ۥ- T &4Kd MAST CELLS IN TESTICULAR BIOPSIES OF INFERTILE MEN WITH "MIXED ATROPHY" OF SEMINIFEROUS TUBULES Davor Je`ek, Ljerka Banek, Anton Hittmair*, Ru`ica Pezerovi}-Panijan, Trpimir Golu`a and Wolfgang Schulze+ Institute of Histology and Embryology, Medical Faculty University of Zagreb, Croatia *Institute of Pathology, University of Innsbruck, Austria +Department of Andrology, University of Hamburg, Germany Running title: Mast cells and male infertility Key words: mast cells, testis, biopsy, male infertility Corresponding author: Dr. Davor Je`ek Majstora Radovana 24 HR-10000 Zagreb CROATIA Phone: + 385 1 317 097 Fax.: + 385 1 424 001 e-mail: davorsymbol 195 \f "Symbol"mef.hr Abstract Mast cells in bilateral testicular biopsies of thirty patients with a mixed atrophy of seminiferous tubules were analysed. Seven biopsies from vasectomy patients served as controls. Regarding their characteristic location within testicular tissue, two groups of mast cells could be distinguished, both in control and infertile patients: interstitial mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and peritubular mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in number and volume of mast cells in infertile patients when compared to controls. Although the both cell groups (i.e. interstitial and peritubular mast cells) in the analysed biopsies of infertile patients increased significantly in their number and volume, it appeared that peritubular mast cells increased with a higher rate than interstitial mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsens scoring. It is concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis. Introduction Mast cells form a heterogeneous cell population which can be found in a wide range of tissues and organs. In the adult human testis, mast cells are predominantly localised in interstitial compartment (Kerr, 1991). However, a small proportion of these cells can be found in the lamina propria of seminiferous tubules (Hermo & Lalli, 1978). Histometric studies have shown that number of mast cells in the testis and epididymis increases slightly during infancy, decreases during childhood, and then increases again at puberty. Interestingly, during adulthood, these cells appear to decrease progressively in the connective tissue of the both, testis and epididimyis (Nistal et al., 1984). Several reports have pointed out an association between increased number of mast cells and male infertility. In 1981, Maseki et al. reported on mastocytosis in infertile males. Salomon et al. (1982) described immune complex orchitis with abnormal basement membrane structures and numerous mast cells in the interstitial tissue of some patients. A relationship between alcohol abuse and the presence of mast cells was presented in another two reports (Haider et al., 1985; Nistal et al., 1986). The morphometric study of Agrawal et al. (1987) pointed out the expanded mast cell density in cases of idiopathic male infertility. Analysing testicular biopsies from such patients, Hashimoto et al. (1988) demonstrated increased presence of mast cells in the lamina propria of seminiferous tubules. A histochemical study on mast cell heterogeneity in the testis of patients suffering from idiopathic male infertility presented a rise in the chondroitin sulfate-positive mast cells (Nagai et al., 1992). Mixed atrophy of seminiferous tubules represents a testicular disorder characterized by tubules with various degrees of degeneration of the germ epithelium, ranging from full spermatogenesis up to "Sertoli cells only" and seminiferous tubule fibrosis within the same biopsy (Sigg & Hedinger, 1981). The presence of mast cells is changeable (Je`ek et al., 1996). This particular disorder is frequently found in biopsies from patients undergoing evaluation for idiopathic male infertility and azoospermia. Interestingly, patients with mixed atrophy often benefit from testicular sperm extraction (TESE) (Schulze et al., 1997; Je`ek et al., 1998). Although all the above mentioned reports indicate the connection between mast cells and male infertility, there is a lack of data on mast cells concerning their exact location (i.e. the relation with the interstitial tissue and seminiferous tubules) in the testis of infertile patients. In addition, there are scarce morphometric data on the same subject. Thus, our study had several objectives: a) to characterise mast cells in testicular biopsies from patients with "mixed atrophy" in respect with their location b) to evaluate these cells by morphometry c) to correlate morphometric data with the testicular volume and the status of spermatogenesis (Johnsensymbol 162 \f "Symbol"s scoring). Materials and methods Patients and biopsy processing Bilateral testicular biopsies from thirty patients with idiopathic infertility and azoospermia (aged 22.5 - 46 y. old; mean = 36 y.), who consulted an andrologist during the year 1996, were retrieved from the archive (Department of Andrology, University of Hamburg). Prior to biopsy, every investigated patient gave his written and informed consent to surgery and biopsy examination. A histological review of the slides of all chosen cases confirmed the diagnosis of "mixed atrophy" of testicular tissue. Control biopsies were provided from 7 men (31 - 46 y., mean = 37 y.) undergoing a vasectomy reversal (vasectomy period: 1-12 y.). The biopsies were obtained and processed according to the procedure described by Holstein et al. (1994). The excised tissue was immediately fixed by 5.5% glutaraldehyde in a 0.05 M phosphate buffer (pH = 7.1-7.4, 800 mosmol). After 3 hours of fixation, the testicular tissue was postfixed in a 1% solution of osmium tetroxide (OsO4) in a saccharose-phosphate buffer for another 2.5 hours (Holstein & Wulfhekel, 1971). The tissue was then dehydrated in ascending concentrations of alcohol, rinsed twice for 15 min. in propylene oxide and embedded in Epon 812. Serial semithin sections (section thickness - d = 1 m) were obtained by an ultramicrotome (Reichert, Austria) and stained with 1% toluidine blue for diagnostic purposes and morphometric (stereological) analysis. Ultrathin sections (d = 70 nm) were mounted on copper grids, contrasted with lead citrate and uranyl acetate and examined by a transmission electron microscope Zeiss 902A (Centre for Electron Microscopy, Medical Faculty University of Zagreb). The volume of each analysed testis was determined by orchidometer and/or ultrasound examination. Morphometric (stereological) analysis The biopsies (which were round or oval, 2mm x 2mm in size) were embedded in a random fashion. Each tissue block was trimmed, oriented (also in the random fashion) and prepared for cutting. Sixty serial semithin sections were made per block, mounted on six slides (each slide bearing ten sections) and stained with toluidine blue as mentioned previously. A non-biased test grid according to Weibel (1979) with 144 test points was inserted into the eyepiece of two binocular microscopes (Carl Zeiss, Jena). The first and the sixth slide was used for a morphometric measurement applying a physical dissector principle (Sterio, 1984; Gundersen et al., 1988; Bolender et al., 1993; Bertram, 1995). Sections were observed at a magnification of x1.000 where a test surface area (At) was 0.0144 mm2 for each analysed microscopic field. The distance between the analysed sections was determined by section thickness (d = 1 symbol 109 \f "Symbol"m). For each biopsy, 200 microscopic fields were analysed. Only the cells with a visible nucleus were considered. During measurement, two distinctive mast cell populations regarding their localisation could be clearly discerned: "interstitial" mast cells and "peritubular" mast cells. Thus, following stereological variables have been assessed: number of "interstitial" and "peritubular" mast cells per mm3 of the testis tissue (Nv); number of "interstitial" and "peritubular" mast cells per the entire testis (N); volume of "interstitial" and "peritubular" mast cells per mm3 of the testis tissue (Vv); volume of "interstitial" and "peritubular" mast cells per the entire testis (V); number of "interstitial" mast cells per mm3 of testis tissue (Nvi); number of "interstitial" mast cells per the entire testis (Ni); volume of "interstitial" mast cells per mm3 of the testis tissue (Vvi); volume of "interstitial" mast cells per the entire testis (Vi); number of "peritubular" mast cells per mm3 of testis tissue (Nvp); number of "peritubular" mast cells per the entire testis (Np); volume of "peritubular" mast cells per mm3 of the testis tissue (Vvp); volume of "peritubular" mast cells per the entire testis (Vp). In order to calculate the above-mentioned relative variables, appropriate formulae for a numerical and a volume density (semithin sections) have been applied (Weibel, 1979; Gundersen et al., 1988; Bolender et al., 1993; Bertram, 1995). The total number and the volume of mast cells was determined by multiplying the testis volume with the numerical and the volume density, respectively (Weibel, 1979). Scoring Estimation of the status of spermatogenesis was accomplished for each seminiferous tubule (average number of tubules per biopsy: 25-30) according to a modified Johnsensymbol 162 \f "Symbol"s score (De Kretser & Holstein, 1976) as follows: 10- full spermatogenesis; 9- many late spermatids, disorganised tubular epithelium; 8- few late spermatids; 7- no late spermatids, many early spermatids; 6- no late spermatids, few early spermatids, arrest of spermatogenesis at the spermatid stage; 5- no spermatids, many spermatocytes; 4- no spermatids, few spermatocytes, arrest of spermatogenesis at the primary spermatocyte stage; 3- spermatogonia only; 2- no germ cells, Sertoli cells only; 1- no seminiferous epithelial cells, tubular sclerosis. Biopsy score was derived as an average of scores for individual tubules. Statistical analysis Statistical analysis was performed using a Microstat bio-statistical package programme. Individual data of stereological assessment were processed by a personal computer. In order to determine statistically significant differences between the investigated groups as well as a correlation between the testis volume, score and calculated stereological variables, a difference between two group means (pooled estimate of variance) and correlation matrix were used. The values of p<0.05 were considered as statistically significant. Results General morphology of the testicular tissue Testicular biopsies from vasectomy patients displayed normal spermatogenesis and spermiogenesis, a regular structure and thickness of the lamina propria of seminiferous tubules and usual morphology of the interstitial tissue. In infertile patients, a variety of degenerative changes of seminiferous tubules could be found. A small number of tubules in such biopsies was deprived of seminiferous epithelium and fibrosed, thus forming "tubular shadows". Some of the tubules were lined with Sertoli cells only and, occasionally, with rare spermatogonia. The vast majority of the tubules had seminiferous epithelium in various degrees of degeneration. The spermatogenic cells consisted mostly of spermatogonia, spermatocytes and early spermatids, while the late spermatids were scarce or absent. The thickness of the tubular lamina propria was frequently significantly enlarged, including the basement membrane. Despite these dramatic changes a small number of seminiferous tubules seemed to have preserved their normal structure, reaching almost full spermatogenesis with just few degenerating late spermatocytes. In the cytoplasm of Leydig cells, many vacuoles and lipid droplets could be observed. Sometimes the interstitial tissue also was fibrosed and contained blood vessels with the thickened wall. Mast cells Within the interstitial tissue of control biopsies, mast cells were easily recognised by their numerous and abundant granules. Most of the cells were found adjacent to small blood vessels and/or "intermingled" with neighbouring Leydig cells (Figs. 1,5). Cells had predominantly spheroid or oval shape, but some were elongated (Figs. 1,5). Because of their typical location (rather distant from seminiferous tubules) we provisory designated these cells as "interstitial" mast cells. A small population of mast cells also could be found in the close proximity of seminiferous tubules or even incorporated into the tubular lamina propria itself. Such cells were named "peritubular" mast cells and were often spindle shaped, elongated or seldom ovoid. In testicular biopsies of infertile men, mast cells were observed with a much higher frequency. The majority of these cells was again found in the interstitial tissue, i.e. around the blood vessels and between Leydig cells and other interstitial cells ("interstitial" mast cells) (Figs. 2,3). However, quite a significant number of these cells was noticed either close to seminiferous tubules, "attached" to the lamina propria or embodied into the lamina propria, i.e. between the layers of myoid cells (Figs. 4,7.). When compared to controls, such "peritubular" mast cells were abundant. In the cases of the pronounced degeneration of testicular interstitial tissue (including interstitial fibrosis) mast cells were found quite frequently, surrounded by an enlarged collagen network and ground substance (Figs. 9,10). In general, the more intensive was the degeneration of seminiferous tubules, the more mast cells could be encountered in the section. However, it should be pointed out that in some cases with a severe damage of spermatogenesis, these cells were less numerous than expected (even at the inspection of serial semithin sections). Mast cells in the both investigated groups (vasectomy and infertile patients) shared the same electronmicroscopical morphological properties. Their nucleus contained mostly euchromatin with some large "patches" of heterochromatin and sometimes prominent nucleolus (Fig. 5). The cytoplasm was filled with granules limited by a unit membrane. Granules had a material of a high or moderate electron density. Most of the "interstitial" cells had granules with a lattice/dot-like pattern and occasional crystalline substructures or a dense core (Figs.6,10). However, in certain cells (predominantly "peritubular" mast cells) granules had a multilayered, scroll-like or reticular architecture (Fig. 8). Throughout the cytoplasm, some cisternae of rough (and rarely smooth) endoplasmic reticulum could be seen. Mitochondria and glycogen were found scattered within the cell. On their surface, cells bore small cytoplasmic projections which extended either towards the neighbouring collagen network and ground substance (Fig. 5) or to the neighbouring cells (Fig. 8). Sometimes the cytoplasmic extensions of "peritubular" mast cells established discreet membrane-to-membrane contacts with the surrounding myoid cells (Fig. 8). Results of the quantitative histological analysis Infertile patients had testes of significantly lower volume when compared to controls (p<0.0001). Score analysis displayed a severe impairment of spermatogenesis in infertile men in comparison with the control group (p<0.0001) (Table 1). Stereological analysis demonstrated a significant increase in number and volume of mast cells per mm3 of the testis tissue (Nv, Vv) as well as per the entire testis (N, V) in the group of infertile patients when related to controls (values of p reaching from <0.05 to <0.001)(Table 2). Thus, the Nv ratio of mast cells between the control and infertile group (Nv controls / Nv infertile) was approximately 1:5. As for number of mast cells per the entire organ (N), the ratio (N controls / N infertile) was 1:3.5, which demonstrates a significant increase in the presence of mast cells in infertile patients. Within the normal testis, the ratio between number of "interstitial" and "peritubular" mast cell per mm3 of the tissue (Nvi / Nvp) was 4:1 in favour of "interstitial" mast cells. In testicular biopsies with the "mixed" atrophy of seminiferous tubules, the both mast cell groups displayed a significant increase in number and volume per mm3 of the testis tissue (Nvi, Vvi, Nvp, Vvp), as well as per the entire organ (Ni, Vi, Np, Vp) when related to controls (Table 2). However, the Nvi / Nvp ratio has been 1.6:1, which shows a significant rise in number of the "peritubular" mast cells. When the correlation between the assessed stereological variables, the testis volume and score is considered, a significant negative correlation was found between number and volume of mast cells per mm3 of the testis tissue (Nv,Vv, Nvi, Vvi, Nvp, Vvp), the testis volume and score (p<0.01) (Table 3). As for the absolute values, a significant negative correlation was found between number of mast cells per the entire testis (N) and the testis volume (p<0.001), as well as volume of peritubular mast cells per the entire testis (Vp) and score (p<0.05) (Table 3). In general, the higher the number and the volume of mast cells the smaller the testis volume and score. Discussion The heterogeneity of mast cells within the testis i.e. the existence of those cells which have heparin and those that have chondroitin sulfate as a chief proteoglycan in their granules was described in the histochemical study of Nagai et al. (1992). The heparin-positive cells would correspond to connective tissue mast cells and the chondroitin sulfate-positive cells to mucosal mast cells (Bienenstock et al., 1982; Aldenborg & Enerback, 1985). In idiopathic male infertility, chondroitin sulfate positive cells were reported to be significantly increased (Nagai et al., 1992). The results of our study demonstrated that, in addition to differences in their granules structure and content, mast cells in the human testis could be divided into two groups regarding their typical site within the testicular tissue: "interstitial" mast cells and "peritubular" mast cells. The ultrastructural data of our survey have shown that "interstitial" mast cells (besides their characteristic location i.e. adjacent to blood vessels and/or between neighbouring Leydig and other interstitial cells), have mostly granules of lattice/dot-like structure, which is an electron microscopic feature of connective tissue mast cells (Craig et al., 1989; Schwartz, 1993). In contrast to "interstitial" mast cells, "peritubular" mast cells were found in the close proximity of seminiferous tubules or in the tubular lamina propria itself. According to our electron microscopic data, such cells have had predominantly granules of multilayered, scroll-like or reticular pattern, that is the major ultrastructural characteristic of the mucosal mast cells (Craig et al., 1989; Schwartz, 1993). Morphometric data in the present study demonstrate that in the normal healthy human testis the dominating mast cell group are "interstitial" cells, while "peritubular" mast cells are less numerous. However, in the case of male idiopathic infertility, the both mast cell groups increase significantly in their number and the volume. Our stereological data have shown that, although "interstitial" mast cells remain larger in number, the increasing rate of "peritubular" mast cells seems to be much higher, as if they have been "attracted" to seminiferous tubules. There are several factors that could contribute to such increase of "interstitial" and "peritubular" mast cells. It is well known that degenerating seminiferous tubules often display an enlarged lamina propria with an augmented extracellular matrix between myoid cell layers (DeKretser et al., 1975; Davidoff et al., 1990). Human mast cells attach to the components of the extracellular matrix like laminin, fibronectin and vitronectin. These cells express integrin receptors and are sensitive to stem cell factor (SCF), which all can contribute to the adhesion of mast cells to the extracellular matrix (Walsh et al., 1991; Sperr et al., 1992). Therefore, the increased amount of extracellular matrix within the tubular lamina propria could promote the adhesion of mast cells. In addition, paracrine factors of myoid, Sertoli and spermatogenic cells could assist to this process. It has been recognised that the maturation of human mast cells is dependent upon at least two factors: interleukin-3 (IL-3) (Ihle et al., 1983) and SCF (Huang et al., 1990; Zsebo et al., 1990). It can be presumed that paracrine factors from seminiferous tubules identical or alike to IL-3 and SCF (particularly in the case of disturbed spermatogenesis) could be released from the tubule and could enhance the activation and adhesion of mast cells to the tubular lamina propria. Myoid cells may play an important role in this process, since we have demonstrated gentle membrane-to-membrane connections between the "peritubular" mast cells and myoid cells. Previous studies on testicular human mast cells have pointed out a significant correlation between these cells and the male idiopathic infertility (Salomon et al., 1982; Agrawal et al., 1987; Hashimoto et al., 1988; Nagai et al., 1992). Moreover, a one-year application of a mast cell blocker (tranilast) has resulted in an appearance of sperms in the ejaculate of one azoospermic patient (Yamamoto et al., 1994). However, no attempt has been made to characterise the "interstitial" and the "peritubular" cells and to provide morphometric data on these cells, particularly in the case of the "mixed atrophy" of seminiferous tubules. As already pointed out, these patients seem to be good candidates for the successful treatment of male infertility by TESE (Schulze et al., 1997; Je`ek et al., 1998). In our study, we have found the strong negative correlation between the number and the volume of mast cells and the testicular volume and score. In general, the more intensive the damage of seminiferous tubules was, the lower was the testis volume and score. However, it should be pointed out that there were exceptions to this rule. Few cases presented heavy impairment of spermatogenesis and yet, the presence of mast cells was not increased as it would have been expected. The reason/s for this remain to be explained. In conclusion, we have analysed the mast cells in the biopsies of patients with the "mixed atrophy" of seminiferous tubules. Two distinctive mast cell groups regarding their location within the testicular tissue could be distinguished: "interstitial" and "peritubular" mast cells. In the control biopsies, "interstitial" cells were the chief mast cells while "peritubular" mast cells were only occasionally observed. In the biopsies of infertile patients, there was significant rise in number and volume of the both, "interstitial" and "peritubular" mast cells. Although "interstitial" cells again predominated (in number and volume) over the "peritubular" cells, the morphometric analysis pointed out that the increasing rate of "peritubular" cells was higher than that of "interstitial" mast cells. The present study demonstrates the close relation between the increase of mast cells and male infertility. Possible reasons for the increased presence of mast cells in the patients with the "mixed atrophy of seminiferous tubules are discussed. Acknowledgements The authors are indebted to Mrs. E. Schfer and Mrs. I. Tschrner for their skilful technical assistance. The study was supported by the Austrian National Bank (Grant No. 6487), Croatian Ministry of Science and Research (Grants Nos. 108012 and 108900) and Das Partnerschaftabkommen der Med. 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Zsebo LM, Wypych J, McNiece IK (1990) Identification, purification and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell 63: 195-201. LEGENDS FOR FIGURES Fig. 1. Semithin section of the testicular biopsy from vasectomy patient. Between two seminiferous tubules (stars) and tubulus rectus (TR) "interstitial" mast cell (arrowhead) is visible (ls - late spermatids). x400. Bar = 50 symbol 109 \f "Symbol"m. Fig. 2. Abundant presence of mast cells (arrowheads) in testis interstitium of infertile patient (star - seminiferous tubule) (L - Leydig cells). x400. Bar = 50 symbol 109 \f "Symbol"m. Fig. 3. Perivascullary localised mast cell (arrowhead) in testicular biopsy of infertile man. Blood vessels (arrows) display a pathological thickening of their wall (star - seminiferous tubule). x400. Bar = 50 symbol 109 \f "Symbol"m. Fig. 4. Two seminiferous tubules (stars) from infertile patient with a significant enlargement of the tubular lamina propria. In the lamina propria of one tubule, two "peritubular" mast cells (arrowheads) are situated (L - Leydig cells). x400. Bar = 50 symbol 109 \f "Symbol"m. Fig. 5. Control biopsy from the vasectomy patient. Interstitial" mast cell (M) is typically localised between neighbouring Leydig cells (L) and blood vessel (C - capillary) (stars - Reinkesymbol 162 \f "Symbol"s crystals). x6.000. Bar = 2 symbol 109 \f "Symbol"m. Fig. 6. Detail of the mast cell illustrated in Fig.5. In the vicinity of the nucleus (N), many characteristic granules (arrowheads) could be seen. Occasional cytoplasmic projections (large arrow) are observed on the cell surface. The mast and endothelial cell (E) are divided by a small network of collagen fibres (c) and ground substance. On its surface, the endothelial cell bears numerous pinocytotic vesicles (small arrows). x24.000. Bar = 1 symbol 109 \f "Symbol"m. Fig. 7. Testicular biopsy from infertile patient with mixed atrophy. In this case, "peritubular" mast cell (M) is incorporated into the thickened lamina propria of one seminiferous tubule. The fraction of another such cell is indicated by an arrow. Spindle-shaped myoid cells (parts of these cells are depicted by arrowheads) surround the mast cell together with the augmented collagen network (C) and ground substance. x6.000. Bar = 2 symbol 109 \f "Symbol"m. Fig. 8. Mast cell from the previous figure, detail. Around the nucleus (N), multitudinous granules (arrowheads) with a complex multilayered (scroll and reticular-like) structure could be observed. Little cytoplasmic projections of the mast cell (large arrows) stretch out towards myoid cells (M) or establish tiny membrane-to-membrane contacts (small arrows). x24.000. Bar = 1 symbol 109 \f "Symbol"m. Fig. 9. Part of the biopsy from the infertile patient with a fibrosis of interstitial testicular tissue. Between some interstitial cells (I) and enlarged collagen fibres (C) and ground substance, a mast cell (M) could be identified. x6.000. Bar = 2 symbol 109 \f "Symbol"m. Fig. 10. Detail of the mast cell depicted in Fig. 9. Despite the interstitial fibrosis, the ultrastructure of this cell is well preserved, demonstrating a lot of granules (arrowheads) and other organelles (m - mitochondrion). x24.000. Bar = 1 m. Tables Table 1. Testis volume and status of spermatogenesis evaluated by Johnsensymbol 162 \f "Symbol"s score in control and infertile patients. Table 2. Number and volume of mast cells in control and infertile group of patients (Nv - number of "interstitial" and "peritubular" mast cells per mm3 of the tissue; N - number of "interstitial" and "peritubular" mast cells per entire testis; Vv - volume of "interstitial" and "peritubular" mast cells per mm3 of the tissue; V - volume of "interstitial" and "peritubular" mast cells per entire testis; Nvi - number of "interstitial" mast cells per mm3 of the tissue; Ni - number of "interstitial" mast cells per entire testis; Vvi - volume of "interstitial" mast cells per mm3 of the tissue; Vi - volume of "interstitial" mast cells per entire testis; Nvp - number of "peritubular" mast cells per mm3 of the tissue; Np - number of "peritubular" mast cells per entire testis; Vvp - volume of "peritubular" mast cells per mm3 of the tissue; Vp - volume of "peritubular" mast cells per entire testis). Table 3. Correlation between the analysed stereological variables, testis volume and score. Table 1. Variable / Group CONTROLS Mean symbol 177 \f "Symbol" SE INFERTILE Mean symbol 177 \f "Symbol" SE  Testis volume symbol 91 \f "Symbol"cm3symbol 93 \f "Symbol" 17.322 symbol 177 \f "Symbol" 0.222 11.628 symbol 177 \f "Symbol" 0.668****  Score 9.228 symbol 177 \f "Symbol" 0.089 5.300 symbol 177 \f "Symbol" 0.398****  **** - p<0.0001 Table 2. Variable / Group CONTROLS Mean symbol 177 \f "Symbol" SE INFERTILE Mean symbol 177 \f "Symbol" SE  Nv symbol 91 \f "Symbol"mm-3symbol 93 \f "Symbol" (x102) 3.791 symbol 177 \f "Symbol" 0.593 19.782 symbol 177 \f "Symbol" 2.361***  N symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x109) 8.211 symbol 177 \f "Symbol" 1.026 21.449 symbol 177 \f "Symbol" 2.331**  Vv symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x10-4) 3.271 symbol 177 \f "Symbol" 0.519 12.442 symbol 177 \f "Symbol" 0.135**  V symbol 91 \f "Symbol"mm3symbol 93 \f "Symbol" (x106) 5.354 symbol 177 \f "Symbol" 0.883 13.064 symbol 177 \f "Symbol" 1.360**   Nvi symbol 91 \f "Symbol"mm-3symbol 93 \f "Symbol" (x102) 3.084 symbol 177 \f "Symbol" 0.636 12.307 symbol 177 \f "Symbol" 1.072***  Ni symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x109) 5.059 symbol 177 \f "Symbol" 1.095 13.730 symbol 177 \f "Symbol" 1.354**  Vvi symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x10-4) 2.477 symbol 177 \f "Symbol" 0.553 7.928 symbol 177 \f "Symbol" 0.794**  Vi symbol 91 \f "Symbol"mm3symbol 93 \f "Symbol" (x106) 4.065 symbol 177 \f "Symbol" 0.930 8.683 symbol 177 \f "Symbol" 1.013*   Nvp symbol 91 \f "Symbol"mm-3symbol 93 \f "Symbol" (x102) 0.728 symbol 177 \f "Symbol" 0.131 7.498 symbol 177 \f "Symbol" 1.593**  Np symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x109) 3.117 symbol 177 \f "Symbol" 1.154 7.739 symbol 177 \f "Symbol" 1.424  Vvp symbol 91 \f "Symbol"mm0symbol 93 \f "Symbol" (x10-4) 0.792 symbol 177 \f "Symbol" 0.111 4.394 symbol 177 \f "Symbol" 0.844*  Vp symbol 91 \f "Symbol"mm3symbol 93 \f "Symbol" (x106) 1.286 symbol 177 \f "Symbol" 0.175 4.388 symbol 177 \f "Symbol" 0.667*  * - p<0.05; ** - p<0.01; *** - p<0.001 Table 3. 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