Computer-aided sperm analysis: past, present and future.

Computer-aided sperm analysis (CASA) system has been accepted and used commonly as a routine semen analysis instrument in hospital clinical laboratories worldwide. However, technicians in clinical laboratories have little informed knowledge about the principles of CASA system and the sources of analysis errors. In this review, we focus on the concept of CASA, the development course of CASA technology, the clinical application of CASA systems and the factors influencing the accuracies of results, such as frame rate, sperm counting chambers affiliated to the CASA system, algorithms and sperm concentration. These factors and lack of internal quality control may result in huge errors of the CASA between systems and laboratories. It is therefore necessary to perform the standardisation and quality control for CASA.

Comparison of the semen analysis results obtained from two branded computer-aided sperm analysis systems.

The study evaluated the comparability of two branded computer-aided sperm analysis (CASA) systems commonly used in andrology laboratories in China. The same semen sample was analysed using two branded CASA systems (WLJY-9000 and CFT-9200) by one well-trained technician. Results of semen analysis obtained from two branded CASA systems were then compared. The accuracy of counting results of CASA systems was evaluated using latex bead solutions with known concentrations of (35 ± 5) × 106 ml⁻¹ and (18 ± 2.5) × 106 ml⁻¹. There were significant differences in all parameters (P < 0.01) except for LIN and WOB. The counting results of CFT-9200 were close to the standard solutions [(38.86 ± 3.79) × 106 ml⁻¹ and (19.03 ± 1.99) × 106 ml⁻¹], while those of WLJY-9000 were underestimated [(28.53 ± 2.06) × 106 ml⁻¹ and (14.62 ± 0.95) × 106 ml⁻¹]. But the coefficient of variation of WLJY-9000 was lower than that of CFT-9200 (7.22%, 6.50% vs. 9.82%, 10.46%). It is concluded that factors such as parameter settings and evaluation algorithms could significantly affect the results obtained from these two branded CASA systems. Great attention should also be paid to the quality control in semen analysis with CASA.

Examination of the semen quality of patients with uraemia and renal transplant recipients in comparison with a control group.

To examine the semen quality of patients with uraemia and renal transplant recipients, 40 patients with uraemia and 40 renal transplant recipients were included. According to their interval of post-transplantation, renal transplant recipients were subdivided into group A (22) < or =2 years and group B (18) >2 years. A total of 40 healthy men with normal fertility were included as the controls. Semen samples from all subjects were collected and analysed. The fertility index (FI) value was calculated. The FI value of the normal fertility men was 13.02 (14.26), that of the renal transplant recipient groups A and B were 5.53 (8.30) and 9.27 (22.49) respectively, while the FI of the patients with uraemia was 0.23 (0.76). Compared with the uraemia group, the FI values of renal transplant recipient group either group A or group B were significantly better (P < 0.01). However, compared with the normal control group, the FI values of renal transplant recipient group A were lower (P < 0.01), while there was no significant difference between group B and the control group (P > 0.05). In conclusion, the FI of renal transplant recipients was recovered close to the level of healthy men with normal fertility 2 years after transplantation.

Is flow cytometry really adapted to the determination of sperm concentration?

It has been reported that flow cytometry can be used as a reference procedure to determine sperm concentrations in quality control schemes in andrology laboratories, but there are no convincing quality control data. To understand comprehensively whether flow cytometry can be used to determine sperm concentration, sperm concentrations of 85 human semen samples were detected using three different methods, namely flow cytometry, computer-assisted semen analysis (CASA) and manual counting with a cell-VU chamber. The bead concentrations of both low [(18+/-2.5)x10(6)/mL] and high [(35+/-5)x10(6)/mL] pre-calibrated standard latex bead solutions were also determined with flow cytometry. The results showed that bead concentrations of both low and high pre-calibrated standard latex bead solutions counted five times with flow cytometry were (21.37+/-0.85)x10(6)/mL and (45.95+/-1.76)x10(6)/mL, respectively. Coefficient variances (CVs) and relative errors (REs) were 4%, 15.51% and 3.84%, 31.3% for low and high latex bead solutions, respectively. The overall correlation between values measured with flow cytometry and values measured with the cell-VU chamber and the CASA system was significant. However, flow cytometry overestimated the sperm concentration by 109% compared to the results with the cell-VU chamber. Moreover, for the azoospermic samples analysed, the sperm concentration was estimated at 0.12 (range from 0.04 to 0.24)x10(6)/mL. In conclusion, the data demonstrated that flow cytometry can result in an overestimation of both bead counting and sperm concentration, suggesting that flow cytometry is an inappropriate method for sperm counting, especially in the case of azoospermia.

Preliminary investigations on the standardisation and quality control for the determination of gamma-glutamyltranspeptidase activity in seminal plasma.

The present study was designed to assess the effects of centrifugation velocity, standing time after dilution, freezing-thawing and chymotrypsin on the determination of gamma-glutamyltranspeptidase (gamma-GT) activities in seminal plasma, and to establish an instruction for the standardisation and quality control for the determination of gamma-GT within the same laboratory and among different laboratories. The gamma-GT level and sperm concentration of each of 72 samples of seminal plasma obtained by centrifugation at 1000 g for 10 min or 3000 g for 15 min were assayed. In addition, gamma-GT activities in diluted seminal plasma with different standing time and in samples with or without chymotrypsin were measured. The results showed that there was a significant difference of gamma-GT levels in seminal plasma obtained by centrifugation at different velocities (P < 0.001), and that gamma-GT activities in seminal plasma measured after standing for 30 min after dilution were notably lower than those measured immediately after dilution (P < 0.001). However, the data indicated that both chymotrypsin and freezing-thawing had no apparent effect on the determination of seminal gamma-GT. In conclusion, standing time after dilution and centrifugation velocity should be standardised, and frozen seminal plasma could serve as quality control products for the determination of gamma-GT activity among different laboratories.

The effect of chymotrypsin on the determination of total alpha-glucosidase activity in seminal plasma and the correlation between alpha-glucosidase level and semen parameters.

To evaluate the effect of chymotrypsin on the examination of alpha-glucosidase activity in seminal plasma, thirty-nine samples of fresh liquefied semen with or without chymotrypsin and forty-eight samples of fresh un-liquefied semen with chymotrypsin were determined for the total alpha-glucosidase activity in seminal plasma. The total alpha-glucosidase level of each sample was assayed by the method of glucose oxidase. The correlations between alpha-glucosidase level and semen parameters, including semen volume, pH, sperm concentration, grade a and b motility and total motility, were analyzed with SPSS 11.0 software. The results showed that chymotrypsin had no effect on seminal alpha-glucosidase activity determination. Chymotrypsin could improve the liquefaction for un-liquefied semen, and there was no significant difference of alpha-glucosidase activity between liquefied and un-liquefied semen samples. There were significantly positive correlations between seminal alpha-glucosidase activity (U/ml) and sperm concentration (r = 0.338, p = 0.015) and between total alpha-glucosidase activity (U/ejaculate) and semen volume (r = 0.677, p = 0.000). However, there was no significant correlation between alpha-glucosidase level (U/ml) and semen volume, pH, sperm motility or grade a and b motility (r = -0.234 approximately 0.077, p = 0.099 approximately 0.993). The data indicated that chymotrypsin could be added into the un-liquefied semen samples for alpha-glucosidase activity determination, and there were different correlations between seminal alpha-glucosidase level and various semen parameters.

Standardization and quality control for the determination of alpha-glucosidase in seminal plasma.

We attempted to provide an instruction for the standardization of alpha-glucosidase level determination and quality controls within the same laboratory and among different laboratories. Each of 51 semen samples was divided into two aliquots, centrifuging at 1000 g for 10 min or 3000 g for 15 min. The alpha-glucosidase level and sperm concentration of each sample were assayed. The alpha-glucosidase level in seminal plasma obtained at 3000 g for 15 min centrifugation was significantly lower than that at 1000 g for 10 min (p = 0.001). An additional 6 samples of seminal plasma with or without phenylmethylsulfonyl fluoride (PMSF), obtained at 3000 g for 15 min centrifugation, were frozen for 20 days, and each of 6 samples was determined for their alpha-glucosidase levels after thawing every other day. There was no significant difference between alpha-glucosidase levels in seminal plasma regardless of the presence of PMSF. The alpha-glucosidase level increased with the length of abstinence period. In conclusion, centrifugal velocity and abstinence time should be standardized in the alpha-glucosidase determination. Frozen seminal plasma may serve as the sample for quality control among clinical laboratories.

Inhibitory effects of human seminal plasma on an ELISA used to detect anti-sperm antibodies: implications for the determination of sperm quality.

The in vitro inhibitory effect of human seminal plasma on an ELISA used to detect anti-sperm antibodies have been observed. The mean inhibition rate of seminal plasma samples from 75 men was 61.5+/-23.1%. The inhibition rate of 29 samples from normal sperm group was 71. 14+/-18.25%, while that of 46 samples from the abnormal sperm group was 55.43+/-23.98%. The results show that human seminal plasma from semen with high quality sperms possesses a high inhibitory rate to anti-sperm antibody reactions, suggesting its efficiency for immunosuppression of humoral immune reactions. Its possible implications are discussed.

At the crossroad of conception and infection: initiatives for immunoglobulin-based contraceptive R & D.

Better understanding of the immunological mechanisms implying the insemination and the infertility of some men and women is needed and crucial to the development of an effective immunocontraceptive method. To provide good protection against conception or infection, and avoid any possible and unexpected complications which immunocontraceptive "vaccine" may arise of, it seems the right time for scientists to create a virtually new thinking for this extremely urgent and important issue. This conceptual article describes our original thoughts of the future development of immunocontraceptives, preferably, based on immunoglobulins rather than vaccines, against human sperm specific antigens and seminal plasma immunosuppressive factors. Its general correctness, advantages and feasibility for fertility regulation and prevention of infection are discussed.

A speculative view of immune recognition.

The speculation that immunologically reactive haptens must be those attached to carriers' immunodominant epitopes suggests a clearer mechanism by which the mysterious hapten-carrier phenomena are generated. This review focuses on the molecular biological nature of immune recognition of hapten-protein antigens both by the T-cell and the B-cell. T and B lymphocytes specifically recognize one determinant of the same antigen molecule in two different ways and in different circumstances. The B-cell recognizes an antigen by the preliminary antigen receptors on the cell's surface, at the time it is still intact, interiorizes it and presents the processed antigenic peptide after an antigen processing procedure. In contrast, the T-cell recognizes a hidden antigenic determinant, together with portions of the MHC on the presenting cell. The immune memory is mainly directed to the hidden internal determinant of an antigen. Some aspects of the clonal selection theory of antibody formation are also discussed at the modern molecular level.