Stability of sperm characteristics in men with disturbances in sperm quality

Transcription

1 Infernational Journal of Andrology, 1989, 12. pages Stability of sperm characteristics in men with disturbances in sperm quality K. PURVIS, A. TOLLEFSRUD and H. RUI Andrology Laboratory, Institute of Pathology, The National Hospital, Oslo, Norway Summary Sixty-one men referred to our laboratory for semen analysis, and subsequently judged to exhibit some form of sperm pathology, were asked to return for a second analysis, not less than 2 months after the first, in order to assess the stability of the pathological changes observed. In almost half of the cases, the referring physician had, on his own initiative, started hormone or antibiotic treatment. The sperm parameters studied included sperm count, sperm motility judged by laser-doppler spectroscopy, and sperm morphology and viability. The motility characteristics included percentage motile, their average velocity, and percentage swimming in a progressive manner, and their progressive velocity. In untreated subjects, there was no significant difference between the first and second analysis in any of the sperm parameters measured. This was also true for both oligozoospermic individuals (<20 X lo6 sperm/ml) and the group with higher sperm concentrations. All parameters were highly correlated on the two occasions. The average coefficients of variation of the paired observations were highest for sperm count (approximately 25%) and lowest for sperm velocities and the proportion of abnormal and viable cells in the ejaculate (1-9%). No major differences in the extent of variation could be detected between the low and high sperm density groups. In general, the unsystematic antibiotic and hormone regimens (clomiphene or androgen) used by the referring physicians had no discernable effect on any aspect of sperm quality, indicating the need for more controlled and standardized programmes of treatment. Keywords: sperm motility, sperm count, sperm morphology, pathology, antibiotic treatment, hormone treatment. Introduction In normal fertile men there can be considerable within-individual variation of sperm quality on different occasions. However, there is little information on the variability of sperm parameters in men with sperm pathology, an important consideration for the diagnosis of the infertile condition and for the predictive value of the findings of the first investigation. In the present study, a random group of men, who on the first visit to our laboratory had shown disturbances in sperm quality, Correspondence: Dr K. Purvis, Institutt for Patologi, Rikshospitalet, 0027 Oslo 1, Norway. 171

2 172 K. Purvis, A. Tollefsrud and H. Rui were asked to return for a second examination not less than 2 months after the first. In some of the subjects, the referring physician had taken the initiative to begin hormone or antibiotic treatment in the interim period. This also provided an opportunity to evaluate the effects of these uncontrolled and unsystematic treatments. Materials and methods Subjects Sixty-one men, partners of barren couples under investigation for their infertility, were used in the study. Their ages ranged between 25 years and 35 years. A semen sample was provided in the laboratory after an abstinence period of at least 3 days. After the initial examination, they were asked to return for a second evaluation after a period of at least 2 months. The mean interval was 3.5 months (range = 2-12 months). The disturbances in sperm quality noted included: different grades of asthenozoospermia, teratozoospermia, oligozoospermia or different combinations of these in the presence of significant quantities of leucocytes (> 1 x 10h/ml). In the interim period, in over half of the cases (34), the referring physician had initiated treatment of varying duration with antibiotics or hormones, or in some cases, a combination of the two. Table 1 summarizes the different treatments which were initiated. The treatment regimen chosen reflected the personal preference of Table 1. Treatment of patients with sperm pathology by the referring physician Treatment No treatment Antibiotic treatment Tetralysal* (0.3 g x 2 for 21 days) Tetralysal (0.3 g for days) Tetralysal (0.3 g for 20 days) Doxylin* (10-21 days) Trimetoprim (160 mg X 2 for 3 months) Vibramycin* (100 mg for 20 days) Doxylin/Flagylt Bactrimt Hormone treatment Clomiphene (SO mg for 3 months) AndroxoneS: (40 mg x 3 for 3 months) Androxone/Clomiphene Antibiotidhormone combination Tetralysal (0.3 g x 2 for days followed by Clomiphene 50 mg for 3 months) *Tetracycline preparations. t Metronidazol. STrimethoprim-sulphamethoxazole, $Testosterone undecanoate. Number S 2 1 2

3 Stability of sperm parameters 173 the referring doctor. Antibiotic treatment was invariably started when the first examination had revealed the presence of leucocytes in significant numbers in the ejaculate. Quantitation of motility After a liquefaction period of 30 min at room temperature, 40 pl aliquots of the ejaculate were transferred to a Lazymot sperm motility analyser (BTG, Dusseldorf, FRG) and the following motility characteristics computed: (a) the percentage of sperm exhibiting all forms of movement, (b) the average velocity of all motile sperm, (c) the proportion of motile sperm swimming in a progressive fashion (with a velocity exceeding 15 ym/sec), and (d) the average velocity of those swimming progressively with a significant speed (> 15 pm/sec). The apparatus uses a method based on the laser-doppler technique and quantifies movement by measuring alterations in the frequency of light reflected from the surface of the sperm. At least five replicate measurements were made on each sample. The mean coefficients of variation were 5.0, 2.4, 4.6 and 1.3% for percentage motility, average velocity, percentage progressive and progressive velocity, respectively. The velocity limit of 15 ym/sec chosen to define the progressively motile group is programmed into the lazymot apparatus and cannot be adjusted. Quantitation of other parameters Immediately after liquefaction, the viscosity of the semen samples was assessed by calculating the time (in seconds) for drop formation to occur from a capillary of standard size (50 ~ 1; Accupette Pipets, Dade Diagnostics Inc., Miami, U.S.A.). Aliquots of the sample were also subjected to vital staining using eosin Y and nigrosin as described by Blom (1950), and were stained with Harris haemotoxylin for the assessment of morphology. Sperm with undamaged oval heads, undamaged and uncoiled tails, and acrosomes covering one-half to one-third of the sperm head were classified as normal. Abnormal sperm were defined as microcephalic, macrocephalic, bicephalic, bicaudal and possessing a coiled tail or a deformed or abnormally small acrosome. As the type of morphological abnormality does not appear to change with sperm count (Singer et al., 1980), the total number of abnormal forms in an ejaculate were computed and used in the various statistical analyses. Sperm counts were made using a Burker chamber after diluting and immobilising the sperm in 5% chloramine-t (1:lO). The concentration of peroxidase-positive cells (leucocytes) in the samples was determined as described elsewhere (Comhaire, Verschrgen & Vermeulen, 1980). Statistical analysis Since the various sperm characteristics were not normally distributed (data not shown), all data were logarithmically transformed prior to the analyses, a modification which had been shown previously (personal observations) to normalize the sperm variables. A paired t-test was used to test the differences between values from the two semen samples. Spearman Rank correlation analyses were carried out on the combined data from the untreated individuals, and coefficients of variation for the various sperm characteristics were also computed.

4 174 K. Purvis, A. Tollefsrud and H. Rui Results Untreated subjects Table 2 summarizes the sperm characteristics of the oligozoospermic subjects (< 20 x lo6 cells/ml) and subjects with a higher sperm count but with astheno- and/or teratozoospermia on the two occasions. Analysis by paired t-test failed to demonstrate a significant difference between the two occasions in either group. The coefficients of variation and correlation coefficients of the duplicate measurements for the various sperm parameters are shown in Table 3. In general, variables other than sperm count were more stable in the oligozoospermic subjects than in men with higher sperm counts. As indicated, the parameters with the least variability between the two occasions were the proportion of cells with abnormal morphology and the sperm velocities. In the oligozoospermic group, the viscosity of the ejaculate and the proportion of living sperm were also relatively stable features. All sperm parameters were highly correlated with each other on the two occasions, when all data were pooled. Table 2. Sperm variables of oligozoospermic subjects (<20 X 10' spermiml) and subjects with >20 X 10' spermiml with astheno- or teratozoospermia on two occasions, compared to men with normal sperm quality Parameter Oligozoospermic (n =8) Normozoospermic (n =19) Normal (ti =99) Sperm density (10"iml) Total count (10") Abnormal forms (%) First Second First Second occasion occasion occasion occasion ( ) ( ) ( 15.%45.O) ( ) (S ) ( ) 51.8 ( ) 65.9 ( ) 5.4 ( ) 4.0 ( ) ( ) Living sperm (%) ( ) ( ) Viscosity (sec) 5.4 ( ) 5.3 ( ) Volume (ml) (3.65.8) ( ) ( ) Motile (%) ( ) ( ) ( ) Mean velocity (ymisec) ( ) ( ) (31.&38.0) Progressive (%)* ( ) ( ) ( O) Progressive velocity ( ymlsec) ( ) ( ) (59.e66.6) 68.6 ( ) ( ) 53.2 ( ) 57.0 ( ) 4.4 (3.65.4) 3.9 ( ) 51.8 (45 Lk59.7) 32.4 ( ) 18.0 ( ) 61.1 ( ) ( ) ( ) 32.2 ( ) 77.1 ( ) 4.4 (4.C-4.8) 3.9 (3.64.2) 76.7 ( ) 41.8 ( ) 36.1 ( ) 68.8 ( ) Values are geometric means after logarithmic transformation, with 95% confidence limits in parentheses. *Percentage of sperm swimming with a velocity exceeding 15 ymisec.

5 Stability of sperm parameters 175 Table 3. Coefficients of variation and correlation coefficients of sperm variables in untreated subjects with either <20 X 10' spermsiml. or >20 X 10' spermsiml, with sperm pathology on two occasions at least 2 months apart Parameter Mean coefficients of variation <20 X 10" spermiml >20 X 10' spermlml Correlation coefficients of combined group (n =27) Sperm density (X IO'lml) $ Total count ( x 10') Abnormal forms (76) $ Living sperm (%) t Viscosity (sec) t Volume (ml) $ Motile (Ye) t Mean velocity (pmlsec) * Progressive (%)* $ Progressive velocity (Kmlsec) t *Percentage of sperm swimming with a velocity exceeding 15 pmlsec. t P < $P< Table 4. Sperm variables of subjects before and after antibiotic or hormone treatment Parameter Antibiotic treatment (n =18) Hormone treatment (n =7) Sperm density ( x 1O'iml) Total count (X 10') Abnormal forms (%) Living sperm (Yr) Viscosity (sec) Volume (ml) Motile (5%) Mean velocity (pmisec) Progressive (%)* Progressive velocity (pmisec) Before After Before After 47.9 ( ) ( ) 44.6 ( ) 69.2 ( ) 3.7 ( ) 3.8 ( ) 42.9 ( ) 30.3 ( ) 15.0 ( ) 58.1 ( ) 35.3 ( ) ( ) 50.5 ( ) 60.3 ( ) 4.2 ( ) 3.7 ( ) 40.7 ( ) 31.0 ( ) 17.3 ( ) 60.7 ( ) 5.1 ( ) 26.7 ( ) 70.6 ( ) 77.0 ( ) 4.3 ( ) 5.2 ( ) 15.4 ( ) 22.0 ( ) 4.8 ( ) 46.9 ( ,8) 4.8 ( ) 23.2 ( ) 54.3 ( ) 70.6 (63.%78.0) 4.8 ( ) 5.0 ( ) 11.7 ( ) 21.6 ( ) 4.2 ( ) 50.3 ( ) ~~ ~~ ~~ ~~~~ Values are geometric means after logarithmic transformation, with 95% confidence limits in parentheses. *Percentage of sperm swimming with a velocity exceeding 15 pm/sec.

6 176 K. Purvis, A. Tollefsrud and H. Rui Treated subjects Antibiotic treatment of those men with leucocytes present in the ejaculate, and hormone treatment of those with severe grades of oligo-, astheno-, or teratozoospermia, failed to significantly alter the sperm characteristics between the two occasions (Table 4). In 67% of the men who had undergone antibiotic treatment no leucocytes could be detected in the ejaculate on the second occasion. In one subject there was a reduction in leucocyte count, but in the remaining subjects the concentration was unchanged in spite of the treatment. There was no association between the type of antibiotic used and the disappearance of leucocytes from the ejaculate. Discussion Several studies have attested to the variability of sperm count within fertile individuals (Hotchkiss, 1941; Freund, 1962; Schwartz ef al., 1979). Coefficients of variation of over 50% have been reported for repeated sperm counts over a period of 1 year (Overstreet, 1984), and Working & Levine (cited by Working, 1988) reported coefficients of variation as high as 89% for sperm counts from over 500 semen samples from 159 men. Within-individual variation in the percentage of motile cells appears generally to be lower, with coefficients of variation of 27% (Katz, Overstreet & Pelfrey, 1982) and 35% (Working & Levine, cited by Working, 1988). In contrast, the proportion of sperm exhibiting abnormal morphology has been considered by some to be so stable that it can be used to identify individuals (MacLeod, 1964). Information on within-individual variation in sperm quality has largely been obtained from healthy men, fertile donors, men undergoing vasectomy or men with unspecified fertility. Read & Schneiden (1978) reported marked variation in sperm count in infertile men, but this was using a Coulter counter which, as the authors admitted, can be prone to error in the presence of cell debris. In contrast to many of the findings described above, the present study revealed that, in men exhibiting abnormalities in one or more aspects of sperm function or morphology, there is a remarkable constency in all sperm characteristics, although to varying degrees. Of the variables studied, it appeared that sperm velocities, the proportion of living and abnormal sperm, and the viscosity of the seminal plasma appeared to be the most stable parameters. However, all the parameters measured were correlated significantly on the two occasions. These findings are in close agreement with Poland ef al. (1986) who concluded that sperm count, motility and morphology were highly stable over time and could be used as identifying characteristics. Hartman, Schoenfeld & Copeland (1964) came to a similar conclusion with regard to the proportion of abnormal cells in the ejaculates of infertile men. In men with depressed sperm quality, the degree to which the results of a second semen analysis could be predicted was closely related to the extent to which the sperm quality deviated from normal on the first occasion (Comhaire et al., 1987). Therefore, men with a sperm concentration of <5 x 106/ml, with a sperm motility <29%, and with abnormal forms >60% had a 69.4, 70.2, and 90.2% chance, respectively, of exhibiting a sperm quality in the same range on the second occasion. With increasing sperm qualities which were still outside normal

7 Stability of sperm parameters 177 standards, the chance of falling in the same narrow range of abnormal quality was gradually reduced. A fertility evaluation usually takes place after several years of attempts to obtain a child. In many cases, a selection has therefore already taken place and poor sperm quality has become a stable feature if responsible for the infertile status. Under such circumstances, it appears that the results of the first investigation can, with a high degree of certainty, predict the status on the second occasion. It should, however, be emphasized that in our own studies, the semen samples were collected under standardized conditions at the laboratory with the same periods of abstinence. In addition, sperm motility was assessed objectively rather than subjectively which is acknowledged to give variable results. When subjects were given antibiotic or hormone treatment by the referring physician, no major change in any of the variables could be detected. The small number of individuals involved mean that no firm conclusions can be reached as to the efficacy of the preparations in improving sperm quality, although it does imply that, in general, poor sperm quality is relatively resistant to major changes. However, in the case of antibiotic treatment, the study does underline the differences in choice of both the preparation and the treatment regimes employed when it is left to the referring physician. A more standardized regime of antibiotic treatment over extended periods may be necessary before an alteration in sperm quality can be detected. On the other hand, if a chronic infection of the sex glands is responsible for the poor sperm quality and infertility, especially after such a long delay between the assumed start of the infection and first consultation, the intlammatory changes in the reproductive tract may be of a more permanent nature and too late to reverse. The present study indicates that: (a) poor sperm quality in men attending an infertility clinic is stable, and can in the majority of cases be predicted after the first semen examination, (b) the sperm velocities and the percentage of abnormal sperm in the ejaculate appear to be the most stable parameters between the two occasions, and (c) there is a great need to standardize preparations and treatment regimes by referring physicians. Acknowledgment Financial support from the Norwegian Council for Science and the Humanities (NAVF) is acknowledged gratefully. References Blom, E. (1950) A one-minute live-dead sperm stain by means of eosin nigrosin. Fertility and Sterility, 1, Comhaire, F. H., de Kretser, d., Farley, T. M. M. & Rowe, P. J. (1987) Towards more objectivity in diagnosis and management of male infertility. International Journal of Andrology. 7(suppl). 36. Comhaire, F. H., Verschrgen, G. & Vermeulen, L. (1980) Diagnosis of accessory gland infection and its possible role in male infertility. Internafional Journal of Andrology. 3, Freund, M. (1962) Interrelationships among the characteristics of human semen and factors affecting semen-specimen quality. Journal of Reproduction and Fertility, 4,

8 178 K. Purvis, A. Tollefsrud and H. Rui Hartman, C., Schoenfeld, C. & Copeland, E. (1964) Individualism in the semen picture of infertile men. Fertility and Sterility, 13, Hotchkiss, R. (1941) Factors in stability and variability of semen specimens. Journal of Urology, 45, Katz, D. F., Overstreet, J. W. & Pelfrey, R. J. (1982) Integrated assessment of the motility, morphology and morphometry of human spermatozoa. In: Human Fertility Factors (eds P. Spira and P. Jouannet), pp Inserm, Paris. MacLeod, J. (1964) Human seminal cytology as a sensitive indicator of the germinal epithelium. International Journal of Fertility, 9, Overstreet, J. W. (1984) Laboratory tests for human male reproductive risk assessment. Teratogen Carcinogen Mutagen. 4, Poland, M. L., Moghissi, K. S., Giblin, P. T., Ager, J. W. & Olsen, J. M. (1986) Stability of basic semen measures and abnormal morphology within individuals. Journal of Andrology, 7, Read, M. D. & Schneiden. H. (1978) Variations in sperm count in oligozoospermic or asthenozoospermic patients. Andrologia, 10, Schartz, A,, Laplanche, A,, Jouannet, P., & David, G. (1979) Within subject variability of liuniaii semen in regard to sperm count, volume, total number of spermatozoa and abstinence. Journal of Reproduction and Fertility, 37, Singer, R., Sagiv, M.. Barnet. M., Segenreich, E., Allalouf, D., Landau, B. & Servadio, C. (1980) Motility, vitality and percentages of morphologically abnormal forms of human spermatozoa in relation to sperm counts. Andrologia, 12, Working, P. K. (1988) Human male reproductive toxicology: a comparison to common animal models. CIIT Activities, 8, No Received 3 October 1988; accepted 12 December 1988