Evaluation of 'sround cells' in semen analysis: a comparative study
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(2) `Round cells' in semen analysis taking into account that some of these cells are in¯ammatory cells while others are degenerated spermatids. Attempts have been made to differentiate neutrophils from degenerated spermatids using the peroxidase ortho-Toluidine Blue technique (Endtz, 1972; Nahoum and Cardozo, 1980). Nevertheless, there is no speci®c histochemical staining method with regard to lymphocytes and macrophages in semen, neither is there any speci®c staining reaction for prostatic epithelial cells, cells originating from the seminal vesicle or from the transitional or squamous epithelial cells. A decline in semen quality among fertile men during the last 20 years has been reported (Auger et al., 1995; Adamopoulos et al., 1996). Although highly sophisticated methods are sometimes used to assess the motility characteristics of human spermatozoa, e.g. time-exposure and computerized video-micrography (Overstreet et al., 1979) and other computer-aided sperm analysis (WHO, 1992, 1999) the reason for this decline in semen quality is not completely understood. Exposure to several physical and chemical agents has been suspected of affecting human semen quality (Giwercman et al., 1993). Changes in life style with regard to sexual, smoking and drinking habits have occurred during the last 60 years and a more promiscuous sex life has probably increased the risk of sexually transmitted diseases. It is noteworthy that, in spite of all hypotheses mentioned above, very little attention has been paid to the so-called `round cells' in semen samples. These `round cells' could be of in¯ammatory origin but they could also re¯ect an excessive degeneration of the late spermatogenesis. In this respect the differentiation of the `round cells' needs to be taken into consideration. A review of the present stage of knowledge in this particular ®eld is therefore justi®ed.. 405. Krausz et al., 1992; Aitken and Baker, 1995) and this ®nding has been recon®rmed (Comhaire and Vermeulen, 1995). Therefore, the differentiation of the `round cells' into either cells of spermatogenic or non-spermatogenic origin has an important clinical relevance.. `Round cells' of spermatogenic origin In order to provide a better comparison between the testicular cells and the round cells of non-spermatogenic origin the authors would like to give a short summary of the normal spermatogenesis and a comparison between the different types of round cells as presented in the semen samples stained by the routine methods. The evaluation of unstained live spermatogenic cells is carried out using a speci®c classi®cation scheme and is described in detail elsewhere (Cremades et al., 1999; Sousa et al., 1999). As indicated in Figure 1, male germ cells originate from initial cells that divide repeatedly to give rise in the male to spermatogonia. Thus, mitotic divisions of some of the primitive spermatogonia result in separate cells that contribute to the maintenance of the stem cell pool. Later, by a ®nal division, some spermatogonia give rise to two cells which begin to increase in volume and to develop a speci®c chromatin pattern. These cells are the primary spermatocytes and have a chromosome set up of 44 + XY. By the next division, each primary spermatocyte gives rise to two daughter cells (secondary spermatocytes) with a chromatin set-up of 22 + X or 22 + Y per cell, in the ®rst meiotic division. These cells, having two different chromatin set-up, undergo a second meiotic division giving rise to four cells, the spermatids. Two spermatids have a chromosome set-up of 22 + X and two spermatids a chromosome set-up of 22 +Y. From this stage no. Characteristics of the `round cells' in semen samples The `round cells' observed in the semen samples could be either of spermatogenic origin or varying types of cells of nonspermatogenic origin such as epithelial cells, neutrophils or lymphocytes. Some spermatogenic cells, as well as some leukocytes, are usually present in every semen sample. In the routine semen analysis, using the Papanicolaou technique as staining method, the differentiation of these so called `round cells' into either spermatogenic cells or leukocytes is sometimes dif®cult. Degenerated spermatids having two or more pyknotic nuclei can easily be falsely diagnosed as polymorphonuclear leukocytes. According to the WHO recommendations (WHO, 1987, 1992, 1999) the normal ejaculate should contain <5 3 106 round cells/ml while the number of leukocytes should be <1 3 106/ml. The pathological in¯uence of an elevated number of neutrophils in the semen is still a matter of dispute (Kiessling et al., 1995). The presence of an increased number of leukocytes may, however, be associated with an in¯ammatory reaction of the male genital tract (Zalata et al., 1995) and the possibility that the presence of the leukocytes in the semen interferes with the fertilizing ability of the spermatozoa cannot be excluded (Sukcharoen et al., 1995). The unfavourable in¯uence on spermatozoa of hydrogen peroxide produced by the polymorphonuclear leukocytes has been clearly shown (Aitken et al., 1992;. Figure 1. Schematic representation of the spermatogenesis in mammals. In all but the earliest spermatogonial division the cytokinesis is considered to be incomplete and clones of germ cells are formed. Theses cells are considered to be joined by intercellular bridges (Fawcett, 1975)..
(3) 406. E.Johanisson et al.. more division takes place and each spermatid starts the transformation into a spermatozoon. During the transformation to a fully developed spermatozoon, the nucleus of the spermatid undergoes a gradual condensation of its chromatin. During the ®rst step of this condensation (usually called the Sab spermatid), the spermatid nucleus is usually spherical in shape and still has diameter of >5 mm in early spermatids (Tesarik, 1997; Aslam et al., 1998). The condensation of the chromatin then gradually transforms the Sa spermatid into a Sd spermatid that has the size of a mature sperm head. In this case it is not possible to distinguish spermatozoa which have lost their tails, from Scd spermatids.. Interestingly, groups of developing germ cells are joined together by intercellular bridges, that give rise to an incomplete cytokinesis in the mitotic and meiotic divisions of the spermatogenesis; these bridges have been observed under electron microscopy (Dym and Fawcett, 1971; Huckins and Oakberg, 1971). The cytokinesis of the spermatogonia is always incomplete and lengthening chains of cells can be found interconnected by bridges. The same phenomenon applies to the subsequent meiotic divisions of the spermatocytes. During spermatogenesis, a random degeneration of individual cellular units may take place without compromising the survival of the remainder of the cell chain. It has been reported that up to. Figure 2. Histological staining (haematoxylin/eosin) of testicular tissue from a normal adult man of fertile age. (a) Seminiferous tubule. Note the basal lamina (BL), the spermatogonia (Spg), the primary spermatocytes (Spc I), the secondary spermatocytes (Spc II), the spermatids (Spt) and the spermatozoa (Spz). Scale bar = 50 mm. (b) Higher magni®cation of the histological section shown in Figure 2a. Note the basal lamina (BL), the spermatogonia (Spg), the primary spermatocytes (Spc I), the secondary spermatocytes (Spc II), the spermatids (Spt) and the spermatozoa (Spz). Scale bar = 20 mm. (c) High magni®cation of the testicular tissue shown in Figure 2a. Note the spermatogonia (Spg) next to the basal lamina (BL), some primary spermatocytes (Spc I) and some spermatozoa. Scale bar = 20 mm. (d) The periphery of two seminiferous tubules showing an histologically normal spermatogenesis. Note the spermatogonia (Spg) and the primary spermatocytes (Spc I). Scale bar = 20 mm. (e) Part of a seminiferous tubule that has been depleted of most primary and secondary spermatocytes suggesting no active meiotic activity in this area. The spermatogonia (Spg) are lined up next to the basal lamina and a few spermatocytes (Spc I) and spermatozoa (Spz) are observed in the lumen. Scale bar = 20 mm. (f) Part of a seminiferous tubule mainly showing Sertoli cells (Sc). A few spermatogonia (Spg), spermatids (Spt) and spermatozoa are seen. Scale bar = 20 mm..
(4) `Round cells' in semen analysis. 407. Figure 4. In¯ammatory cells in blood and semen smears. (a, c) Blood smears stained with May±GruÈnewald±Giemsa. (b, d) Blood smears stained with Papanicolaou. (e) Semen smear stained with Papanicolaou. Scale bar = 10 mm. (a) Neutrophil (Nt) and lymphocyte (L) observed in a blood sample stained according to the May±GruÈnewald±Giemsa technique. (b) Neutrophil and lymphocyte in Papanicolaou stained blood smear. When compared with the same cells in Figure 4a no difference in size and shape was observed. (c, d) A monocyte (M) observed in a blood sample stained by May±GruÈnewald± Giemsa and another according to the Papanicolaou technique. Note the thin cytoplasmic border and the mononuclear appearance. (e) A macrophage observed in a semen sample and stained according to the Papanicolaou technique. The cell has attracted several spermatozoa, out of which some sperm heads are intruded in the cytoplasm.. Figure 3. Papanicolaou staining of semen smears containing immature germ cells. Scale bar = 10 mm. (a) The cells illustrated in this ®gure are sometimes classi®ed as `round cells' which include also in¯ammatory cells. However, all the cells of this ®gure represent cells of the spermatogenesis from primary spermatocytes (Spc I ) to some Sab and Scd spermatids (Spt ab, Spt cd). Please note that the nuclei of the Spt cd have lost their elongated shape, probably due to degeneration, and they are also observed in the periphery of the cytoplasm. (b) This cell which sometimes is classi®ed as a `round cell' represents a primary spermatocyte (Spc I). (c) Some Scd spermatids (Spt cd) and one secondary spermatocyte (Spc II). See comments made under 3a. (d, e) These pictures show multinucleated and mononucleated Sab and Scd spermatids (Spt ab and Spt cd). See comments under 3a. Probably the division of the secondary spermatocyte was never completed. (f) This picture shows the difference in size between the pyknotic nucleus of a mature squamous epithelial cell (Et) and the nucleus of some small Scd spermatids. Note that most spermatids (cd) nuclei are placed in the periphery of the cytoplasm.. 25% of germ cells normally degenerate in the course of the spermatogenesis (Greep et al., 1976). In this respect the presence of double-headed mature spermatozoa is noteworthy. This phenomenon might be a consequence of an incomplete cytokinesis when two sperm heads are found inside the same cytoplasmic residue. By electron microscopic studies it has been established that cohorts of developing germ cells remain interconnected by cellular bridges throughout the spermatogenesis (Dym and Fawcett 1971). The cytoplasmic connections of the germ cells have important implications for the dynamics of the synchronized production and release of spermatozoa. The displacement of germ cells towards the lumen of the seminiferous tubule during spermatogenesis and spermiogenesis is passive, i.e. the sperma-. tocytes are not motile. Spermatogonia are linked to the basal lamina and remain in the basal layer; when they divide, the daughter cells are released to the upper part of the epithelium where Sertoli cells establish ®ne contacts with them and send cytoplasmic projections that partially surround them. When primary spermatocytes divide into secondary spermatocytes, the daughter cells are further displaced toward the lumen and thus a passive process of space occupation by new cells is responsible for the shift of germ cells toward the lumen. It is probable that the complex migration from the spermatocytes to the spermatids and ®nally to free spermatozoa could not take place without cell±cell communication among the Sertoli cells. The cytoplasmic bridges interconnecting developing germ cells may explain the synchronization of the meiotic and mitotic divisions. Indeed, segments of seminiferous tubules exhibit synchronized spermatogenesis. As shown in Figure 2 (a±f), the spermatogonia are found in the periphery of the seminiferous epithelium next to the basal lamina. In the testicular tissue of a normal fertile man the spermatogonia are then followed by the layers of primary and secondary spermatocytes and ®nally by spermatids and spermatozoa moving towards the centre of the seminiferous tubule. As shown in Figure 2 (d±f), the cell morphology in histological sections may vary from one tubule to another. In some tubules, cells representing the full spermatogenesis are found (Figure 2d). Other tubules are depleted of spermatocytes and spermatids (Figure 2e), perhaps re¯ecting the cohort release of spermatozoa. Still other tubule areas seem to be composed mainly of Sertoli cells attached to the basal lamina (Figure 2f). The possibility that such sections represent the transition of the seminiferous tubules into collecting ducts cannot be excluded..
(5) 408. E.Johanisson et al.. The complex migration of the germ cells into the lumen of the seminiferous tubules often results in the release of immature germ cells in the semen sample (Figure 3a±f). In case of damage to the seminiferous tubules, e.g. injury to the Sertoli cells, the connections structuring the ordered epithelium are lost resulting in the premature release of excessive numbers of immature germ cells. During their transport through the ductus deferens such spermatids are likely to undergo degenerative changes. For instance, during normal spermatogenesis, the nuclei of Sc and Sd spermatids are elongated, this characteristic can be lost due to degenerative change. With Papanicolaou staining, degenerated cells take on an acidophilic pink colour, clearly a sign of modi®cation of the chemical composition of the cytoplasm. A high number of spermatocytes in the semen is seldom accompanied by a large number of mature spermatozoa and severe disturbances of the spermatogenesis can be expected in such cases. On the other hand, degenerated spermatids are frequently observed also in the presence of mature spermatozoa in the semen. Large Sab and small Scd spermatids are illustrated in Figure 3. In Figure 3f, the size of the spermatids is compared with that of the nucleus of a normal mature squamous epithelial cell, the diameter of which is 3mm. It is probable that some of the nuclei of the Scd spermatids have lost their elongated shape, probably due to degeneration, e.g. pyknotic nuclei and the acidi®cation of the cytoplasm shown by the pink Papanicolaou staining (WHO, 1988) (Figure 3a,c). The central location of the nucleus of the Scd spermatid could also be questioned due to degeneration (Figure 3d). Abnormalities of the development of the spermatids into mature spermatozoa are dif®cult to detect by ordinary light microscopy. However, by using biopsy material of the human testis embedded in Epon and processed for electron microscopy, abnormal changes of the differentiation of the spermatids has been detected in 50% of all cases studied (Holstein and Schirren, 1979). The results of a previous study (Holstein and Schirren, 1979) suggest that degenerative changes of the spermatids have already started before they pass though the epididymis and ductus deferens. The following abnormalities in human spermatids, based on advances in ultrastructural research, have been reported by Holstein and Schirren: (i) spermatids with giant acrosome vesicles; (ii) spermatids having no acrosome; (iii) malformations of the main piece; (iv) spoon-like nuclei in spermatids; and (v) multinucleated spermatids. These malformations observed in biopsy material may be accompanied by infertility. Unfortunately, the light microscopic examination of testicular biopsies does not allow the detection of all the malformations reported by Holstein and Schirren (1979). Multinuclear spermatids are commonly found in the semen samples and need to be carefully distinguished from neutrophils and lymphocytes. Spermatids that have failed to complete development can be found in semen as multinucleated syncytium-like structures, usually containing three or four round nuclei that are located in the periphery of the cell (Figures 6a, 7c). According to the presentation of these cells in the smear, these independent nuclei could be confounded with the lobes of the multi-lobated nuclei of neutrophils. This is the most dif®cult cell to distinguish in semen, as the bridges that connect the lobes of. the neutrophil nucleus, and that are absent in multinucleated spermatids, are not always easy to observe on the microscope. The speci®city of the neutrophils for the peroxidase orthoToluidine Blue stains could indeed be of great help in the discrimination between the multinucleated spermatids and the polymorphonuclear leukocytes.. `Rounds cells' of non-spermatogenic origin Epithelial cells, polymorphonuclear granulocytes, macrophages, lymphocytes and various epithelial cells are usually referred to this category. In the present study, attempts have been made to compare the leukocytes found in the semen samples with those appearing in a normal blood smear (Sandoz, 1952).. Characteristics of the polymorphonuclear granulocytes Out of the in¯ammatory cells observed in the blood smears as well as in the semen samples the polymorphonuclear leukocytes are the most commonly found. They are characterized by segmented nuclei (Figure 4a). The diameter of the neutrophils is usually 14 mm and when stained according to the Papanicolaou method (Figure 4b), no difference could be found in the size and shape of the neutrophils observed in the blood smears when compared with those found in the semen samples.. Morphological characteristics of the lymphocytes The lymphocytes are small cells having an diameter of ~8±12 mm; they are never multinucleated (Figure 4a). This is an important observation which helps in the differentiation between these cells and the spermatids. Furthermore, the lymphocytes usually have a small cytoplasmic border with a bluish-green colour after Papanicolaou staining (Figure 4b). In contrast, the spermatids present in the semen samples are usually degenerated and the cytoplasm has taken on a pink colour after Papanicolaou staining (Figure 3).. Morphological characteristics of the monocytes/ macrophages The macrophages have a diameter of 16±20 mm (Figure 4c). These cells usually have a kidney-shaped nucleus, which clearly helps in the differentiation between the macrophages and the degenerated and less degenerated spermatids. The cytoplasm of the macrophages is usually blue±greyish after Papanicolaou staining and numerous cytoplasmic vacuoles can be observed; sometimes these vacuoles contain degenerated cellular debris. The macrophages are often multinucleated, but the nuclei are never pyknotic or degenerated in the same way as observed in degenerated multinuclear spermatids. Sometimes macrophages present in semen are seen that have phagocytosed spermatozoa and the sperm heads can be seen in the cytoplasm (Figure 4e). Macrophages are chemotactically attracted into the seminiferous tubules when germ cells degenerate, in case of in¯ammatory response or of obstruction. When macrophages come in contact with spermatozoa they do not recognize them as self and phagocytose them. The clear morphological difference between.
(6) `Round cells' in semen analysis the macrophages and the degenerated and less degenerated spermatids however allows for an easy differentiation between these cell types.. Cells originating from the prostatic glands and the seminal vesicles In routine semen samples the prostatic glandular epithelial cells are rarely found. However, if the semen sample is made after a prostatic massage, a number of prostatic epithelial cells are frequently found together with the spermatozoa. The prostatic epithelial cells are usually well-differentiated cuboidal or columnar cells appearing in clusters or as single cells (Johannisson and Eliasson, 1978) (Figure 5a). Cell nuclei are small and round and similar in size to the lymphocytes. Clumps of chromatin are sometimes found in the nucleus but in normal prostatic epithelium no abnormal distribution of the chromatin is observed and the cytoplasm is ®nely granulated. Cells originating from the seminal vesicles are cuboidal or columnar in shape (Figure 5b). They sometimes appear in clusters but they can also be observed as single cells. The shape of the nucleus can vary from small and round to an irregular shape which could be misinterpreted as suspiciously malignant. The regular distribution of yellow pigment granules in the cytoplasm is however signi®cant for the cells originating from the epithelium of the seminal vesicles.. 409. Transitional epithelial cells and squamous epithelial cells The transitional epithelial cells are usually originating from the bladder and urethra. In the semen samples these cells are ¯at, cuboidal or oval not unlike parabasal squamous epithelial cells (Figure 5c±d). The cytoplasm is usually in the ratio of 2:1 when compared with the nucleus and it takes a green±bluish colour after Papanicolaou staining. The nuclei are round or slightly oval with ®ne regular distribution of the chromatin. The transitional cells can be derived from the basal layer (Figure 5c) or the super®cial layer (Figure 5d). Squamous epithelial cells usually represent a variety of cell types re¯ecting the maturation of the cells. Mature squamous cells have a large polygonal shape of their cytoplasm, which is usually ¯at, transparent and eosinophilic after Papanicolaou staining. The nuclei are small and pyknotic with a diameter of ~3 mm (Figure 3f). The `intermediate' squamous epithelial cells have a polygonal basophilic cytoplasm and vesicular nucleus with a ®ne regular chromatin distribution. The younger squamous epithelial cells are the parabasal cells. They have an oval shape and the cytoplasm takes a strongly basophilic colour in the Papanicolaou stain. The nuclei of these cells contain chromatin granules or `chromocenters'. The diameter of the nuclei of the parabasal cells is similar to that of the transitional cells.. Figure 5. Papanicolaou staining of various epithelial cells. Scale bar = 10 mm. (a) Prostatic cell derived from an ejaculate. The cell is well preserved and has a slightly elongated cytoplasm and a round chromatin-dense nucleus. (b) A cluster of cells originating from the epithelium of seminal vesicles. The nuclei of these cells have a triangular shape, occasionally, secretory granules can be found in the cytoplasm. (c) The ®gure illustrates two cells derived from the basal layer of the transitional epithelium, together with a Scd spermatid and a spermatozoon. The relationship between the nucleus and the cytoplasm of the epithelial cells is in favour of the nucleus. (d) Transitional cell in ®nal stage of maturation, derived from the upper layer of the transitional epithelium. Note that the size of the cytoplasm is largely increased when compared with the basal layer..
(7) 410. E.Johanisson et al.. Based on the observation of the leukocytes in the blood smear and in the semen samples it can be concluded that the Papanicolaou staining procedures do not interfere with the size of the leukocytes neither in the blood smear nor in the semen samples.. Figure 6. Papanicolaou staining of a (a) semen smear and of a (b) blood smear. Scale bar = 10 mm. (a) Multinucleated small spermatids (Spt cd) and one large spermatid (Spt ab) stained according to the method of Papanicolaou. They could easily be falsely diagnosed as neutrophils. (b) The picture shows three neutrophils with segmented nucleus stained according to the technique of Papanicolaou.. How to differentiate between spermatogenic and non-spermatogenic cells When stained according to the method of Papanicolaou, the multinucleated spermatids (Figure 6a) appearing in the semen samples are sometimes dif®cult to differentiate from neutrophils (Figure 6b) having a segmented nucleus. Figure 7 shows the comparison between neutrophils with segmented nucleus and small spermatids when stained by the peroxidase reaction. The size between the small spermatids and the neutrophils is more or less the same. In view of the dif®culty of distinguishing between the intrasegmentary bridges of the neutrophils and the multinucleated small spermatids in Papanicolaou stained specimens, the speci®city of the peroxidase reaction of the neutrophils is important. The discrimination between the two cell types can be done on the basis of their peroxidase content. For the time being at least, two rapid and simple histochemical methods are available for the detection of peroxidase-positive cells. The ®rst method was described by Endtz (1972). In this method, a drop of the working solution of the Leucoscreen kit (FertilPro, Lotenhulle, Belgium) is mixed with one drop of semen and after settlement the mixture is observe under a coverslip in ordinary light microscope or under phase contrast. The neutrophil polymorphonuclear leukocytes all contain brown or brownish± yellowish granules, being by this reaction peroxidase positive. The second method was described by Nahoum and Cardozo (1980). The reagents and the details of the method are described in WHO laboratory manual for the examination of human semen and semen±cervical mucus interaction (WHO, 1987, 1992, 1999). As for the ®rst method, the peroxidase staining was exclusively positive for the neutrophils which. Figure 7. Semen smears stained with Papanicolaou (a, c) or with peroxidase-ortho-Toluidine Blue (b, d, e, f). Scale bar = 10 mm. (a) Some neutrophils in a sperm sample stained according to the technique of Papanicolaou. (b) A positive peroxidase staining, showing abundant brownish granules in the cytoplasm of the neutrophils, easily allows to distinguishing neutrophils from multinucleated spermatids. (c) One degenerated multinucleated Scd spermatid stained according to the Papanicolaou technique. This cell could be falsely taken as a neutrophil. (d) A negative peroxidase staining immediately clari®ed that the type of cell shown in Figure 7c does not belong to the neutrophils in the semen sample. (e) This ®gure shows the difference in peroxidase staining between spermatids and neutrophils. (f) This ®gure illustrates the negative peroxidase staining in cells of spermatogenic origin. These cells are comparable in size and shape to those shown in Figure 7e..
(8) `Round cells' in semen analysis contained dark brown granules. In our hands, the method of Nahoum and Cardozo (1980) has also been used in the blood smears to distinguish polymorphonuclear leukocytes from the monocytes. In these cases a peroxidase positive reaction was found in all polymorphonuclear neutrophils while the lymphocytes and the macrophages were cells being peroxidasenegative. A similar ®nding was observed in the semen samples. The polymorphonuclear granulocytes in the semen samples were all positive to the peroxidase staining method whereas multinucleated spermatids, monocytes and various epithelial cells were all negative to the peroxidase staining. The differentiation between spermatids and in¯ammatory cells like the lymphocytes and the macrophages can be done on a cytological basis comparing the size and morphological shape of the cell types which is evident in most cases as outlined in the preceding sections. In addition, as multinucleated spermatids are likely to be degenerated in semen, they can also be distinguished by their pink cytoplasm in Papanicolaou-stained smears. Additional speci®c tests could also be used to distinguish spermatids from somatic cells, such as ¯uorescence in-situ hybridization enzyme markers or speci®c antibodies recognizing leukocyte antigens, e.g CD45 (WHO, 1999). However, such sophisticated techniques might not be always available and they might be redundant as they would provide in most instances the same information as careful morphological evaluation.. The importance of the differentiation between `round cells' of spermatogenic and nonspermatogenic origin in semen samples In view of the increase in sexually transmitted diseases, particularly in younger age groups of men, it is important to differentiate between degenerated spermatids and in¯ammatory cells. An interaction between the presence of leukocytes and the functional competence of human spermatozoa to fertilize in vitro has clearly been shown by Sukcharoen et al. (1995). The addition of leukocytes to human sperm suspensions has been reported to result in a loss of motility (Kovalski et al., 1992). By the lack of de®nition, the simple indication of the presence of `round cells' does not yield suf®cient information to the physician to know whether or not the patient has an in¯ammatory condition of the male genital tract. Accordingly, the patient runs the risk not be treated in an appropriate way. It is strongly recommended that the mentioning of `round cells' should be omitted from all records of the analysis of sperm samples and replaced by information like `degenerated spermatids or spermatocytes' or identi®ed by the type of in¯ammatory cells (neutrophils, lymphocytes, macrophages, etc). For the clinical evaluation it is of particular importance to distinguish between the cells of spermatogenic and nonspermatogenic origin when no mature spermatozoa are present in the semen. Indeed, the complete absence of spermatogenic cells suggests an obstructive cause to the azoospermia, that may be corrected by surgery. On the other hand, the presence of spermatogenic cells in semen samples having few if any mature spermatozoa, suggest a testicular malfunction where the impaired spermatogenesis could be of endocrinological origin.. 411. Furthermore a number of new data in the literature are supporting the hypothesis that the presence of leukocytes in the semen is an important factor interfering with the fertilizing potential of human spermatozoa (Fedder, 1996). This has previously been shown in vitro (Sukcharoen et al., 1995) as well as in ejaculates of oligozoospermic patients (Aitken et al. 1992). Also, Krausz et al. (1992) emphasized the susceptibility of human spermatozoa to peroxidative damage due to the contamination of leukocytes in the semen. A differentiation of the `round cells' into cells of spermatogenic and non-spermatogenic origin is a conditio sine qua non for a correct semen analysis. The lumping of all `round cells' into one group (as suggested in many recommendations for semen analysis) highly increases the risk of misinforming the treating clinicians.. Acknowledgements We are grateful to Dr Annelise Wohlwend for providing us with histological sections of human testicular tissue.. References Adamopoulos, D.A., Pappa, A., Nicopoulou, S. et al. (1996) Seminal volume and total sperm number trends in men attending subfertility clinics in the Greater Athens area during the period 1977±1993. Hum. Reprod., 11, 1936±1941. Aitken, R.J. and Baker, H.W.G. (1995) Seminal leukocytes: passengers terrorists or good samaritans? Hum. Reprod., 10, 1736±1739. 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