Lack of impact of semen quality on fertilization in assisted conception.

BACKGROUND: Defective semen quality is one of the commonest causes of infertility. The diagnosis of male fertility depends upon a descriptive evaluation of human semen, however a normal semen analysis does not necessarily indicate satisfactory fertility potential. AIMS: (i) to examine the semen quality of patients undergoing treatment by assisted conception, (ii) to explore relationships between semen quality and treatment outcomes, and (iii) to look at inter-laboratory variation in the assessment of semen quality. METHODS: Semen quality in patients undergoing assisted conception treatment between 2001 and 2004 was reviewed. Data on female age, egg numbers and fertilization outcomes was obtained by case note review. RESULTS: The thresholds used to direct patients towards IVF or ICSI treatment were comparable with the normal values promulgated by WHO, with the exception of morphology. Semen quality was not predictive of fertilization rates. When the results of independent measurements of the same sample were compared, there was diagnostic disagreement in between 10%-29% of samples. CONCLUSIONS: The conventional criteria of semen quality are used to determine treatment strategy for couples undergoing assisted conception but are not reflected in fertilization rates, emphasising the limited utility of the conventional criteria of semen quality in the assessment of sperm function. There remains significant inter-laboratory variation in the results of semen analysis.

The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated.

Image analysis techniques have been used to demonstrate that progesterone induces a rapid calcium transient in the acrosomal domain of greater than 90% of human spermatozoa (n = 2354). These results are at variance with previous reports, suggesting that progesterone receptors are only expressed on a small subpopulation of these cells, by virtue of their ability to bind fluorescent probes incorporating progesterone 3- (O-carboxymethyl) oxime conjugated to BSA. In the present study, we could confirm that such probes only bound to a small proportion of human spermatozoa (3.01 +/- 0.29%; n = 7557) although 91.79 +/- 1.8% of the same sperm populations exhibited a calcium transient in response to progesterone. These results indicate that the binding of labeled progesterone conjugates to human spermatozoa does not reflect the size of the progesterone responsive population; the response elicited by this steroid is essentially ubiquitous. Progesterone action was shown to involve an influx of extracellular calcium via mechanisms that did not involve voltage sensitive- or second messenger operated-channels, phospholipase C, or G proteins. Despite previous evidence suggesting that progesterone action might involve a GABAA receptor/chloride channel, neither GABA nor the GABA agonist muscimol had any effect on intracellular calcium concentrations in human spermatozoa or influenced their functional competence. The only factor that disrupted the responses of human spermatozoa to progesterone was this steroid itself. Progesterone exposure induced a prolonged period of refractoriness to further stimulation that influenced the capacity of these cells to generate calcium transients, and their ability to exhibit a biological response to changes in intracellular calcium. There are implications in these results for our understanding of the extragenomic action of progesterone on human spermatozoa and the clinical manipulation of this system for the assessment and suppression of human sperm function.

Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function.

Sensitive techniques have been developed for monitoring superoxide dismutase (SOD) activities in human sperm preparations. In contradiction to the protective role normally assigned to SOD, populations of defective spermatozoa recovered from the low density region of Percoll gradients were found to have three times more SOD than functionally competent preparations pelleting in high density Percoll. SOD activity was negatively correlated with the movement characteristics of human spermatozoa and their capacity for oocyte fusion, and positively associated with the induction of peroxidative damage. SOD activity was also highly correlated with other markers of the cytoplasmic space, creatine kinase (CK), and glucose-6-phosphate dehydrogenase (G-6-PDH). We conclude that while SOD may play a physiological role in maintaining a balance between O2.- and H2O2, high levels of this enzyme are associated with impaired sperm function because (a) the human spermatozoon is highly susceptible to the cytotoxic effects of H2O2, (b) O2.- is an important mediator of normal sperm function, and (c) high SOD activities reflect errors in spermatogenesis associated with germ cell exfoliation and the retention of excess residual cytoplasm by the spermatozoa.

Seminal fluid analysis and sperm function testing.

The diagnosis of male infertility is a rapidly developing field of investigation. The traditional descriptive approach to semen analysis is still at the heart of the diagnostic workup of male patients and important advances have been made in the standardization of the procedures used to construct the conventional semen profile, as embodied in the WHO handbook. Significant improvements have also been made in the techniques used to assess the quality of sperm motility. While this was once an entirely subjective exercise, the introduction of CASA systems to take objective measurements of the trajectories of human spermatozoa has revolutionized this form of analysis. The speed and accuracy of such systems has permitted detailed analyses of the relationships between sperm movement and sperm function, which has greatly enhanced the diagnostic power of this form of descriptive analysis. Notwithstanding the importance of the descriptive approach to semen analysis, it has also been recognized that to achieve an accurate diagnosis of male infertility such criteria should be supplemented with assays designed to reveal the functional competence of the spermatozoa. Bioassays have therefore been developed to measure such functions as the penetration of cervical mucus, sperm-zona interaction, the acrosome reaction, and sperm-oocyte fusion. All of these assays have been shown to generate information predictive of the fertilizing potential of human spermatozoa in vivo and in vitro. Despite their diagnostic value, the time, expense, and expertise required to run such functional assays has meant that they have not been widely used by infertility specialists. These functional assays are of value, however, in fundamental studies designed to elucidate the biochemical basis of defective sperm function. The first fruits of this research effort are now beginning to appear in the identification of a number of cytoplasmic markers for defective sperm function and the realization that lipid peroxidation plays a key role in the etiology of male infertility. In the wake of these fundamental studies will flow a new generation of biochemical tests for the diagnosis of defective sperm function and, it is hoped, the development of rational therapies with which to treat this condition.

The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation.

Capacitation had no effect on the ability of progesterone to elicit a rapid calcium transient in the acrosomal domain of human spermatozoa but had a marked influence of the ability of this steroid to induce a biological response. The development of this responsiveness to progesterone appeared to be redox regulated in that it was promoted by the stimulation of reactive oxygen species generation and inhibited by the presence of antioxidants, including catalase and membrane permeant thiols. The ability of redox conditions to influence the biological responsiveness of human spermatozoa did not involve changes in the dynamics of the calcium transients induced by progesterone but was causally linked with clear differences in tyrosine phosphorylation. We conclude that the ability of human spermatozoa to respond to the calcium transients induced by progesterone depends on a background of phosphotyrosine expression that can be profoundly influenced by the redox status of the spermatozoa during capacitation.

The value of adenosine triphosphate (ATP) measurements in assessing the fertilizing ability of human spermatozoa.

A study was undertaken to examine the relationship between adenosine triphosphate (ATP) levels and the fertilizing capacity of human spermatozoa in vitro and in vivo. The concentration of ATP in semen was found to be positively correlated with the ability of sperm to fuse with zona-free hamster oocytes. However, it was also demonstrated that a large part of this relationship depends upon the relationship between semen ATP concentrations and sperm number. Measurements of ATP levels in cryostored ejaculates used in an artificial insemination by donor program revealed that such measurements were not able to distinguish fertile from infertile ejaculates. However, among fertile donors, ATP levels did seem to contribute useful information on relative fertility. It is concluded that ATP measurement has a limited role in the laboratory evaluation of sperm function.

Protective effect of antioxidants on the impairment of sperm motility by activated polymorphonuclear leukocytes.

OBJECTIVE: To test the ability of antioxidants to reduce the loss of sperm motility caused by reactive oxygen species generated by polymorphonuclear leukocytes (PML). DESIGN: Standardized preparations of leukocyte-contaminated semen were created by suspending known concentrations of purified spermatozoa and PML in seminal plasma. After the stimulation of reactive oxygen species generation with phorbol ester, the spermatozoa were washed and incubated in culture medium before an analysis of their movement. The ability of antioxidants to counteract the free radical-induced loss of sperm motility observed under these circumstances was assessed. SETTING: An institutional research laboratory. PARTICIPANTS: The semen was obtained from normal volunteers. INTERVENTIONS: The following were tested: vitamins C and E, dimethylsulfoxide, catalase, hypotaurine, N-acetylcysteine, and reduced glutathione. MAIN OUTCOME MEASURES: Reactive oxygen species generation was monitored by luminol-dependent chemiluminescence. Sperm motility was assessed manually and by computer-aided semen analysis. RESULTS: Consistent impairment of sperm motility and average path velocity was observed in the presence of activated PML. This effect was reduced by the concomitant presence of glutathione, N-acetylcysteine, hypotaurine, and catalase. CONCLUSION: Antioxidants can protect against the damaging effect of leukocyte-derived reactive oxygen species on sperm movement and may be of clinical value in assisted conception procedures.

Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination.

OBJECTIVE: To determine the relationships between sperm function tests and fertilization of human oocytes in vitro. DESIGN: Analysis of infertile patients undergoing IVF therapy. SETTING: Diagnostic Andrology Laboratory and Assisted Conception Service. PATIENTS: Forty-one couples who underwent IVF-ET therapy were studied. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to numerous criteria of semen quality, including the conventional semen profile, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, sperm morphology, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS: Significant correlations were observed between fertilization rates and several attributes of the sperm preparations, including elements of sperm function (acrosome reaction), movement (percentage motile, hyperactivation, the amplitude of lateral sperm head displacement), morphology (normal morphology, midpiece defects, multiple anomalies index), nuclear normality (acridine orange staining), and reactive oxygen species generation (chemiluminescence induced by phorbol ester and FMLP). In a stepwise multiple regression analysis, an accurate prediction of fertilization rates was obtained using a multiple regression equation incorporating six variables of which sperm morphology and FMLP-induced chemiluminescence were the most informative. CONCLUSIONS: A set of criteria have been identified that accurately predict the fertilizing potential of human sperm suspensions in vitro and that place particular emphasis on sperm morphology and the degree of leukocyte contamination.

Field trial of a diluent for the transportation of human semen at ambient temperatures.

OBJECTIVE: To examine the ability of a citrate-yolk buffer extender to preserve human semen samples at ambient temperatures over a 25- to 30-hour period. DESIGN: Human semen samples were diluted 1:1 with citrate-yolk buffer or homologous seminal plasma and transported at ambient temperature between two distant locations (London to Edinburgh, United Kingdom). Various criteria of semen quality then were assessed before and after 25 to 30 hours storage and transportation in these diluents. SETTING: An institutional research laboratory and a private fertility clinic. PATIENT(S): Samples were provided by 21 donors of unknown fertility and 7 asthenozoospermic patients. INTERVENTION(S): The diluent used to preserve human semen comprised an egg yolk buffer supplemented with fructose and citrate. MAIN OUTCOME MEASURE(S): Aspects of semen quality assessed included movement, hyaluronate penetration, viability, acrosome reaction, and reactive oxygen species generation. RESULT(S): The deterioration of semen quality at ambient temperatures could be prevented by the presence of citrate-yolk buffer, permitting the accurate analysis of oxidative stress and human sperm function, 25 to 30 hours postejaculation. CONCLUSION(S): Citrate-yolk buffer offers considerable promise as a medium for the ambient temperature storage and transportation of human semen.

A simple and sensitive nonradioactive method for the detection of urinary human chorionic gonadotropin and diagnosis of early human pregnancy. II. Single-unit test.

A simple, sensitive, and reliable single-unit nonradioactive method for the detection of human chorionic gonadotropin (hCG) in concentrated urine and the diagnosis of early pregnancy is reported. This unit, presently termed the Ayerst pregnancy test kit (APTK), consists of four components: a sampler-filter paper cone, an ultrafilter-concentrator to which a vial holder is attached, a support stand with a mirror, and an immunologic reagent vial. In the APTK, 5 to 6 ml of urine were sampled, filtered, and concentrated, and the hCG in the retentate was detected by Ayerst immunologic reagents [APTK(AY)] and by the Pregnosticon "All In" [APTK(P)]. Some of the unconcentrated urine samples (0.1 ml) were also tested in hemagglutination inhibition tests (HIT) using Ayerst [HIG(AY)] and Pregnosticon "All In" [HIT(P)] reagents. Urine samples from pregnant, nonpregnant (ovulating and nonovulating), perimenopausal, and menopausal women were tested. It was found that the APTK(AY) and APTK(P) were significantly more sensitive and reliable than the HIT(AY) and HIT(P) in detecting low levels of urinary hCG for early diagnosis of pregnancy. The sensitivity and specificity of the APTK(AY) were better than those of the APTK(P). The APTK(AY) give significantly more correct positive and negative results than the other tests performed simultaneously. The APTK(AY) is simpler and safer than the serum radioimmunoassays and radioreceptor assay presently used to detect low levels of hCG for the early diagnosis of pregnancy and other hCG-producing states.

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.

A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.

Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.

The ability of human spermatozoa to exhibit sperm-oocyte fusion in response to the ionophore, A23187, was examined in relation to the capacity of these cells to generate reactive oxygen species. In 70 fertile control donors, there was an overwhelming pattern of high levels of sperm-oocyte fusion associated with low levels of reactive oxygen species production. By contrast, 88% of the 74 oligozoospermic patients exhibited less than 25% oocyte penetration in response to A23187 and 58% exhibited no penetration whatsoever. Of the 40 oligozoospermic patients who failed to respond to A23187, nine had low levels of reactive oxygen species production in association with impaired liquefaction of seminal plasma. Of the remainder, 17 (55%) exhibited defective sperm function together with elevated production of reactive oxygen species. These observations, which are the first to describe a biochemical defect in the spermatozoa of oligozoospermic patients, may carry significant implications for the etiology and treatment of this condition.

DNA integrity in human spermatozoa: relationships with semen quality.

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

Distress and concerns in couples referred to a specialist infertility clinic.

OBJECTIVES: The aims of this study were to examine emotional distress and infertility-related concerns in male and female members of couples referred to a specialist infertility clinic and to determine changes in these over time. METHODS: A prospective cohort study with a 6-month follow-up. Emotional distress was measured using the Hospital Anxiety and Depression Scale, and concerns by a specially designed questionnaire. RESULTS: The response rate achieved was 38%. At baseline, 25.7% of women and 8.9% of men had scores of greater than 10 on the Hospital Anxiety and Depression Scale (HADS) Anxiety subscale, and 2.7% of women and 1.8% of men had scores of greater than 10 on the HADS Depression subscale. At 6-month follow-up the HADS scores were substantially unchanged. Females reported a significantly greater infertility-related concerns regarding life satisfaction, sexuality, self-blame, self-esteem and avoidance of friends compared with males. CONCLUSIONS: The prevalence of emotional disorder identified was low. There were gender differences in the nature of the specific concerns reported. The degree of distress and concerns did not change significantly over time. There are a minority of patients, mainly females, with clinically significant distress and infertility-related concerns amongst patients attending infertility clinics who deserve psychological attention.

Declining sperm quality: a review of facts and hypotheses.

A substantial body of evidence has accumulated in recent years suggesting that human semen quality may be deteriorating. This has been associated with evidence of other changes in male reproductive health, including increases in congenital malformations and testis cancer in men, and associated problems in wildlife. Unfortunately the evidence remains inconclusive. It has been suggested that these changes may be due to environmental xenoestrogens acting during development. Although there is now a large quantity of data indicating that this is a plausible hypothesis, evidence of causality, rather than association, remains to be provided. The potential importance of these changes for human health is considerable, and urgent research is required to clarify the situation.

Epidemiology and aetiology of male infertility.

Infertility is a common problem, affecting perhaps one couple in six, the majority of whom now seek medical care. Although diagnostic problems make it difficult to establish the extent of the male partner's contribution with certainty, a number of studies suggest that male problems represent the commonest single defined cause of infertility. The World Health Organization has proposed a scheme for the diagnostic classification of male infertility, based upon a standardized approach to clinical assessment and to the assessment of semen quality. Some of these classifications are now controversial, and many are descriptive, rather than aetiological. Increasingly, the importance of occupation, environmental and particularly genetic factors in the causation of male infertility is being recognized.

Glutathione as a treatment for male infertility.

The excessive generation of reactive oxygen species (ROS) by abnormal spermatozoa and by contaminating leukocytes has been identified as one of the few defined aetiologies for male infertility. As a consequence, work has begun on evaluating the role of antioxidants in the management of these patients. Glutathione plays a significant role in the antioxidant defences of the spermatogenic epithelium, the epididymis, and perhaps in ejaculated spermatozoa. The use of antioxidants in vitro appears to be of value in preserving fertilizing capacity, although no clinical data are available. Glutathione administered in vivo to patients who may have infertility secondary to excessive oxidative stress appears to act at the epididymis and during spermatogenesis, to improve the function of ejaculated spermatozoa. However, fertility studies have not yet been conducted. Controlled studies of glutathione and other antioxidants in patients with defined ROS pathology are urgently required.

Male reproductive health: cause for concern?

A substantial body of evidence has accumulated in recent years that human semen quality may be deteriorating. This has been associated with evidence of other changes in male reproductive health, including increases in congenital malformations and testicular cancer in humans, and similar problems in wildlife. Unfortunately, the evidence remains inconclusive. It has been suggested that these changes may be due to environmental xeno-oestrogens acting during development. Although there is now a large quantity of data indicating that this is a plausible hypothesis, evidence of causality, rather than association, remains to be provided. The potential importance of these changes for human health is considerable and urgent research is required to clarify the situation.

The predictive value of computer-assisted semen analysis in the context of a donor insemination programme.

In this paper we examine the value of both conventional and computer-assisted semen analysis (CASA) using the Hamilton-Thorn HTM-S 2030 in predicting the in-vivo fertility of cryopreserved donor semen. Semen samples were examined prospectively and data on the conventional criteria of semen quality, sperm morphometry and movement were collected. Of 61 ejaculates identified, 33 achieved pregnancies ('successful') and 28 failed to do so ('unsuccessful'), despite insemination into at least four different normal female recipients. When the post-thaw semen profiles were compared, no differences were observed between the two groups in respect of the conventional criteria of semen quality determined by conventional laboratory techniques; however, there were differences in respect of both morphometry and movement characteristics determined by the HTM-S. When multiple logistic regression was used to examine the ability of the variables measured to predict the achievement of pregnancy, the conventional criteria of semen quality were of no value (chi 2 = 6.67, P = 0.353). However, the CASA assessment successfully predicted outcome in 86.9% of cases (chi 2 = 44.3, P = 0.0021). It was concluded that CASA assessment is of significant value in predicting the ability of an ejaculate to achieve pregnancy.