Spermatid injection

Reconsidering spermatid injection

Y. Prapas1, A. Chatziparasidou2,4, P. Vanderzwalmen3, M. Nijs3, N. Prapas1, B. Lejeune3, G. Vlassis1 and R. Schoysman3

1 Fourth Department of Obstetrics and Gynaecology, Aristoteleian University of Thessaloniki, Thessaloniki, 2 Iakentro, IVF Medical Center, Agiou Vasiliou 4, 54250 Thessaloniki, Greece and 3 SIMAF Van Helmont Hospital, Vilvoorde, Belgium

New developments in the treatment of male infertility offer the possibility for even azoospermic men with impaired spermiogenesis to become biological fathers. The combination of intracytoplasmic sperm injection (ICSI) with techniques such as microepididymal sperm aspiration (MESA), fine needle aspiration (FNA), testicular sperm extraction (TESE) and testicular sperm aspiration (TESA), in most cases yield tiny (but sufficient) spermatozoa to be used on the day of oocyte retrieval. However, there are cases where no spermatozoa can be found, and in these cases, some scientists have attempted spermatid injection based on the data already available in the rodent model (mouse: Ogura et al., 1993, 1994; rabbit: Sofikitis et al., 1994) after it was first suggested (Edwards et al., 1994Go). This new approach has given encouraging results, and has led to fertilization and embryo formation (Vanderzwalmen et al., 1995Go) and to 35 pregnancies (Tesarik et al., 1995Go, 1996Go; Fishel et al., 1995Go, 1997Go; Tesarik and Mendoza, 1996aGo; Amer et al., 1997Go; Antinori et al., 1997aGo,bGo; Araki et al., 1997Go; Vanderzwalmenet al., 1997Go; Barak et al., 1998Go; Barros et al., 1998aGo,bGo; Bernabeu et al., 1998Go; Kahraman et al., 1998Go; Sofikitis et al., 1998aGo).

Spermatids are immature haploid germ cells that have not yet undergone all the biochemical and morphological changes that accompany spermiogenesis through to the formation of spermatozoa. Today, 4 years after the first baby was born as a result of spermatid injection (Tesarik et al., 1995Go), the clinical outcome of spermatid injection remains low when using round cells and there are many concerns about the suitability and the safety of this technique (Fishel et al., 1996Go, 1997Go; Tesarik and Mendoza, 1996bGo; Kahraman et al., 1996Go; Silber and Johnson, 1998Go; Sousa et al., 1998; Tesarik et al., 1998aGo,bGo; Vanderzwalmen et al., 1998Go; Aslam et al., 1998aGo; Sofikitis et al., 1998bGo).

When only spermatids are available, using those that are at the elongation phase give normal fertilization and implantation rates (Vanderzwalmen et al., 1997Go; Kahraman et al., 1998Go). At this developmental stage, the elongating spermatids are easy to recognize among the other cell types in a wet preparation. However, the isolation and morphological identification of spermatids does not reveal anything about spermatid viability, even when elongating and elongated spermatids are selected. According to a recent study (Aslam et al., 1998bGo), 95% of the isolated spermatids remain viable for at least 12 h, but in severe pathological cases, germ cell apoptosis in men with complete and incomplete spermiogenesis failure has been found (Tesarik et al., 1998cGo). However, the high pregnancy rates obtained with elongating and elongated spermatids suggest that most of the cells are viable and genetically normal.

When round spermatids are used instead of elongating or elongated spermatids, the clinical outcome is dramatically lower, and this treatment is still under consideration. Nevertheless, there have been eight pregnancies reported that were achieved as a result of round spermatid injection, three using spermatids from ejaculates (Tesarik et al., 1995Go, 1996Go; Tesarik and Mendoza, 1996aGo; Barak et al., 1998Go) and five using spermatids extracted from testicular treatment biopsies (Antinori et al., 1997aGo; Vanderzwalmen et al., 1997Go; Kahraman et al., 1998Go), with one cycle using frozen–thawed cells (Antinori et al., 1997bGo). However, in all these cases, the male patient did present in his past history the presence of elongated spermatids or spermatozoa in the ejaculate or in the diagnostic testicle biopsy, with no elongated spermatids or spermatozoa having been recovered at the moment of treatment. In contrast, in all cases where the male patient showed a complete absence of elongated spermatids or spermatozoa in previous ejaculates or in the diagnostic testicle biopsy, no pregnancy has been obtained so far using round spermatids (Amer et al., 1997Go; Vanderzwalmen et al., 1997Go; Barros et al., 1998aGo,bGo; Bernabeu et al., 1998Go; Kahraman et al., 1998Go; Sofikitis et al., 1998aGo).

Perhaps the future should be the in-vitro culture of round spermatids to the elongation phase. Recent reports (Aslam and Fishel, 1998Go; Tesarik et al., 1998cGo) have indicated that round spermatids can develop flagellae and elongate after in-vitro culture under special media, either supplemented with sera or recombinant follicle stimulating hormone (rFSH). To date, there has been only one report of a pregnancy after culture of round spermatids (Tesarik et al., 1999Go). In spite of the fact that in-vitro culture of round spermatids is a tentative approach, it is our opinion that, before using in-vitro matured spermatids for clinical trials, those cells should be firstly experimentally studied to see if they are viable (Trypan Blue exclusion staining), if they present normal chromosomal contents [fluorescent in-situ hybridization (FISH)], if they are able to fertilize oocytes (experimental injections and determination of normal fertilization and cleavage rates), and if the embryos produced are genetically normal (by FISH). Since this approach seems to be very promising, in the interest of many patients it is urgent that other groups begin research programmes in an attempt to find the appropriate medium that could successfully mature round spermatids up to the elongated stage in vitro or even to mature spermatozoa at a reasonable rate. Also, the research on in-vitro culturing of round spermatids should study the optimum temperature for maturation, the reason why spermatogenesis in vitro seems accelerated, and whether the whole spermatogenic population can be used instead of isolated cells.

We are aware that spermatid injection is a novel way of treating male infertility and that there are risks concerning its clinical application, since spermatids are precursor cells that still need to go through changes in order to give mature functional forms. At present there is no way to ensure that the spermatids selected for injection are alive and that they are not undergoing apoptosis. Neither is there any way of ensuring that the selected immature haploid germ cells are genetically normal (Hendry et al., 1976Go). For this reason, we stress the need for couples entering the spermatid injection programme to be informed in detail of all the risk factors concerning the application of this technique, and to apply amniocentesis when pregnancies are obtained, although most of the babies born up to the present moment appear normal. Long-term follow up of those babies born is still recommended.

According to the results of our spermatid injection programme, two pregnancies were established after elongating spermatid injection. One was delivered uneventfully whereas the other had to be terminated as the amniocentesis results showed trisomy of chromosome 9.

Presently, it is known that a diagnostic testicle biopsy showing Sertoli cell-only syndrome or maturation arrest at the primary spermatocyte level does not preclude the possibility of finding spermatozoa or elongated spermatids for ICSI treatment (Amer et al., 1997Go; Vanderzwalmen et al., 1997Go; Barros et al., 1998aGo,bGo; Bernabeu et al., 1998Go; Kahraman et al., 1998Go; Sofikitis et al., 1998aGo). For this reason, a diagnostic testicle biopsy should be taken only as a prognostic factor and the result should not be an exclusion criterion for the patients. Similarly, there are diagnostic testicular biopsies showing foci of complete spermatogenesis or foci of elongated spermatids, with no spermatids being recovered during the treatment biopsies. From the published literature, elongated spermatids have been recovered from Sertoli cell-only syndrome and maturation arrest (at primary spermatocyte or round spermatid stage) cases, with excellent results regarding fertilization and pregnancy rates. Thus, a trial should always be offered to azoospermic patients. Whenever spermatids are found, only the more mature forms should be used. However, if only round spermatids are found and if the patient's previous history has never shown elongated spermatids or spermatozoa in the ejaculate or in the diagnostic testicular biopsy, then the couple should be advised about the very low possibility of obtaining a pregnancy using such cells (Tesarik et al., 1999Go), and thus in-vitro fertilization (IVF) with donor spermatozoa should be offered as an alternative, with the final decision being made by the patients.

Notes

4 To whom correspondence should be addressed Back

This debate was previously published on Webtrack 74, June 25, 1999

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