Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF

STUDY QUESTION. Are significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER. Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY. Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects. STUDY DESIGN, SIZE AND DURATION. Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS. Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE. Patch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION. For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS. These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized.

Indoor environment and the impact on the health of pre-exisiting asthmatics at work: the development of a risk management framework

This thesis examines the spatial and temporal variation in nitrogen dioxide (NO2) levels in Guernsey and the impacts on pre-existing asthmatics. Whilst air quality in Guernsey is generally good, the levels of NO2 exceed UK standards in several locations. The evidence indicates that people suffering from asthma have exacerbation of their symptoms if exposed to elevated levels of air pollutants including NO2, although this research has never been carried out in Guernsey before. In addition, exposure assessment of individuals is rarely carried out and research in this area is limited due to the complexity of undertaking such a study, which will include a combination of exposures in the home, the workplace and ambient exposures, which vary depending on the individual daily experience. For the first time in Guernsey, this research has examined NO2 levels in correlation with asthma patient admissions to hospital, assessment of NO2 exposures in typical homes and typical workplaces in Guernsey. The data showed a temporal correlation between NO2 levels and the number of hospital admissions and the trend from 2008-2012 was upwards. Statistical analysis of the data did not show a significant linear correlation due to the small size of the data sets. Exposure assessment of individuals showed a spatial variation in exposures in Guernsey and assessment in indoor environments showed that real-time analysis of NO2 levels needs to be undertaken if indoor micro environments for NO2 are the be assessed adequately. There was temporal and spatial variation in NO2 concentrations measured using diffusion tubes, which provide a monthly mean value, and analysers measuring NO2 concentrations in real time. The research shows that building layout and design are important factors for good air flow and ventilation and the dispersion of NO2 indoors. Environmental Health Officers have statutory responsibilities for ambient air quality, hygiene of buildings and workplace environments and this role needs to be co-ordinated with healthcare professionals to improve health outcomes for asthmatics. The outcome of the thesis was the development of a risk management framework for pre-existing asthmatics at work for use by regulators of workplaces and an information leaflet to assist in improving health outcomes for asthmatics in Guernsey.