1 Laboratorio de Bioquímica and 2 Servicio de Ginecología, Hospital Galdakao, Barrio Labeaga, 48960 Galdakao, Vizcaya, Spain
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Abstract |
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Key words: automated semen analysis/semen quality/sperm motility index/sperm quality analyser
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Introduction |
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In the last few years, several automated systems known as computer-assisted semen analysis (CASA) have become available. These systems, besides analysing the conventional parameters, supply a description of sperm kinematic movements (Boyers et al., 1989; Holt et al., 1994
). A number of reports have demonstrated the prognostic and diagnostic value of these systems (Aitken et al., 1985
; Feneux et al., 1985
; Jeulin et al., 1986
; Barratt et al., 1993
; Aitken et al., 1994
; MacLeod and Irvine, 1995
). However, CASA instruments are not ready-to-use robots, and their reliability depends largely on the training, ability and experience of the user. In addition, the high cost of this equipment places it out of reach of most routine laboratories (Knuth et al., 1987
; Comhaire et al., 1992
; Holt et al., 1994
; Kraemer et al., 1998
).
The sperm quality analyser (SQA) is a simple device with no training requirements that performs an indirect analysis of sperm movement. The SQA has a photoelectric cell that detects the optic density fluctuations caused by the motility of spermatozoa. The analogue signal registered is converted digitally to provide the sperm motility index (SMI) value (Bartoov et al., 1991), which is affected by the number of spermatozoa and by the type of motility. Recently, a new version of the same device has appeared (SQA-IIB), incorporating a series of algorithms to calculate the traditional parameters of semen assessment on the basis of SMI measured value. There are several evaluations of the first model (Johnston et al., 1995
; Shibahara et al., 1997
; Mahmoud et al., 1998
) comparing SMI parameter with the traditional parameters and the result of several assisted reproduction techniques. At present, only one evaluation of this new model has been reported (Makler et al., 1999
) where the three parameters obtained from the traditional method and the SQA-IIB were compared. The aim of the current study was to evaluate this new model and to study its applicability to basic semen analysis, which has not been done yet.
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Materials and methods |
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Semen analysis
After semen liquefaction for 30 min, sperm concentration, motility and morphology were determined at room temperature. Selected sperm suspensions were also analysed after the preparation process.
Sperm concentration and motility were measured objectively by means of the multiple exposure photographic method (MEP) described by Makler (Makler et al., 1984), using a phase contrast microscope, stroboscope and Polaroid photographic camera. The integration of photographs was done with the Andros software (Micron Espana, Barcelona, Spain) on digitized tablet connected to a computer. The Andros software is presented as a menu with nine options. With the aid of a digital pencil the number of motile and immobile spermatozoa and the trajectories of motile spermatozoa were entered. The semen volume and the number of counted squares are then introduced in the corresponding option in order for the Andros system to calculate the following data: effective distance (ED) mean/s, maximum and minimum ED, average velocity, index of directionality and percentage of motility grades a, b, c and d. Criteria for the different grades of sperm motility were established as a function of the ED/s: grade a (>26 µm/s), grade b (1626 µm/s), grade c (<16 µm/s) and grade d (immobile) (Arán et al., 1990
). The derived variable `concentration of progressively motile spermatozoa' (CPMS) was calculated by multiplying the sperm concentration by the sum of motility grades a and b (progressive motility) expressed as percentages.
Morphology was evaluated on air-dried smears, fixed and stained by the quick-stain technique (Diff Quick; Quick-Panoptic, Amposta, Spain). World Health Organization criteria (WHO, 1992) were used to consider a spermatozoon as morphologicaly normal or abnormal. The result was expressed as percentage of spermatozoa with normal morphology examining at least 200 spermatozoa.
Sperm preparation
Semen was centrifuged at 200 g for 15 min through discontinuous Percoll gradients (40/80%) (Pharmacia, Uppsala, Sweden) and washed with Ham's F-10 medium (Biochrom), Berlin, Germany) supplemented with bovine serum albumin (BSA; Merck, Darmstadt, Germany). The spermatozoa were resuspended in 0.5 ml Ham's F-10 medium/BSA.
Sperm motility index determination
SMI determinations were performed at room temperature after routine analysis using the sperm quality analyzer (SQA-IIB; Medical Electronic Systems Ltd, Migdal Haemed, Israel). The semen sample was aspirated into a specifically designed capillary tube and then introduced into the analyser. The SQA has a photoelectric cell that detects the variations in optical density (OD) caused by the motility of spermatozoa. The SQA does not analyse the morphological defect (head problem, tail problem, acrosome staus, etc.). It only indicates what percentage of spermatozoa is normal. This is done not directly but through analysis of motion, i.e. if cell metabolism (energy) seems normal but motion is not progressive (circular, zig-zags, shaking only, etc.), the unit concludes that there is some defect in morphology. The measurement is performed over a 40 s period comprising four 10 s measurement periods, offering a mean of these four readings as a final result of SMI.
The new version (SQA-IIB) integrates a series of mathematical algorithms supplying the total sperm concentration, percentage of progressive motility, percentage of normal forms and the total functional sperm concentration (TFSC). The only parameter measured was the SMI, while the other four parameters are calculated. In order to compare the concentration of progressively motile spermatozoa obtained by two methods, we calculated SQA-CPMS multiplying sperm concentration by percentage of progressive motility provided by SQA-IIB.
According to the manufacturer's instructions semen samples with SMI <80 were abnormal, 80160 were doubtful and >160 were normal. With SMI readings of <20, corresponding to very low quality semen, the SQA-IIB did not provide the parameters sperm concentration, percentage of progressive motility and normal morphology.
Intracapillary imprecision assay
In order to evaluate the stability of readings within the same capillary tube, six samples with different quality semen were analysed (samples 1 to 6) in 2 min intervals, repeating the measurement 20 consecutive times.
Intercapillary imprecision assay
Three semen samples (A, B and C), with low, medium and high values of SMI, were analysed, assaying 20 different capillary tubes in each case.
Statistical analysis
A descriptive table was printed to show the median and range of the data obtained from the 89 semen samples and the 53 Percoll selected spermatozoa using the two methods compared. The imprecision study of the measurements performed by the SQA-IIB both intracapillary and intercapillary included the mean and coefficient of variation (CV) for each parameter. In the comparison study between methods, linear regression analysis was used to compare SMI with CPMS from the laboratory method. On the other hand, the Spearman correlation coefficient was applied to study the relationship between the studied parameters using the laboratory method and the SQA-IIB. Two-by-two tables were used to group semen samples according to the SMI test in relation to CPMS, and the sensitivity and specificity of the new method were determined.
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Results |
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The linear regression analysis of the relationship between SMI and CPMS indicated that 73.2% of the variance of SMI could be accounted for by the CPMS alone. The linear regression was: CPMS = 0.13xSMI 1.54, R2 = 0.732.
In order to study the equivalence between CPMS and SQA-CPMS, correlation and linear regression analysis were performed. A correlation coefficient of 0.87 (95% CI: 0.820.92) was found and the linear regression was: CPMS = 0.41x(SQA-CPMS) + 6.1, R2 = 0.714.
Detection limit and linearity
In five semen samples the SQA-IIB showed an SMI value less than 20: two cases with zero value were azoospermic and in the other three, for an SMI equal to 0, 13 and 18, CPMS values of 0.45, 0.78 and 0.42x106/ml respectively (mean = 0.54x106/ml) were obtained. Establishing the limit at 0.54x106/ml CPMS we found only one case with a value lower than this limit (0.42x106/ml) where the SMI reading was 22, very close to the reading limit of the SQA-IIB.
Since high correlation between SMI and CPMS with Percoll selected spermatozoa disappeared with values higher than 75x106/ml, a limit of 75x106/ml for CPMS was established corresponding to an SMI of 500. A SMI value higher than 500 was not found in unprocessed semen and only one semen sample had a CPMS value of 79.2x106/ml with an SMI of 432. In the case of Percoll selected spermatozoa, five cases out of 31 with CPMS lower than 75x106/ml had an SMI value higher than 500, and otherwise only two out of 21 cases with CPMS higher than 75x106/ml had an SMI value lower than 500.
Diagnostic usefulness of SMI
Because of the excellent correlation existing between SMI and CPMS, the diagnostic usefulness of the SMI was studied. The population was divided into two groups using as threshold value a CPMS of 10.4x106/ml, the value corresponding to the 10th percentile of the reference values established in our laboratory analysing semen from men with proven fertility within 2 years before the analysis.
Table V shows the two-by-+two tables grouping the values using threshold values recommended by the manufacturer (80 and 160). For an SMI threshold value of 80, the sensitivity was 80% and the specificity was 98%. When a threshold value of 160 was used, the sensitivity increased to 96% and the specificity remained at 84%.
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Discussion |
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Several quality control programmes implemented in the last 10 years revealed that the results were highly scattered among different laboratories, among different technicians from the same laboratory and even within the same technician. Thus, very different CV are found in the literature. In intra- and inter-technician studies, sperm concentration ranged from 6 to 12%, motility from 5.5 to 28.5% and morphology from 5.1 to 27.7%. Comparing different spermatozoa counting chambers, CV moved from 4.3 to 26.3% and in external quality control studies CV from 8 to 37% were found. The lowest CV were found in intra-technician studies, related to observer ability and training. Higher values in inter-technician studies, reaching clinically significant differences were found. Also, unacceptably high values were observed in multicentric and external quality control studies that rendered the results from different laboratories not interchangeable (Dunphy et al., 1989; Menkveld et al., 1990
; Neuwinger et al., 1990
; Cooper et al., 1992
; Matson et al., 1995; Mahmoud et al., 1997
).
A high variation was found in CASA systems, mostly because these results depended on the user training and expertise. Clements (Clements et al., 1995) obtained for the HamiltonThorn motility (HTM) analyser a coefficient of variation for concentration of 9.5%, for fast motility 11.8% and for slow motility 67.3%.
Our experience with the SQA-IIB showed, in the imprecision assay with a single capillary tube, a good repeatability for the SMI value; the CV ranged from 3.9 to 11.3%, values that also remained low for the calculated parameters included: sperm concentration, percentage of motile spermatozoa and morphology. These low CV found in the intracapillary assay demonstrated the sample stability in the capillary for at least 40 min. One of the six semen samples showed a very high variation in the readings performed in the same capillary tube, with a CV value for SMI of 75.7%. This sample had an immunological factor causing instability in the measurements. Every basic semen study has to include a test to investigate the presence of antisperm antibodies (WHO, 1992) and, if a positive result is observed, the use of the SQA-IIB would be ruled out.
In the intercapillary study, slightly higher CV values were found for the SMI: from 7.7 to 18.8%. This imprecision resulted from the variation due to the instrument itself and that introduced by the user when charging the capillary tube; however, it did not reach the high variations published for the traditional parameters and methods (Matson, 1995; Johnson et al., 1996
; Mahmoud et al., 1997
). The results for overall imprecision in the current study, including that from intra-assay, inter-assay and inter-observer, were 7.4% for sperm concentration (mean = 91.6x106/ml), 13.41% for progressive motility (mean = 35.64%) and 16.13% for CPMS (mean = 32.72x106/ml), which corresponded to a high SMI value.
The linearity of the SQA-IIB with Percoll selected spermatozoa was studied, as a way to achieve high concentrations of progressively motile spermatozoa. It was found that this limit was achieved with a CPMS value equal to 75x106/ml corresponding to an SMI value of 500. With higher values the excellent correlation disappeared (r = 0.39). Since similar correlation coefficients were found between SMI and CPMS in unprocessed semen and selected spermatozoa (r = 0.87 and 0.86 respectively), the same value was applied to unprocessed semen. None of the 89 unprocessed semen samples had an SMI value higher than 500 and only one case exceeded the CPMS value of 75x106 ml, which was 79.2x106/ml, very close to the limit. The results were different to those of Bartoov (Bartoov et al., 1991) who published a linearity corresponding to 40x106/ml motile cells without specifying the motility type. The data presented here established that the SQA-IIB readings were linear up to SMI values of 500 with selected spermatozoa. However, it would be necessary to confirm this linearity limit with unprocessed semen samples with high CPMS.
Another subject of the evaluation was the detection limit. A SMI limit of 20 is given by the manufacturer. With SMI values lower than 20 the remaining parameter values are zero. SMI readings between 0 and 20 would indicate the presence of scarce motile spermatozoa, as it was observed in two cases showing SMI of 18 and 13 with CPMS of 0.42 and 0.78x106/ml respectively. SMI readings equal to zero would correspond to an absence of motile spermatozoa or azoospermia, a situation that was not fulfilled in one case in which a CPMS value of 0.45x106/ml was found for an SMI equal to zero. This would invalidate the use of the SQA-IIB in post-vasectomy controls, since there can be cases with the presence of spermatozoa and a zero SMI result; however, the manufacturer noted that in these cases repeating the reading up to five times in the same capillary tube, at least one reading would be different to zero. The opposing case was not found in this study, i.e. absence of spermatozoa and an SMI value not equal to zero. With the five semen samples with an SMI value lower than 20 a CPMS limit of 0.54x106/ml was established, below which the SQA-IIB would not be able to distinguish the presence or absence of motile spermatozoa. The number of semen samples used to establish this limit was very low, but more cases were not available, so more data would be required in order to ascertain this with more precision.
The correlation study performed on unprocessed semen samples between the SQA-IIB and the laboratory method showed that the best relationship was established between the SMI and CPMS (r = 0.87), slightly lower than the correlation found by other authors (r = 0.92) (Johnston et al., 1995). This high correlation would be expected since the SQA-IIB measures moving spermatozoa.
When comparing SMI with sperm concentration and progressive motility percentage lower correlation coefficients were found, 0.75 and 0.64 respectively. This was due to the fact that in both parameters non-motile spermatozoa were involved and the SQA-IIB system was unable to detect them. Similar correlation coefficients were found when comparing sperm concentration and percentage of progressively motile spermatozoa with both methods (r = 0.75 and 0.64). This would also be expected since the SQA-IIB calculates both parameters from the SMI, repeating the same concentration and percentage of motility values for the same SMI value. However, the experience in the andrology laboratory shows that it is not possible to establish a mathematical relationship between concentration and motility. In relation to morphology the correlation coefficients found were drastically reduced, r = 0.45 with SMI and r = 0.37 between both methods. So, the SQA-IIB was found to be unsuitable for obtaining the main parameters in the analysis of semen, i.e. spermatozoa concentration, motility and morphology.
The study between the derived variables CPMS and SQA-CPMS showed a good correlation (r = 0.87), but the linear regression analysis demonstrated no identity between them, with a slope of 0.41. So, the same parameters were not being measured. The SQA-CPMS would have a similar significance as the SMI had; however, it might introduce confusion with the true CPMS parameter.
The results of the current study agreed with those obtained by Makler (Makler et al., 1999), who observed important deviations among the three traditional parameters measured by both methods and rejected the use of SQA-IIB in routine semen analysis. The only objective of Makler et al. (1999) was to compare the three main sperm parameters obtained according to the standards of the WHO and those measured by the SQA-IIB, but they did not go any further in evaluating SQA-IIB. Seventeen out of 43 semen samples studied by Makler were manipulated. The manufacturer states that with these types of samples the only parameter to be considered is the SMI. In the current study technical characteristics of SQA-IIB were evaluated and 89 unprocessed semen samples compared. The satisfactory imprecision and the good correlation observed between SMI and CPMS encouraged further investigation its clinical usefulness in the male factor study.
The sensitivity and specificity of both SMI threshold values supplied by the manufacturer (80 and 160) were investigated. Thus a CPMS threshold value of 10.4x106/ml was used, the value corresponding to the 10th percentile of reference values established in our laboratory from semen from men with proven fertility within 2 years before the analysis. Considering the SMI value of 80, 80% sensitivity and 98% specificity were obtained, values different from those obtained by Bartoov (Bartoov et al., 1991), who found a 93% sensitivity and 85.5% specificity, but using a threshold value for sperm concentration of 20x106/ml. Using the SMI value of 160 a 96% sensitivity and 84% specificity was achieved in the current study. Therefore, using 160 as threshold value to differentiate fertile and subfertile semen, only one semen with CPMS of 7.6x106/ml remained misclassified (Table V
). This suggested that it would only be necessary to assess sperm concentration and motility by microscopy in those semen samples showing SMI lower than 160. CPMS is a parameter that can be used to differentiate between fertile and subfertile semen (Hinting et al., 1988
). It has been related to success in intrauterine insemination (Brasch et al., 1994
). Also it has been demonstrated to be able to discriminate between in-vitro fertile and unfertile semen (Comhaire et al., 1988
). CPMS was also recommended by the WHO manual in its latest edition (WHO, 1999) as derived variable. All this, together with the high correlation found between SMI and CPMS, and the good performance of the instrument, permits consideration of the SMI value as a screening test for the global measurement of concentration and progressive motility of spermatozoa.
In conclusion, the SQA-IIB was a very easy to use, manageable and cheap device, that required no trained personnel, was not time consuming and showed a good performance. The SMI parameter allowed discrimination, with high sensitivity, between fertile and subfertile semen samples according to CPMS. For all these reasons, it is concluded that SMI was a good screening test to rule out oligozoospermia and asthenozoospermia in the male factor study in the context of sterility clinics, requiring the assessment of sperm concentration and motility by microscopy in all those semen samples showing an SMI value lower than 160 and higher than 500. It should be noted that it was not a valid method for evaluating morphology, in which case WHO methods would have to be followed (WHO, 1999).
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Acknowledgments |
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Notes |
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References |
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Submitted on January 26, 2000; accepted on April 26, 2000.