1 Andrology Department, Cairo University Hospital, Cairo, 2 Adam International Clinic, Guiza and 3 AI/ET Department, Animal Reproduction Research Institute, Guiza, Egypt
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Abstract |
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Key words: azoospermia/intracytoplasmic sperm injection/May-Grünwald-Giemsa stain/spermatids/sperm extraction
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Introduction |
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In order to predict the likelihood of successful TESE, several studies have been performed to determine the value of various parameters, including testis size (Tournaye et al., 1996), FSH concentration (Kahraman et al., 1996
), previous testicular histopathology (Tournaye et al., 1997
) and, more recently, seminal anti-Müllerian hormone (AMH) (Fenichel et al., 1999
). There was no strong predictor for successful TESE except testicular histopathology. Recently, it has been reported that spermatozoa can be found in all patients, with detectable round spermatids in testis biopsy (Silber and Johnson, 1998
). Despite a strong association of testicular spermatids with TESE confirmed, and a prospective study correlating TESE with morphological, biophysical and endocrine profiles in men with functional azoospermia, the correlation between histological patterns and TESE was found to be poor (Ezeh et al., 1998
). Nonetheless, a good correlation was found between the TESE result and the detection of seminal fluid spermatids, using immunofluorescent localization. However, this technique is sophisticated and requires special skills; hence its routine laboratory application is not straightforward.
Consequently, prediction of TESE outcome, based on seminal characteristics, would be very beneficial in patients with NOA as it may circumvent the need for any preliminary diagnostic testicular biopsy, with its recognized hazards. The presence of ejaculated spermatids indicates that spermatogenesis must have reached the post-meiotic stage in at least part of the testis, and so the possibility of finding testicular spermatozoa without performing diagnostic biopsy is increased. The aim of the current study was to identify seminal spermatids by using the inexpensive, commercially available May GrünwaldGiemsa (MGG) stain, and to assess its predictive value for successful TESE in comparison with serum FSH concentration, testicular volume and histopathology.
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Materials and methods |
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Technique of MGG staining
The seminal fluid or testicular biopsy material was centrifuged, and the pellet smeared onto a glass slide and left to air-dry for 12 h. The smear was then stained using a stepwise procedure (Ludwig and Frick, 1987). The smear was fixed for 15 min in methanol, and transferred without blotting to diluted MayGrünwald solution for 15 min. (A stock 0.3% MayGrünwald solution was prepared by grinding the powdered stain in methanol with a pestle and mortar, and filtering after 23 days. For use, the stock solution was diluted 1:1 with phosphate buffer, pH 7.2, the dilute solution being discarded after 1 day.)
Excess stain was drained off on filter paper (without blotting), and the smear was then transferred to dilute Giemsa solution for 30 min. (Giemsa was prepared by dissolving 0.6 g powder in 50 ml methanol, adding 25 ml glycerine, and filtering after 23 days. For use, the stock solution was diluted 1:9 in phosphate buffer, pH 7.2.)
The smear was transferred to phosphate buffer, pH 7.2, rinsed for 1020 s, and then dried and mounted. Smears were examined under an oil immersion lens (x1000) for the presence of spermatocytes and round spermatids according to morphological criteria and staining properties. Primary spermatocytes are broad cells, and have a large central nucleus 89 µm in diameter in which the chromosomal spindle and a large central nucleolus 12 µm in diameter can be recognized. They are very fragile cells, and are easily damaged by preparation techniques. Initial (round) spermatids are small round cells with a condensed nucleus (sometimes two nuclei) with a continuous rim of cytoplasm and an acrosomal vesicle appearing either as an empty dot or having a pink coloration. At earlier stages, the acrosomal vesicle may not be present; thus, its presence confirms the identification of spermatid (Gandini et al., 1999) (Figures 1 and 2
).
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Testis biopsy
Under general anaesthesia, a vertical incision was made in the median scrotal raphe. One testis was delivered outside the wound, and inspected together with the epididymis. A biopsy was taken from the upper pole of the testis and sent for immediate sperm detection. If no spermatozoa were identified after thorough examination of the entire specimen, a second biopsy was taken from the lower pole. If still no spermatozoa were found, biopsies were taken from the medial and then lateral aspects of the testis. Two further biopsies were taken from the upper and lower poles and fixed in Bouin's fixative for histopathological examination. After adequate haemostasis, the tunica were closed, and the other testis was delivered through the same incision, whereupon the same steps were repeated. If at any time spermatozoa were seen, the procedure was terminated and the wound closed in layers with 3/0 Vicryl sutures.
TESE
TESE was performed by mincing and shredding of the biopsy specimen (Verheyen et al., 1995) in HEPES-buffered Earle's balanced salt solution, using sterile glass slides and forceps under laminar flow in a Petri dish. The minced tissue was examined under an inverted microscope (Hoffman optics) using x400 magnification. If no spermatozoa were seen after checking the entire dish, the contents were transferred to a 5 ml Falcon tube after removing gross testicular debris, centrifuged at 400 g for 5 min, and the pellet was re-examined for the presence of spermatozoa. The result was interpreted as positive or negative TESE according to the presence or absence of at least one live spermatozoon; this was either spontaneously motile, became motile after pentoxifylline stimulation, or was shown to be viable by the hypo-osmotic swelling test (HOST).
Statistical analysis
2 and unpaired Student's t-tests were used as appropriate. Statistical significance was accepted at a P value of < 0.05.
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Results |
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There was no statistically significant difference in testicular volume between patients with retrievable spermatozoa and those without (Table II). Furthermore, no lower limit for testicular volume to exclude the presence of spermatozoa was identified, where spermatozoa could be retrieved from testes of <5 ml volume.
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Discussion |
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Testicular volume was found to have a poor predictive value for successful TESE, as topographical variations in testicular pathology can occur independently of testicular size. Thus, large testicular volume does not indicate successful TESE in patients showing SCO pattern in their testicular histopathology. Conversely, in patients showing maturation arrest pattern, a low testicular volume does not preclude successful TESE (Devroey et al., 1995). On the basis of the current results, testicular volume is seen to be an inaccurate parameter, as spermatozoa were successfully retrieved from 44% of normal-sized testes, 42% of moderate-sized testes, and from 25% of small-sized testes, the latter value being especially high for such a population of patients. Furthermore, no lower limit of testicular volume excluded the presence of testicular spermatozoa.
In the current study it was possible to retrieve spermatozoa from 62% of patients with normal FSH, from 59.4% with mildly elevated FSH, and from 30.8% with markedly elevated serum FSH. No upper limit for FSH could be detected that excluded the presence of testicular spermatozoa. The sensitivity of FSH in predicting successful TESE is low at normal levels, but with higher concentrations of FSH the sensitivity increases while the specificity decreases. These results support the findings of many investigators who showed that serum FSH concentration has a poor predictive value for successful TESE (Chen et al., 1996; Kahraman et al., 1996
; Jezek et al., 1998
; Ezeh et al., 1999
). This is because FSH concentration is not related to sperm count or production, but it is related inversely to the total number of testicular germ cells present. Hence, the only relationship between serum FSH concentration and the more advanced stages of spermatogenesis is an indirect one, and depends strictly on the primary inverse relationship between FSH and spermatogonia (Silber et al., 1996
). Therefore, patients with complete maturation arrest could be expected to have normal FSH concentrations, but no spermatozoa could be retrieved from the testis, whereas patients with SCO pattern in most of the testis and with small foci of active spermatogenesis are most likely to have a very high FSH concentration, with spermatozoa retrievable on biopsy (Silber et al., 1996
).
Histopathology of testicular biopsy sections is considered the best predictive parameter for successful TESE, especially with visualization of mature testicular spermatids. Although its value in functional azoospermia is questionable (Tournaye et al., 1996, 1997
), visualization of testicular spermatids at histopathological examination was found to provide a correct prediction in 77% of cases (Ezeh et al., 1999
).
Correlation of TESE with the histopathological picture showed that spermatozoa were retrievable from 85% of cases with round spermatid arrest, from 44.4% in C1 arrest, from 65% with mixed pathology, from 24.2% in SCO syndrome and from 25% in cases with tubular sclerosis. Thus, the chances of sperm retrieval improves with more advanced stages of spermatogenesis, and detection of round spermatids in histopathology can predict successful TESE in about 74% of the current cases. However, spermatozoa could still be retrieved from cases without any spermatids seen histopathologically. Consequently, histopathology alone cannot be considered as an accurate parameter for prediction when spermatids are not observed, even in cases of round spermatid arrest, when spermatozoa could not be detected in 15% of cases. This fact confirms our previous description of the entity of complete spermiogenesis failure, where round spermatids were detected in the testis without finding testicular spermatozoa (Amer et al., 1997). The finding also confirms that testicular biopsy for histopathology is not completely representative of the whole testis, there being variation between both sides, and also from site to site within the same testis (Amer et al., 1999
).
Immature germ cells may be present in the ejaculate of normospermic, oligozoospermic and azoospermic patients. The quantity of these germ cells increases as the sperm count decreases, and is not related to serum FSH concentration (Tomlinson et al., 1992; Tesarik and Mendoza, 1996
). It has also been reported that the presence of round spermatids in testis biopsy indicates the presence of mature spermatozoa, and it was claimed that no case with round spermatids has complete absence of spermatozoa (Silber and Johnson, 1998
). Furthermore, the number of testicular spermatids has been shown to correlate with sperm count in men with oligozoospermia (Silber and Rodriguez, 1981
), and with testicular spermatozoa in men with non-obstructive azoospermia (Mulhall et al., 1997
; Silber et al., 1997
). In a recent study (Ezeh et al., 1999
), it was reported that identification of seminal spermatids by an immunofluorescence method utilizing mouse monoclonal antibodies could predict testicular sperm retrieval (100%); however, testicular spermatozoa could still be extracted from patients without detectable seminal spermatids in 25% of cases. Thus, the sensitivity was 100% while the specificity was 75%. Various methods ranging from Percoll centrifugation and direct microscopy of cells in the ejaculates (Tesarik et al., 1995
; Tesarik and Mendoza, 1996
; Gandini et al., 1999
) to immunological methods such as staining with monoclonal antibodies to the acrosomal moiety (Moore et al., 1987
) and antiacrosin antibodies (Tesarik and Mendoza, 1996
) have been used to identify spermatids in the human ejaculate. These techniques are rather tedious, expensive and require special facilities, however. A more simple technique was described by using MayGrünwaldGiemsa stain to identify various types of testicular and ejaculated germ cells (Ludwig and Frick, 1987
; Foresta et al., 1992
; Gandini et al., 1999
). This method has been found to be simple, effective and inexpensive, and also to correlate with other methods of spermatogenic cell identification such as Papanicolaou (Pap) staining and immunofluorescence techniques (M.Amer et al., unpublished data).
Evidence is accumulating that deletion of the AZFc region only on the Y chromosome confers a slightly better chance of having usable spermatozoa, either from the ejaculate or from the testis, whereas AZFb-region deletions may predict failure of sperm recovery (Brandell et al., 1998). The current results with MGG showed that identification of round spermatids in semen has a sensitivity of 83.7% and a specificity of 78%, while identification of primary spermatocytes has a sensitivity of 100% and a specificity of only 50%. Thus, this simple, cheap test can predict successful TESE in comparison with the expensive sophisticated methods used to detect Y-chromosome microdeletion and the azoospermia factor. Moreover, the detection of round spermatids in semen using MGG stain as a predictive parameter for successful sperm recovery is more accurate than testicular volume, FSH concentration or even previous histopathology or tested TESE, as the patient may have spermatozoa in previous tested TESE, but no spermatozoa could be retrieved at the time of TESEICSI attempts (Vanderzwalmen et al., 1997
; Amer et al., 1999
). More extensive methods for processing testicular biopsy using collagenase and trypsin inhibitor or erythrocyte-lysing buffer provide a gentle dissolution of the cells from their tissue, and clear the red blood corpuscles. This results in high yields of mature viable spermatozoa, and makes the search for spermatozoa simple and more rapid (Salzbrunn et al., 1996
; Crabbé et al., 1997
; Nagy et al., 1997
). Thus, we expect that microsurgical biopsy to select healthy tubules (Schlegel, 1999
; Amer et al., 2000
), combined with more extensive processing of the biopsy using the above-mentioned techniques, providing more time and patience for the search, will increase the sensitivity and specificity of spermatid identification in predicting TESE outcome and the numbers of false-negative cases will be minimized.
The fact that motile spermatozoa were recovered from cases without detected spermatids (eight patients) in the ejaculate may suggest the presence of partial or intermittent obstruction. We agree with others (Ezeh et al., 1998), that the absence of ejaculated spermatids cannot be used to distinguish obstructive from non-obstructive azoospermia, but that their presence indicate NOA. Another hypothesis which had been advanced (Ezeh et al., 1998
) is that there may be a threshold level of spermatogenesis below which no spermatids could be detected in semen. In support of this hypothesis is a study showing that in order for any spermatozoa to reach the ejaculate, more than six mature spermatids per seminiferous tubule must be present in the histological section of the testis (Silber et al., 1997
). Moreover, MGG staining after a modified discontinuous Percoll gradient technique (Gandini et al., 1999
) may be helpful in detecting spermatids in patients with negative MGG screening test in semen, and retrievable testicular spermatozoa.
The current results indicate that the sensitivity of detecting seminal spermatids is 100% in predicting the presence of testicular round spermatids, and its specificity is 92.8%. This observation may be important in the selection of cases who are candidates for in-vitro culture of spermatogenic cells from testicular tissue in case of complete absence of spermatozoa after an extensive search, as has been recently suggested (Tesarik et al., 1998).
In conclusion, the detection of seminal spermatids using MGG stain offers a good predictive parameter for successful TESE compared with testicular size, serum FSH concentration and histopathological diagnostic testicular biopsy. Additional advantages of the MGG stain are the simplicity, reliability, low cost and commercial availability. Advances in the techniques of TESE and processing combined with extensive search may further improve its sensitivity and specificity in the near future.
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Notes |
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References |
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Submitted on June 12, 2000; accepted on March 20, 2001.