of onion plants killed by D. antiqua from 6% to 30%. Entomopathogenic nematodes reduced D. antiqua damage and increased end of season yield over two field seasons. Applications of entomopathogenic nematodes may be a viable option for reducing D. antiqua populations in conventional and organic systems. Together with other management tactics, like insecticide seed treatments, applications of entomopathogenic nematodes can provide a yield boost and a commercially acceptable level of D. antiqua control.

Democratic decay of 6Be exposed by correlations.

The interaction of an E/A=70-MeV (7)Be beam with a Be target was used to populate levels in (6)Be following neutron knockout reactions. The three-body decay of the ground and first excited states into the α+p+p exit channel were detected in the High Resolution Array. Precise three-body correlations extracted from the experimental data allowed us to obtain insight into the mechanism of the three-body democratic decay. The correlation data are in good agreement with a three-cluster-model calculation and thus validate this theoretical approach over a broad energy range.

The use of novel biochemical markers in predicting spontaneously resolving 'pregnancies of unknown location'.

BACKGROUND: 'Pregnancies of unknown location' (PULs) include viable and failing intrauterine and extrauterine pregnancies. The aim of this study was to evaluate the role of novel biochemical markers in the prediction of spontaneous resolution of PULs. METHODS: Serum samples were taken at the first visit to the pregnancy unit for measuring the traditional markers ß-hCG and progesterone, and for inhibin A, inhibin pro-αC-related immunoreactivity (inhibin pro-αC-RI) and insulin-like growth factor-binding protein 1 (IGFBP-1). Follow-up was continued until the pregnancy had resolved, the location of the pregnancy and viability was determined or treatment was required. Outcomes were dichotomized into 'spontaneous resolution' and 'other outcome' categories. RESULTS: One-hundred and nine cases of PUL were included in the data analysis. Spontaneous resolution occurred in 70% and a further scan was required in 30% to reach a diagnosis. Levels of progesterone and inhibin A were significantly lower (both P < 0.001) and levels of IGFBP-1 significantly higher (P = 0.02) in the pregnancies that spontaneously resolved than in those pregnancies that required further intervention. In decision tree analysis, the novel markers were less useful than progesterone and ß-hCG in predicting spontaneously resolving PULs. Inhibin pro-αC-RI and IGFBP-1 were not useful in the prediction of spontaneously resolving PULs. Inhibin A is more predictive than ß-hCG alone, but serum progesterone is the best single marker and progesterone and hCG together continues to be the best way of predicting spontaneously resolving PULs. CONCLUSIONS: These novel biochemical markers are not clinically useful in predicting spontaneously resolving PULs.

Ground-state proton decay of 69Br and implications for the 68Se astrophysical rapid proton-capture process waiting point.

We report on the first direct measurement of the proton separation energy for the proton-unbound nucleus (69)Br. Bypassing the (68)Se waiting point in the rp process is directly related to the 2p-capture rate through (69)Br, which depends exponentially on the proton separation energy. We find a proton separation energy for (69)Br of Sp((69)Br )= -785(-40)(+34) keV; this is less bound compared to previous predictions which have relied on uncertain theoretical calculations. The influence of the extracted proton separation energy on the rp process occurring in type I x-ray bursts is examined within the context of a one-zone burst model.

Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labeling.

An immunoperoxidase-labeling technique allowing visualization of antibody binding to the cell surface at the electron microscopical level has been employed an an analysis of H-2 and non-H-2 alloantigen expression on the early mouse embryo. The presence of non-H-2 antigenic determinants has been confirmed on eight-cell, morula, and blastocyst stages of development. Contrary to previous reports, however, low levels of H-2 antigen have also been detected on the blastocyst. This is the earliest stage at which H-2 has been shown to be expressed on the fertilized mouse egg and may reflect the greater resolution of the immunoperoxidase technique. Using two different models to study the critical peri-implantation stages, those of experimentally induced blastocyst activation and blastocyst outgrowth in vitro, it has been demonstrated that antigen loss occurs on the trophectoderm at the time of implantation, and that this is not necessarily dependent upon maternal influence. It is suggested that the loss may be an important factor in the prevention of maternal immune rejection during the establishment of the fetal allograft. The two major components of the early postimplantation conceptus display a striking differential in antigenic status. The embryonic sac shows a high degree of peroxidase labeling, while the ectoplacental cone trophoblast is unlabeled. These findings add support to the concept of antigenic neutrality of the early trophoblast and its role in the maintenance of a normal fetomaternal immunological equilibrium.

Mitochondrial 12S rRNA susceptibility mutations in aminoglycoside-associated and idiopathic bilateral vestibulopathy.

The mitochondrial 12S rRNA is considered a hotspot for mutations associated with nonsyndromic (NSHL) and aminoglycoside-induced hearing loss (AIHL). Although aminoglycoside ototoxicity is the most common cause of bilateral vestibular dysfunction, the conceivable role of 12S rRNA mutations has never been systematically investigated. We sequenced the 12S rRNA of 66 patients with bilateral vestibulopathy (BV) with (n=15) or without (n=51) prior exposure to aminoglycosides, as well as 155 healthy controls with intact vestibular function (sport pilots), and compared these to 2704 published sequences (Human Mitochondrial Genome Database). No mutations with a confirmed pathogenicity were found (A1555G, C1494T), but four mutations with a hitherto tentative status were detected (T669C, C960del, C960ins, T961G). Due to their predominant occurrence in patients without aminoglycoside exposure, their detection in controls and a weak evolutionary conservation, their pathogenic role in vestibulocochlear dysfunction remains provisional.

Mitochondrial DNA haplogroups and type 2 diabetes: a study of 897 cases and 1010 controls.

Mitochondria play a central role in the secretion of insulin by pancreatic beta-cells, and pathogenic mutations of mitochondrial DNA (mtDNA) can cause diabetes. The aetiology of type 2 diabetes has a strong genetic component, raising the possibility that genetic variants of mtDNA alter the risk of developing the disorder. Recent studies have produced conflicting results. By studying 897 UK cases of type 2 diabetes and 1010 population-matched controls, it is shown that European mtDNA haplogroups are unlikely to play a major role in the risk of developing the disorder.

Multiple-species natural enemy approach for biological control of alfalfa snout beetle (Coleoptera: Curculionidae) using entomopathogenic nematodes.

Multiple-species natural enemy approach for the biological control of the alfalfa snout beetle, Otiorhynchus ligustici (L.) (Coleoptera: Curculionidae), was compared with using single-species of natural enemies in the alfalfa ecosystem by using entomopathogenic nematodes with different dispersal and foraging behaviors. Steinernema carpocapsae NY001 (ambush nematode), Heterorhabditis bacteriophora Oswego (cruiser nematode), and Steinernema feltiae Valko (intermediate nematode) were applied in single-species, two-species combinations, and one three-species combination treatments at 2.5 x 10(9) infective juveniles per hectare. All nematode species persisted for a full year (357 d). S. carpocapsae NY001 protected the plants from root-feeding damage better than H. bacteriophora Oswego but allowed for higher larval survival than all other nematode treatments. S. feltiae Valko protected the plants better than H. bacteriophora Oswego and controlled alfalfa snout beetle larvae better than S. carpocapsae NY001. H. bacteriophora Oswego allowed for similar root damage compared with control plots but reduced larval populations better than S. carpocapsae NY001. The combination of S. carpocapsae NY001 and H. bacteriophora Oswego provided significantly better protection for the plants than the control (unlike H. bacteriophora Oswego alone) and reduced host larva survival more than S. carpocapsae NY001 alone. The combination S. feltiae Valko and H. bacteriophora Oswego could not be statistically separated from the performance of S. feltiae Valko applied alone.

The effect of dose escalation for large squamous cell carcinomas of the anal canal.

PURPOSE: Chemoradiation allows for organ preservation in patients with anal cancer, but patients with large tumors (> 5 cm) have elevated rates of locoregional recurrence. With conformal radiation techniques, there is interest in dose escalation to decrease local recurrence in patients with large tumor size. METHODS/PATIENTS: The National Cancer Database (NCDB) was used to identify patients with anal cancer from 2004 to 2013 with tumors > 5 cm. Adult patients who received definitive chemoradiation were included. Patients with prior resection were excluded. High dose was defined as greater than or equal to 5940 cGy. Statistical analyses were performed using logistic regression, Kaplan-Meier, and Cox proportional hazards for overall survival (OS). RESULTS: In total, 1349 patients were analyzed with 412 (30.5%) receiving high-dose radiation therapy (RT). 5-year OS was 58 and 60% for high and standard dose RT, respectively (p = 0.9887). On univariate analysis, high-dose RT was not associated with improved OS (HR = 0.998, CI 0.805-1.239, p = 0.9887). On multivariate analysis, high-dose RT (HR = 0.948, CI 0.757-1.187, p = 0.6420) was not associated with improved OS but older age (HR = 1.535, CI 1.233-1.911, p = 0.0001), male sex (HR = 1.695, CI 1.382-2.080, p < 0.0001), comorbidities (HR = 1.389, CI 1.097-1.759, p = 0.0064), and long RT (HR = 1.299, CI 1.047-1.611, p = 0.0173) were significantly associated with decreased OS. CONCLUSIONS: There was no observed difference in OS for dose escalation of anal cancers > 5 cm in this population-based analysis. Differences in local control and salvage therapy cannot be assessed through the NCDB. Whether dose escalation of large tumors may improve local control and colostomy-free survival remains an important question and is the subject of ongoing trials.

Long-term Persistence of Native New York Entomopathogenic Nematode Isolates Across Crop Rotation.

Entompathogenic nematodes are found worldwide in a wide array of soil habitats with a broad host range and significant variation in foraging strategies. The primary use of entomopathogenic nematodes (EPNs) in managed plant systems has been focused on inundative releases in a biopesticide strategy. Little effort has been placed in investigating the use of natural occurring or adapted EPN strains for long-term suppression of pest outbreaks in managed systems. This study examined the potential of EPN isolates from Northern New York (NNY), inoculated at a low level (250 million IJ/ha), which are climate adapted and their persistent characteristics preserved to maintain population levels in agricultural fields (N = 82) for multiple years and across crop rotation (alfalfa:corn:alfalfa). Persistence levels for Steinernema carpocapsae (Weiser) (Rhabditida:Steinernematidae) ranged between 8 and 12% of the soil cores assayed in continuous alfalfa and 1-14% of the soil cores assayed in continuous corn rotated from EPN treated alfalfa. Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) residual persistence level ranged between 17 and 32% in continuous alfalfa and 22-41% in continuous corn rotated from EPN treated alfalfa. Combined EPN level ranged between 27 and 43% of the soil cores in continuous alfalfa and 28-55% in continuous corn rotated from EPN-treated alfalfa. Inspection of individual fields suggested EPN populations established in prior years at the residual soil core level of 18-35% can respond positively to an increase of susceptible hosts in both alfalfa and corn, often increasing their presence to 100%.