Difficulties in distinguishing between a mature spermatid and testicular spermatozoon

R.T. Mansour1, M.A. Aboulgar, G.I. Serour, A. Kamal, N.A. Tawab and I. Fahmy

The Egyptian IVF–ET Center, 85 Maadi Zeraei Road, Maadi, Cairo 11431, Egypt

Dear Sir,

We read with interest the paper by Kahraman et al. (1998). The problem of spermatid identification in unstained wet mounts during intracytoplasmic sperm injection (ICSI), has to be resolved so that different investigators are talking about the same thing. We do not agree with the authors that it is usually difficult to discriminate a mature spermatid from a mature spermatozoon and it very rarely has a twitching tail motility. In our view, what they are considering as a spermatid (Figure 2) is simply a spermatozoon. Based on the Clermont classification (1963), as illustrated by de Kretser and Kerr (1994), elongated spermatids (Sb2 stage) have a dark condensed nucleus which resembles the head of a spermatozoon, and the cytoplasm is shed from around the nucleus surrounding it from one side only, giving it a very characteristic cone shape appearance (Mansour et al., 1998). At this stage, the forming tail is still embedded in the cytoplasm that is shed at one side of the nucleus and it is difficult to identify under x400 magnification (during the actual ICSI work). If we agree on what the authors consider as spermatids, consequently, hundreds of pregnancies that have been already achieved in non-obstructive azoospermia all over the world could have been claimed as spermatid pregnancies. With regard to round spermatids, Figure 1 (Kahraman et al., 1998Go) showed a round spermatid and a spermatozoon in the same field. Since the process of spermatogenesis is complete to the stage of spermatozoa, it would be expected to find different forms of spermatogenetic cells including round spermatids. The clinically significant issue is to be able to inject round spermatids where no other more mature forms are available. Unfortunately, it is extremely rare to find only round spermatids, without any other more mature spermatogenic cells (Mansour et al., 1998).

Notes

1 To whom correspondence should be addressed Back

References

de Kretser, D.M. and Kerr J.B. (1994) The cytology of the testis. In Knobil, E. and Neill, J. (eds), The Physiology of Reproduction. 2nd edn. Raven Press, New York, USA, pp. 1177–1290.

Kahraman, S., Polat, G., Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104–110.[Abstract]

Mansour, R.T. (1998) Intracytoplasmic spem injection: a state of the art technique. Hum. Reprod. Update, 4, 43–56.[Abstract/Free Full Text]


 
S. Kahraman

Bevgi Hospital, Department of Reproductive Endocrinology, and Infertility, Tunus Caddesi, No. 28 Kavaklidere A, Ankara 06680, Turkey

Dear Sir,

I would very much like to reply to Dr. Mansour's comments. However, primarily, I would like to emphasize that only round and elongated spermatids were used for intracytoplasmic sperm injection (ICSI) in our study as is clearly shown in Table II. Ironically, Figure 2 (mature spermatid) was included to assist with the classification of a mature spermatid, not to confuse the reader. At this late stage, these cells can be termed `mature spermatids, late elongated spermatids, or immature spermatozoa'. As this stage is still controversial, these cases were not included in our study.

Ultrastructural observations of the differentiation of spermatids were performed by some authors (Clermont, 1963Go; de Kretser and Kerr, 1994; Holstein, 1976Go). They developed a classification and made a strict distinction between the different developmental stages (acrosome swelling Sa, Sb1, Sb2; nuclear changes, development of the tail Sb2, Sc, Sd1, Sd2, cytoplasm reorganization), involving the beginning of spermiogenesis (Golgi cap and acrosome phase).

Unfortunately, `in situ', without cytological techniques and under a conventional light microscopic technique, it is more diffficult to make a strict distinction between the different stages. The stage from an elongated spermatid to a mature spermatozoa should be defined and more detailed classification is necessary as this is not a direct transition. Although a classification on spermiogenesis was described by de Kretser and Kerr (1994), the transition from an elongated to a mature spermatozoa needs to be further explained as these cells which are in different stages of spermiogenesis are more commonly used for ICSI. For this reason, the term `mature spermatid', first employed by Silber and Lenohan (1995). Furthermore, Vanderzwalmen et al. (1997) used this terminology including an illustration in their excellent study on spermatids. lt is difficult to give a strict terminology for this kind of spermatid, so it is defined as a `mature spermatid (Silber and Lenohan, 1995) or late elongated spermatid or immature spermatozoon' since they appear as the well known spermatozoon (Sd2). Possibly the degree of maturity of the cytoplasm and the nucleus is not totally the same as for the mature spermatozoon. As it was emphasized by Silber, there is little discernable difference between a mature spermatid and a testicular spermatozoon. The only difference really is a technical one that a spermatid is still embedded in the Sertoli cell, and a sperm has been released from the Sertoli cell (personal communication). Moreover, a part of the mature spermatid, which is trapped between the Sertoli cells at the level of the lumen, exhibits a moving tail. Therefore, on a morphological basis, such spermatids could not be included in the results termed as `elongated spermatids' but, as suggested by Silber, as `mature spermatids'.

During a conventional ICSI–testicular sperm extraction (TESE) procedure, after a gentle crushing of the seminiferous tubules between two needles, extremely few isolated spermatozoa in the suspension can be observed, but cells which are still entrapped in the germinal epithelium have protruding tails coming outside. After enzymatic treatment or hard delaceration of the tubules, a large majority of the male gamete cells is released from the Sertoli cell and can be used successfully in a conventional ICSI–TESE programme without making a distinction between the spermatozoon and the mature spermatid.

Furthermore, as suggested by Tesarik et al (1998a), under pathological conditions, the classical spermiation process did not occur due to a poor contact between the male gamete and the Sertoli cells with an earlier release of elongated or even round spermatids. In such a situation it is also impossible to make the distinction between an abnormal spermatozoon and a prematurely released spermatid.

As described by Vanderzwalmen et al. (1997) according to the size of the tail, the spermatids were classified as elongating or elongated. Cells with an oval shape, a visible head but well defined tail were classified in the elongated spermatid group. The mature spermatids, aspirated from the germinal epithelium, appear similar to the ultimate sperm morphology. In their study the injection of mature spermatids (n = 3) was included where the search for cells took >2 h. However, cases with a higher concentration of mature spermatids trapped between Sertoli cells, were not included in this study even if such cells were histologically classified as mature spermatids.

With regard to Figure 1, it was emphasized in the results section that in three of our cases elongated spermatids were used and a few immotile spermatozoa were identified in the basal sample (pellet). Although only two or three spermatozoa were transferred in a droplet, we were unable to find any during the ICSI procedure. In these cases, elongated forms were used for ICSI. Figure 1 demonstrates one of these patients testicular samples with round spermatid and spermatozoa. When patients with very severe testicular dysfunction are treated, the qualitative criteria is more difficult to recognize. This occurs in some cases when some of the tubules show spermatids and the percentage of spermatids in the tubules are extremely low.

Round spermatids can be detected in some patients in whom late spermatids or spermatozoa cannot be found (Amer et al., 1997Go; Tesarik et al., 1998bGo). Similar findings were also reported by other groups (Kahraman et al., 1997Go; Lee et al., 1997Go; Yamanaka et al., 1998). It is currently impossible to determine the relative incidence in which only round spermatids are produced in the testis of patients with non-obstructive azoospermia as the results from different centres are dependent on their individual treatment policy. It cannot be excluded that, in some cases, spermatozoa would be found by sampling more testicular tissue.

In non-obstructive azoospermia, depending on the severity of spermatogenesis it is possible to find either a few or no spermatozoa amongst other immature spermatogenetic cells. However, the localization of residual spermatogenetic foci in the testis is difficult and unpredictable. Acceptable fertilization rates after spermatid injection were obtained when spermatids from a testis highlighted a few spermatozoa in a previous positive biopsy (Vanderzwalmen, 1997).

As the number of spermatozoa was so low, round or mostly more mature forms (elongated) were used in three patients in our study. Therefore, the pregnancies obtained were achieved with only elongated forms where a few spermatozoa were present in their basal sample.

References

Amer, M., Soliman, E., El-Sadek, M. et al. (1997) Is complete spermiogenesis failure a good indication for spermatid conception? Lancet, 350, 116.

Clermont, Y. (1963). The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 35–51.[ISI]

de Kretser, D.M. and Kerr, J.B. (1994). The cytology of the testis. In: The physiology Silber S.J., Lenohan K., (1995) : Sertoli cell surgery: spermatid retrieval and ICSI in azoospermic patient with maturation arrest. Human Reprod. Update, Vol 1, No:6, November(1995).

Holstein, A. (1976). Ultrastructural observations of the differentiation of spermatids in man. Andrology, 8, 157–165.

Kahraman, S., Polat, G., Sozen, E. et al. (1997) Fertilization and pregnancy rates of testicular spermatids in ICSI. [Abstr. no. P-108] Hum. Reprod., 12 (Abstr. Book 1), 171–172.

Lee, D.R., Lim, Y.J., Yoon, H.S. et al. (1997) Application of ICSI or ROSI by testicular biopsy in non-obstructive azoospermia [Abstr. no. P–118] Fertil. Steril. (ASRM Annual Meeting Program Suppl.), 148.

Tesarik, J., Greco, E. and Mendoza, C. (1998a) ROSI, instructions for use: 1997 update. Hum. Reprod., 13, 519–523.[ISI][Medline]

Tesarik, J., Sousa, M., Greco, E. et al. (1998b) Spermatids as gametes: indications and limitations. Hum. Reprod., 13 (Suppl.3) 89–107.[Medline]

Vanderzwalmen, P., Zech, H. and Birkenfeld, A. (1997) Intracytoplasmic injection of spermatid retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 1203–1213.[ISI][Medline]

Vanderzwalmen, P., Nijs, M., Schoysman, R. et al. (1998) The problems of spermatid microinjection in the human: the need for an accurate morphological approach and selective methods for viable and normal cells. Hum. Reprod., 3, in press.

Yamanaka K., Sofikitis N.V., Miyagawa I. Et. al., (1997). Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for non-obstructive azoospermia. J. Assist. Reprod. Genet., 14, 55–62[ISI][Medline]