Biomedical subjects
Publications and source records attributed to R J Aitken.
Consultant-supervised operative experience must be at the core of any training programme. The level of consultant supervision of United Kingdom trainees is largely unknown. In this study, the unique Lothian Surgical Audit database was used to assess consultant supervised training.
As the molecular basis of sperm-egg interaction is resolved, so new opportunities are created for the development of immunological approaches to disrupt the process of conception. Thus, realisation that the zona glycoprotein, ZP3, serves as a specific receptor for spermatozoa, has prompted a detailed examination of its contraceptive potential. In primate models, recombinant ZP3 has been shown to suppress fertility very efficiently, however this efficacy is tempered by the appearance of adverse side-effects involving accelerated primordial follicle depletion and a lymphocytic infiltration of the ovarian stroma. Synthetic peptides encoding B-cell epitopes have been found to circumvent the lymphocyte response although the effectiveness of such reagents in preventing the loss of primordial follicles has not yet been determined. The induction of active immunity against sperm-specific antigens has also been shown to generate long term infertility in both males and females. Molecular and immunological techniques are now being used to produce a rapidly expanding list of unique sperm antigens which are currently being evaluated to determine their potential contribution to the development of safe, effective, contraceptive vaccines.
BACKGROUND: Postoperative death following large bowel surgery is relatively infrequent and no large study has analysed the cause of all deaths comprehensively and critically. METHODS: In-hospital deaths following large bowel surgery in South-East Scotland were reviewed by independent assessors. The audit was confidential but not anonymous. Independent assessors' reports were returned to consultants. RESULTS: The audit documented 187 deaths. The independent assessors noted an adverse event in 78 patients (42 per cent). Twenty-six deaths (14 per cent) occurred following an anastomotic leak. A further 43 deaths (23 per cent) occurred because surgery was delayed (17) or there was undue delay in making the initial diagnosis (12) or recognizing a developing complication (14). Consultants operated on only half the patients classed as American Society of Anesthesiologists grade IV or V, or undergoing a second or subsequent operation. CONCLUSION: Half the patients dying in this study had identifiable deficiencies in their management. There is a clear need for greater consultant input with critically ill patients.
A spectrophotometric assay for the measurement of malondialdehyde and 4 hydroxyalkenals (MA + 4HA) has been evaluated for the detection of sperm pathologies involving oxidative stress. In order to make sensitive measurements of MA + 4HA on human spermatozoa, the stimulation of a lipid peroxidation cascade with a ferrous ion promoter was found to be necessary. The optimal configuration for the promoter was defined (0.64 mM FeSO4 + 20 mM ascorbate for 2 h in Ca2+ and Mg2 free Hanks' balanced salt solution) and the assay used in a series of studies to elucidate the functional significance of MA + 4HA determinations. Such measurements were found to give highly significant correlations (p < 0.001) with the loss of motility induced by oxidative stress created either with a xanthine oxidase, free radical generating system or by prolonged incubation under aerobic conditions. Experiments involving the stimulation and suppression of lipid peroxide release from human sperm suspensions, in concert with a bioassay for cytotoxicity, confirmed the strength and causative nature of these associations. Measurements of lipid peroxidation potential in highly purified, leucocyte-free sperm suspensions revealed the presence of inverse correlations with the motility of the spermatozoa, their viability, their competence for sperm-oocyte fusion and, most significantly, the quality of sperm movement in the original semen samples. Similar negative correlations were observed between sperm function and phorbol ester-stimulated reactive oxygen species generation but, unlike the MA + 4HA determinations, these relationships were obfuscated by the presence of leucocytes. We conclude that the measurement of MA + 4HA in human spermatozoa provides important information on the underlying quality of spermatogenesis and should be of value in the clinical diagnosis of infertility involving oxidative stress and the selection of patients for antioxidant therapy.
Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.
We present the first evidence that genetically damaged human spermatozoa are able to form normal pronuclei in oocytes after intracytoplasmic sperm injection (ICSI). The role of reactive oxygen species (ROS) as a cause of chromatin and DNA damage is well recognized. The same class of molecule can be found in the semen of males with severe infertility, who remained infertile until the advent of ICSI. In this study we have investigated the role of ROS in the induction of chromatin damage, DNA strand breakage and the subsequent ability of spermatozoa to decondense and form pronuclei after ICSI. Spermatozoa from normozoospermic men participating in our research programme were exposed to oxidizing environments created by co-incubation with hydrogen peroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH) or activated white cells. The subsequent ability of the spermatozoa to decondense in vitro was examined using sequential incubations in EDTA, dithiothreitol and sodium dodecyl sulphate, and the amounts of DNA strand breakage were assessed using an in-situ nick translation protocol. Finally, cells exposed to hydrogen peroxide, NADPH and activated leukocytes were microinjected into hamster oocytes, and their ability to decondense and form normal pronuclei was determined. The results indicate that human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage, yet the rate of pronucleus formation is no different from that of untreated control cells. The ability of genetically damaged spermatozoa to achieve normal fertilization following ICSI has implications for the practice of this form of assisted conception therapy.
Before the advent of intracytoplasmic sperm injection (ICSI) semen preparation techniques focused on the need to sustain the fertilizing potential of the spermatozoa particularly by reducing oxidative stress. However, for severely oligozoospermic patients treated by ICSI, sperm preparation protocols are used which aim to maximize sperm recovery rather than sperm function. In this study we have examined the impact of different sperm preparation techniques on oxidative stress, sperm motion and DNA integrity. Reactive oxygen species (ROS) generation was monitored using luminol-dependent chemiluminescence, seminal antioxidant activity was assessed using a total reactive antioxidant potential (TRAP) assay while sperm motility and DNA damage were evaluated using computer assisted semen analysis and in-situ nick translation respectively. The results demonstrate a significant increase in the levels of ROS generated by samples prepared by swim-up from a washed pellet compared with spermatozoa isolated directly from seminal plasma. This oxidative stress was associated with a highly significant increase in the level of DNA damage sustained by the spermatozoa while the quality of sperm motility remained largely unchanged. These results suggest that if repeated centifugation protocols are to be used to prepare spermatozoa, strategies should be developed for minimizing collateral DNA damage.
Progesterone exerts an extragenomic action on human spermatozoa, inducing a rapid calcium transient in the acrosomal domain of these cells and enhancing their potential for fertilization. This response is known to exhibit an absolute dependence on the presence of extracellular bicarbonate, although the mechanisms underlying this interaction are not understood. In this study, bicarbonate was found to exert a dose-dependent impact on the ability of progesterone to promote sperm-oocyte fusion in the absence of any collateral effect on sperm motility. The loss of sperm function in bicarbonate-free medium was associated with a failure to produce reactive oxygen species, an impaired capacity to exhibit redox-associated changes in tyrosine phosphorylation, and an apparent incapacity to generate normal calcium transients on exposure to progesterone. These defects were not related to a cAMP deficiency but were associated with a significant fall in intracellular pH. If cytosolic pH was chemically buffered into the normal range, then the spermatozoa regained every element of their response to progesterone. These results emphasize the importance of an alkaline intracellular milieu for the extragenomic action of progesterone on human spermatozoa and stress the fundamental difference between intracellular and extracellular sources of bicarbonate in maintaining the proton balance within such cells.
Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.
PROBLEM: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. METHOD OF STUDY: Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3(45-64), hZP3(93-110), hZP3(172-190) and hZP3(341-360) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3(45-64) and hZP3(172-190) recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. RESULTS: No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. CONCLUSION: Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.
Capacitation is a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus-oocyte complex. The attainment of a capacitated state is dependent upon an increase in tyrosine phosphorylation and results in the acquisition of responsiveness to physiological agonists such as progesterone and ZP3. In this study we have shown that this capacitation-dependent increase in tyrosine phosphorylation is controlled by a unique redox-regulated, cAMP-mediated, signal transduction cascade. Either stimulation of reactive oxygen species generation or elevation of intracellular cAMP induced increases in phosphotyrosine expression by human spermatozoa and enhanced their responsiveness to progesterone. Ultimate convergence of the redox- and cAMP-regulated pathways was indicated by the ability of the protein kinase A inhibitor, H89, to block both modes of signal transduction. Furthermore, the fact that the redox-regulated pathway could be silenced by catalase, while this enzyme had no effect on the cAMP-mediated response, indicated that oxidant generation must lie upstream from cAMP in the reaction sequence. In keeping with this conclusion, a functional association was demonstrated between the redox status of human spermatozoa and their cAMP content. The continuous production of reactive oxygen species was also shown to be necessary for the protein kinase A-tyrosine phosphorylation axis to remain functional. If the generation of oxidising conditions during capacitation was prevented with 2-mercaptoethanol, 2-deoxyglucose or the flavoprotein inhibitor, diphenylene iodonium, then cAMP could no longer trigger tyrosine phosphorylation. These data support a model for human sperm capacitation as a redox-regulated process, involving a unique sequence of interactive events including reactive oxygen species production, elevation of intracellular cAMP, stimulation of protein kinase A and the induction of tyrosine phosphorylation. This is the first report of such a signal transduction cascade and may have implications for the functional significance of reactive oxygen metabolites in other cell types.
As spermatozoa pass through the epididymis they complete a maturation process that enables these cells to participate in the process of fertilization. Epididymal maturation involves a complex cascade of changes involving the remodelling of the sperm surface, the induction of chromatin condensation, the acquisition of movement, and development of the potential for capacitation. In this review we shall consider how changes in the redox status of mammalian spermatozoa may contribute to the completion of these maturation events. Spermatozoa from all regions of the epididymis exhibit a spontaneous capacity for superoxide anion production which can be enhanced by exposure to NADPH, particularly in the caput region. It is hypothesized that this spontaneous free radical generating activity is mediated by a membrane-bound NADPH oxidase, the function of which is to generate the peroxides that are needed to serve as hydrogen acceptors for phospholipid hydroperoxide glutathione peroxidase in the induction of sperm chromatin condensation. As spermatozoa enter the cauda epididymidis they also express a capacity for hydrogen peroxide (H2O2) generation when released into simple, defined culture media. The onset of this activity is thought to be associated with the induction of sperm capacitation through stimulation of the tyrosine phosphorylation events involved in the attainment of a capacitated state. It is concluded that sperm maturation is a dynamic, redox regulated process, any imbalance in which could lead to the production of spermatozoa that are compromised in terms of their potential for fertilization and the integrity of their DNA.
For the past 15 years, general surgeons in Lothian, UK, have done a computerised audit. Lothian Surgical Audit (LSA) has two components: the first is educational, in the form of a weekly meeting and an annual report; the second is the accurate recording of surgical activity by use of a simple three-digit code. The main message from LSA is that changes in surgical practice typically occur over 3 to 4 years, and require repeated education. Changes attributable to LSA include the establishment of specialist units, improved results after large-bowel surgery, and the demise of outmoded techniques. Audits should have a clear educational commitment, and be carried out long enough to allow for reauditing. The LSA software has been developed at minimal cost and is networked throughout Lothian hospitals. LSA provides clear evidence that regional audit can significantly influence and improve surgical practice. Its success has resulted from its surgeon-driven development, its flexible use in everyday practice, and the confidence it inspires in local surgeons.
Male germ cells at various stages of differentiation from pachytene spermatocytes to mature caudal epididymal spermatozoa were examined for their ability to generate reactive oxygen species (ROS) using sensitive chemiluminescence techniques. In general, spermatozoa were found to spontaneously generate hydrogen peroxide as they progressed through the epididymis, maximal activity being observed on the release of mature cells from the caudal region into a modified Krebs-Ringer's solution. The spontaneous production of hydrogen peroxide rose rapidly during the first 10 min after the spermatozoa had been diluted into culture medium and thereafter stabilized, neither phorbol esters nor A23187 subsequently influencing this activity. Low levels of superoxide generation were also detected in suspensions of epididymal spermatozoa, but did not correlate with maturation status. However, superoxide production could be dramatically enhanced by the addition of exogenous NADPH, in a manner that was closely correlated with the stage of epididymal development being maximal for immature cells recovered from the caput epididymis in all species. Precursor germ cells (pachytene spermatocytes, round and elongate spermatids) similarly generated chemiluminescent signals compatible with the low level generation of ROS. Superoxide generation in these cells could again be stimulated by NADPH, via mechanisms that were inversely related to the stage of germ cell differentiation, the greatest activity being observed in pachytene spermatocytes. These results demonstrate that differentiating male germ cells have the potential to generate ROS, and have implications for the redox regulation of gonadal function and the development of reproductive pathologies involving oxidative stress.
The reduction in doctors' hours and the introduction of specialist training have reduced general surgical training by 60%. This study assessed the implications for a single health board. A questionnaire listing 13 representative operations was sent to 44 trainees and 52 trainers to determine the number of operations a trainee should perform. The total number of operations required for training was compared against the total actually performed across the health board. Operating times for five representative operations were audited prospectively. Trainers and trainees recommended a similar and conservative number of operations. The total number of operations available for training (4913) was 38% less than the number recommended (7946). Trainees required 50-75% more operating time than consultants. To increase the proportion of operations undertaken by trainees from the current 30% to 70% would require an extra 270 theatre days (of pounds 1.3m) yearly. The minimum number of operations required for training must be defined and the proportion of supervised operations undertaken by trainees substantially increased. Service and financial implications will have to be addressed. Action is needed urgently, as the first trainees will become consultants in less than five years.
Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redox regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Aitken et al., 1995). However, the mechanisms by which ROS are generated by these cells are not understood. In this study we have examined the possible significance of NADPH as a substrate for ROS production by human spermatozoa. Addition of NADPH to viable populations of motile spermatozoa induced a sudden dose-dependent increase in the rate of superoxide generation via mechanisms that could not be disrupted by inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide), diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic acid dehydrogenase (sodium oxamate). However, NADPH-induced ROS generation could be stimulated by permeabilization and was negatively correlated with sperm function. Both NADH and NADPH were active electron donors in this system, while NAD+ and NADP+ exhibited little activity. Stereo-specificity was evident in the response in that only the beta-isomer of NADPH supported superoxide production. The involvement of a flavoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and quinacrine. These results indicate that NAD(P)H can serve as an electron donor for superoxide generation by human spermatozoa and present a simple strategy for the production of motile populations of free radical generating cells with which to study the significance of these molecules in the control of normal and pathological sperm function.
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