Sperm functional tests.

Several semen parameters are used to discriminate the fertile male from the subfertile male. The most widely used parameters are sperm concentration, motility, progressive motility, and sperm morphology. Semen analysis is usually applied as described in the World Health Organization manual for semen analysis. In addition to a routine semen analysis, sperm functional tests have been described for many years, which in most cases are regarded as research tools and not part of the routine semen testing in an infertility clinic. In this review we report on the value of four sperm function tests: the sperm penetration assay, the sperm-zona pellucida binding tests, the acrosome reaction, and the hyaluronan binding assay. For each test we describe the current value, the indication for performing the test, how to interpret the results, and its therapeutic implications. Our data show that sperm functional assays are highly predictive of IVF outcome results and have the potential to assist in clinical decision making, especially to avoid the current long-standing treatment with IUI and to direct the patients to intracytoplasmic sperm injection without delay when sperm functional testing fails. We believe that advances in molecular biology techniques will allow us to develop simpler sperm function assays in the near future. This will undoubtedly help clinicians in optimizing male factor infertility diagnosis and treatment.

Semen analysis and sperm function testing.

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

Mobile phone radiation does not induce pro-apoptosis effects in human spermatozoa.

Abstract Recent reports suggest that mobile phone radiation may diminish male fertility. However, the effects of this radiation on human spermatozoa are largely unknown. The present study examined effects of the radiation on induction of apoptosis-related properties in human spermatozoa. Ejaculated, density-purified, highly motile human spermatozoa were exposed to mobile phone radiation at specific absorption rates (SARs) of 2.0 and 5.7 W/kg. At various times after exposure, flow cytometry was used to examine caspase 3 activity, externalization of phosphatidylserine (PS), induction of DNA strand breaks, and generation of reactive oxygen species. Mobile phone radiation had no statistically significant effect on any of the parameters studied. This suggests that the impairment of fertility reported in some studies was not caused by the induction of apoptosis in spermatozoa.

Semenology training programs: 8 years' experience.

OBJECTIVE: To evaluate the immediate effect of hands-on training on the quality of technical skills of laboratory technicians. DESIGN: Retrospective analytical study. SETTING: Academic institutions and private infertility clinics. PARTICIPANTS: One hundred and ten laboratory technicians. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): First, 110 participants from 16 African countries attended 5-day semenology workshops at Tygerberg Hospital. During these workshops the methodology as described by the World Health Organization manual for the analysis of human semen, namely, sperm concentration, motility, vitality, and morphology, formed part of the curriculum. Second, two experienced morphology readers from the Tygerberg group presented 23 1-day sperm morphology workshops in nine other countries. RESULT(S): The semenology workshops indicated a significant improvement in the evaluation of sperm concentration only, whereas pretraining and posttraining results for motility did not differ. Vitality reports did not differ from those of the experienced worker in the first place. Calculation by means of a linear regression model showed a significant decrease in the mean posttraining scores from the pretraining scores for the sperm morphology courses. CONCLUSION(S): Training improved technical skills for the evaluation of morphology and sperm concentration. The evaluation of graded sperm motility seems to be more difficult to teach over a short period.

The clinical significance of sperm-zona pellucida binding: 17 years later.

The development of homologous functional bio-assays for sperm quality assessment has been a focal point of reproductive biologists in order to provide a scientifically- based diagnosis in cases of fertilization failure. The availability of viable oocytes still remains an important limiting factor for laboratories to embark on the methodology of assays that examine sperm-oocyte interaction. The use of zonae pellucidae obtained from oocytes derived from post mortem tissue and failed in vitro fertilization cycles, enhanced the availability of zona material. Sperm-zona pellucida binding has been illustrated to be an essential requisite during human fertilization. This fundamental biological step can be measured under hemizona assay as well intact-oocyte test conditions. The sensitivity and specificity of sperm-zona binding results indicated the assay to be positively and significantly correlated with in vitro fertilization outcome. Furthermore, highly significant correlations were demonstrated between normal sperm morphology, hyperactivated motility, sperm creatine kinase activity and the zona binding capacity of a given sperm sample. It was concluded that andrology testing remains an ever-growing component in the work-up of the infertile couple. We enter the next millennium with many questions that remain to be answered by the hand of efficacious screening techniques and a new formidable therapy in intracytoplasmic sperm injection.

Effect of acute in vivo sildenafil citrate and in vitro 8-bromo-cGMP treatments on semen parameters and sperm function.

OBJECTIVE: To determine the effect of acute in vivo sildenafil citrate (VIAGRA) and in vitro 8-Bromo-cGMP treatments on semen parameters and sperm function. DESIGN: Prospective double-blind, placebo-controlled, crossover, two-period clinical investigation. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Twenty male volunteers with normal erectile function and normal electrocardiogram were recruited. INTERVENTION(S): In vivo: 50 mg of sildenafil citrate (VIAGRA) or placebo was administered p.o., and semen samples were collected 1 hour after administration of the test drug. In vitro: 8-Bromo-cGMP (20 microM; 60 minutes) was added to semen samples. MAIN OUTCOME MEASURE(S): Macroscopic and microscopic seminal parameters were measured and motility studies performed. Various acrosome reaction studies and sperm-zona pellucida binding studies were also done. RESULT(S): Either sildenafil citrate (VIAGRA) or 8-Bromo-cGMP treatments had no effect on both macroscopic and microscopic seminal parameters as well as the acrosome reaction. Sperm-zona pellucida binding results were however increased to 148.75% and 134%, respectively, by these treatments. Various kinematical parameters increased after treatment with the most significant increase detected in the population of rapid cells. CONCLUSION(S): VIAGRA can be used successfully to enhance sperm motility and binding to the oocyte especially during fertility treatments.

African experience with sperm morphology training courses.

This paper reports on the results of a 6-year ongoing training and quality control programme for sperm morphology. The programme consists of a 5-day semenology workshop, during which participants receive intensive training on issues such as sperm concentration, motility, vitality and morphology. Following the workshop, all the participants are enrolled in a continuous quality control (CQC) programme for sperm morphology. These workshops and quality control programmes are presented by the Department of Obstetrics and Gynaecology at Tygerberg Hospital (South Africa) in conjunction with the World Health Organization's (WHO) Special Programme for Research Development and Research Training. Seventy individuals from 11 countries in sub-Saharan Africa have been trained since 1997; 58 of these are still participating in the quality control programme. Using the results from the quality control programme, participants were classified according to their sperm morphology reading skills recorded over an extended period. It is concluded that an external quality control programme can be highly successful, on condition that it is presented continuously with a 3-4 month interval between tests.

Monitoring technologist reading skills in a sperm morphology quality control program.

OBJECTIVE: The value of sperm morphology as a predictor of a man's fertilizing potential has often been challenged because of different classification systems used to distinguish between normal and abnormal spermatozoa. The study aimed to monitor the reading skills of 53 laboratory technologists who are enrolled in a continuous quality control program for sperm morphology assessment. DESIGN: Prospective analytical study. SETTINGS: Academic hospital and academic institution setting. PATIENT(S): Sperm samples from the sperm donor program and andrology technicians from sub-Saharan Africa. INTERVENTION(S): Papanicolaou-stained sperm slides were prepared and shipped on a quarterly basis to participating laboratories. Papanicolaou-prestained sperm morphology slides were used as test material for 21 months. MAIN OUTCOME MEASURE(S): Percentage normal spermatozoa.A new statistical model was developed to record reading skills of the participating technicians. Reading skills were classified as marginal (5.7% of cases), good (11.3% of cases), and excellent (83% of cases). RESULT(S): Participants maintained their morphological reading skills and agreed with the reference laboratory by not exceeding a SD limit of 0.2 to set stringent standards for the program. CONCLUSION(S): Technician proficiency can be monitored using the results of a quality control program. A continuous quality control program can be initiated only after intensive training, because baseline values at the onset of the quality control program serves as an internal reference value.

Relationship between zona pellucida-induced acrosome reaction, sperm morphology, sperm-zona pellucida binding, and in vitro fertilization.

OBJECTIVE: To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm-zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. DESIGN: Prospective analytical study. SETTING: Academic training hospital.Randomly selected couples consulting for infertility. INTERVENTION(S): Acrosome reaction response to solubilized human zona pellucida was recorded. MAIN OUTCOME MEASURE(S): We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). RESULT(S): Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm-zona binding (r = 0.57). CONCLUSION(S): ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.

Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium, and priming effect of progesterone and follicular fluid.

In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.