From Testicular Biopsy to Human Embryo

The diagnostic approach to an infertile couple should be a comprehensive one, with a detailed diagnostic procedure (24). Testicular biopsy, if necessary, should be at the very end of this procedure. Some major indications for testicular biopsy are given in Table 3. During the operation, it is highly recommended to take biopsy from various sites, to freeze several pieces of the tissue and to perform a histological analysis. There is an on-going discussion whether to perform TESA or TESE procedure (10,23,40). In our opinion, TESA should be performed in the case of obstructive azoospermia, when other methods to acquire sperm (i.e. PESA/MESA) have failed. Even with one or two aspirations with an 18-21 gauge needle, a satisfactory sperm yield could be accomplished. However, in the case of non-obstructive azoospermia, TESE procedure is recommended. The open biopsy of testicular tissue can, in such cases, be used for histological diagnosis and biopsy frozen for further consequent TESE/ICSI attempts (15,25,29,30). Examination of seminiferous tubules under the operating microscope and their microdissection during operation could additionally increase sperm yield by TESE (1,26,27,35,36). The comparison between TESA and TESE is given in Table 4
            Another important issue is the accuracy of the histological analysis. Various parts of the testis may have different histological appearance, i.e. preserved or damaged spermatogenesis (12,20). That is why it is crucial to take open biopsy of the testis from at least 3 different sites. Histological analysis of only one biopsy (piece) could be misleading and a wrong diagnosis could be established, since frozen biopsies may have a quite different morphology. In order to increase the accuracy of the histological analysis, a concept of “sandwich” organisation of the testicular biopsy was proposed (15,30). According to this concept, one piece of testicular tissue is subjected to the histological analysis and another to enzymatic digestion/maceration (so-called diagnostic TESE). Data of both analyses are combined in order to put the correct diagnosis (Fig.1a). However, even when such analysis is done, there could be doubts on the morphology of the frozen parts. Therefore, we would like to propose a new way for the analysis of the testicular biopsy which we called “piece per piece”. Namely, each piece of dissected testicular tissue is divided into 2 parts: one part is always histologically evaluated and another part frozen. Histological analysis is made on minimum of 50 serial semi-thin sections per block, thus ensuring sufficient tissue sections for histological evaluation. The diagnostic TESE is avoided because a more precise diagnosis could be done on sections. Usually 3 pieces per testis are frozen and subjected to histology (total = 6 pieces). The above-described concept is illustrated on Fig.1b.  
            Enzymatic digestion contributes to the sperm yield form frozen parts of the biopsy. It was shown that mild concentration of collagenase produces defects in the lamina propria of seminiferous tubules, thus releasing sperm “trapped” in the tubular lumen (25). Additional maceration of the biopsy with sterile needles significantly increases the presence of sperm in the pellet (29,30). If there is no sperm in the sample, elongated or round spermatids could be used for ICSI (38). However, despite some optimistic reports on round spermtid injection into the oocyte, sperm and elongated spermatids are cells of preference. Therefore, the pellet should be carefully observed in order to identify the above-mentioned cells. This may take sometimes 1-2 hours.
            Future investigations are necessary to establish which sperm isolated from the testicular biopsy is the most suitable one for ICSI. Recent studies on testicular sperm DNA damage rouse significant concern (31). New methods for selection of viable and undamaged sperm (or elongated/round spermatid) for ICSI could be very helpful in order to avoid unnecessary ovarian stimulation and TESE/ICSI attempts. More comprehensive preventive measures to reduce infertility should be taken. These include the reduction of pollution (estrogen-like substances), change in the life style and diminishing of stress in developed countries. Improvement of spermatogenic cell cultivation (spermatogonia, spermatocytes, round spermatids) could be applied when sperm are lacking in the testicular biopsy. Investigations on embryonic stem cells, their cultivation and development into sperm are exciting future perspectives for the treatment of infertility. The same goes for the differentiation of embryonic tumour cells. New genetic and protein markers for identifying infertility should be found employing new techniques like DNA multiarray analysis and proteomics.  

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