Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE–ICSI

Sandro Francavilla1,3, Maria Antonietta Bianco2, Giuliana Cordeschi1, Piera D'Abrizio1, Cristoforo De Stefano2, Giuliana Properzi1 and Felice Francavilla1

1 Department of Internal Medicine (Andrology), at University of L'Aquila, Via S. Sisto 22E, 67100 L'Aquila and 2 Centre for Reproductive Medicine, Avellino, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is offered to treat obstructive and non-obstructive azoospermia, but factors that influence the outcome of ICSI are not well defined. METHODS AND RESULTS: The percentage of elongated spermatids with normal chromatin condensation in azoospermic patients submitted for TESE–ICSI was determined. The quantitative analysis could be applied to nine of 19 biopsies classified as incomplete late maturation arrest (LMA) and compared with 10 biopsies with normal spermatogenesis. The percentage of elongated spermatids with normal chromatin was lower in LMA than in normal histology (mean 4.4%, range 0–20, and mean 52.9%, range 40–70 respectively; P = 0.0001). The percentage of elongated spermatids with normal chromatin was negatively correlated with the serum concentration of FSH (r = –0.86, P < 0.0001) and the number of degenerated germ cells per 100 Sertoli cells nuclei (r = –0.68; P < 0.0001), while it was positively correlated with the number of elongating spermatids per 100 Sertoli cell nuclei (r = 0.81; P < 0.0001). The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was higher in cases with normal histology compared with cases of LMA. CONCLUSIONS: These preliminary data suggest that an altered chromatin condensation is a ubiquitous defect in spermatids of non-obstructed azoospermic men submitted for TESE–ICSI.

Key words: chromatin/intracytoplasmic sperm injection/spermatogenesis/TESE/ultrastructure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Intracytoplasmic sperm injection (ICSI) using fresh spermatozoa obtained by testicular sperm extraction (TESE) is well established as a treatment for irreparable obstructive azoospermia (Silber et al., 1995Go; Abuzeid et al., 1997Go). This treatment has been extended to couples where the male partner suffers from azoospermia due to testicular failure, and a pregnancy rate comparable with that obtained from cases of obstructive azoospermia has been reported (Devroey et al., 1996Go; Silber et al., 1996Go; Palermo et al., 1999Go). Non-obstructive azoospermia is associated with a significant risk of failure to retrieve spermatozoa, ranging between 10 and 57% (Devroey et al., 1996Go; Friedler et al., 1997aGo; Schlegel et al., 1997Go; Gnazzawi et al., 1998; Palermo et al., 1999Go; Su et al., 1999Go). The use of frozen–thawed testicular spermatozoa obtained by TESE avoids the risk of lack of spermatozoa available at the time of oocyte retrieval for ICSI, although data on the outcome of ICSI with frozen–thawed testicular spermatozoa in cases of non-obstructive azoospermia are still limited. On the whole, it seems reasonable to assume that the clinical pregnancy rates of fresh and frozen–thawed testicular spermatozoa after ICSI in cases of non-obstructive azoospermia are comparable (Friedler et al., 1997bGo; Palermo et al., 1999Go; Haberman et al., 2000; Kupker et al., 2000Go), but a conspicuous risk of pregnancy loss is observed in the latter (Friedler et al., 1997bGo; Kupker et al., 2000Go).

A major conclusion of results obtained with TESE–ICSI is that the few testicular spermatozoa, yielded in cases of severely deranged spermatogenesis, are potentially competent for syngamy and embryonal and fetal development, although reduced fertilization and implantation rates have been reported (Tournaye et al., 1996Go). On the other hand, altered spermatogenesis seems to result in an increased incidence in sperm chromosome disomy and DNA ploidy after ICSI and in an increased abortion rate of presumably paternal origin (Egozcue et al., 2000Go). Incomplete chromatin condensation has been observed in testicular spermatozoa of men affected by non-obstructive azoospermia (Hammadeh et al., 1999Go), and an abnormal chromatin packaging in ejaculated spermatozoa is associated with infertility or with early miscarriage (Evenson et al., 1999Go) and with low oocyte fertilization after ICSI (Sakkas et al., 1996Go).

In the present study, we investigated the efficiency of spermatogenesis in azoospermic patients submitted for TESE-ICSI, by quantifying the number of elongating spermatids and the number of degenerating germ cells using light microscopy. Quantitative ultrastructural analysis of the nuclei of elongated spermatids was applied to evaluate the condensation of chromatin. The data obtained by light and electron microscopy were compared with ICSI outcome, to explore the relationship between an altered spermatogenic process, abnormal maturation of nucleus in spermatids and the outcome of ICSI. The results showed that an altered spermatogenesis is associated to a diffuse defective maturation of chromatin in elongated spermatids and this was associated with a low delivery rate after ICSI was performed with testicular spermatozoa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
The local institutional human research committee approved the study, and informed consent was obtained from the subjects included in the investigation. From January 1999 to April 2000, 50 patients (age range 25–50 years), infertile from 1–15 years, were submitted to testicular biopsy to obtain spermatozoa for ICSI, since in two consecutive semen analyses performed in our institution, no spermatozoa were recovered, even after prolonged centrifugation. Forty-one patients had a previous history of azoospermia, while nine patients reported the presence of few spermatozoa, at least once, in their ejaculate at previous semen analysis. All patients were thoroughly evaluated by a clinical questionnaire, physical examination and hormonal analysis. Testicular volume was recorded by an orchydometer, and a rectal digital exploration was performed in all patients. The hormonal analysis included the evaluation of blood serum FSH in two pooled samples obtained within 1 h of each other, prolactin and total testosterone. A semen analysis was obtained on the day of sperm retrieval, to confirm azoospermia. Testicular sperm extraction was performed under local anaesthesia, while general anaesthesia was reserved for patients with very small testes (<12 ml). An incision was made on the tunica albuginea in the medial or lateral aspects of the upper pole of the testis, to reduce the risk of cutting the sub-albuginea end-arteries, which are distributed in this region (Jarow, 1990Go). Three to four consecutive specimens, 0.1–0.2 g each, were sampled and placed in a sterile plastic tube (2001 Falcon; Becton-Dickinson, Lincoln Park, NJ, USA) and immediately transferred to the adjacent laboratory. Testicular tissue was placed in a Petri dish (1029 Falcon) containing 3 ml HEPES buffered Earle's medium supplemented with 0.4% human serum albumin (HSA) (Centeon Pharma, Marburg, Germany), and a microscopical examination of the shredded and minced wet preparation was than performed under an inverted microscope (x400) (Diaphot; Nikon Corporation, Tokyo, Japan). If spermatozoa were not observed in any specimen, biopsies were taken from the contra lateral testis. One testicular specimen was sent for histology. This specimen was immediately immersed in cold 0.1 mol/l cacodylate buffer pH 7.4, containing 3% (v/v) glutaraldehyde (AGAR Scientific Ltd, Stansted, Essex, UK) for 2 h at 4°C. Samples were subsequently washed in cacodylate buffer and post-fixed in 1% (w/v) osmium tetroxide in distilled water, dehydrated through graded ethanol and embedded in Epon 812 (AGAR Scientific Ltd).

Preparation of testicular spermatozoa
Each biopsy specimen was rinsed in culture medium, shredded with sterile glass slides and minced with scissors. The resulting suspension was sequentially passed through a 21 G needle to enhance disruption of the tubules and then centrifuged for 5 min at 500 g in a Falcon tube (2095). The pellet was resuspended in 2–4 ml of erythrocyte lysing buffer for 10 min at room temperature (Nagy et al., 1997Go), diluted with 8 ml HEPES-buffered Earle's medium supplemented with 2.25% HSA and centrifuged for 10 min at 500 g. The pellet was resuspended in the same medium before both sperm concentration and motility were immediately obtained. The resulting suspension was transferred in the injection dish to collect viable spermatozoa suitable for microinjection or cryopreserved for ICSI use after thawing. Testicular cells were diluted 1:1 (v/v) by dropwise addition of TEST-yolk buffer medium with glycerol (12% v/v) (TYB; Irvine Scientific, Santa Ana, CA, USA). The freezing procedure was carried out by a computer-controlled slow freezing program and vials were finally stored in liquid nitrogen (–196°C). ICSI was performed with fresh spermatozoa when a previous biopsy had documented the presence of elongating spermatids, or when, after thawing a frozen specimen the day of oocyte retrieval, no viable spermatozoa were recovered. In this case, a new biopsy was immediately obtained.

Oocyte collection and ICSI procedure
Controlled ovarian stimulation was performed in 36 partners (age range 25–42 years) of azoospermic men, using a GnRH agonist to achieve pituitary suppression, and recombinant FSH. Ovulation was induced with 5000–10 000 IU of human chorionic gonadotrophin (HCG) (Profasi, Serono, Rome, Italy). Oocytes were harvested 34–36 h later using transvaginal ultrasound-directed follicle aspiration. Oocytes were left in Universal IVF medium (Medicult, Jyllinge, Denmark) at 37°C in an atmosphere of 5% CO2, 5% O2, 90% N2. Metaphase II oocytes were injected with one single viable spermatozoon, either fresh or thawed at room temperature, into the ooplasm. Injected oocytes were washed and stored in 0.5 ml IVF medium under light mineral oil (Sigma-Aldrich, MI, Italy) in Nunclon multidishes (Nunc, Roskilde, Denmark). Fertilization was confirmed after 16–18 h by the presence of 2 polar bodies and 2 pronuclei (2PN). Embryo cleavage and quality was assessed ~40–44 h after microinjection. Embryos with the best morphology (<30% of their volume filled with anucleate fragments) and the highest cell number were selected for embryo transfer. A maximum of three embryos were replaced in the uterine cavity ~48 h after the ICSI procedure.

Morphological evaluation of testicular tissue
Histology
Thin (1 µm) Epon-embedded sections were stained in 1% toluidine blue and analysed under light microscopy. On the basis of qualitative interpretation, biopsies were classified as described below.

Normal histology Almost all tubules showed >10 elongating spermatids in each cross-tubule section.

Incomplete late maturation arrest (LMA) Tubules showed spermatogenesis progressing through elongated spermatids; the latter, however, were greatly reduced to less than five in each cross-section of seminiferous tubules, and co-existed with tubules where only round spermatids were observed, and tubules where primary spermatocytes were the most mature germ cells. In no case was spermatogenesis arrested in all tubules at the level of round spermatids (Silber and Johnson, 1998Go).

Complete early maturation arrest of spermatogenesis (EMA) All tubules showed arrested spermatogenesis at level of leptotene or pachitene spermatocytes, while spermatids were never observed.

Complete Sertoli-cell-only syndrome (SCOS) All seminiferous tubules showed only Sertoli cells and the lamina propria was thin or focally thickened.

Mixed atrophy The tubules showed a thickened lamina propria associated with a total lack of the seminiferous epithelium (tubule shadow), which co-existed with some tubules with Sertoli cells only and a thickened lamina propria, and some tubules with spermatogenesis progressing also through rare elongated spermatids. Quantitative analysis of testicular biopsies by light and electron microscopy was performed on nine out of 19 cases with post-meiotic arrest. The specimens with LMA selected for the quantitative study were those which allowed us to study at least 20 elongated spermatids with transmission electron microscopy (see following section). The nine biopsies of LMA included one of two cases that resulted in a live birth after ICSI (see Results); the second case was not suitable for quantitative ultrastructural study. The data were then compared with those obtained from 10 specimens with normal histology. The 10 biopsies with normal histology were selected among those with a very homogeneous pattern of preserved spermatogenesis, and also included four cases that resulted under delivery of live births after ICSI (see Results). Toluidine blue-stained sections were examined with a x63 oil immersion objective lens and a x12.5 eyepiece. Cell counting on coded slides and ultrastructural evaluation were performed by S.F., without any knowledge of clinical data and of the number of spermatozoa retrieved by TESE. The counting method was similar to that proposed previously (Rowley and Heller, 1971Go). Longitudinal and cross-sections of seminiferous tubules with a lumen were used for scoring. Approximately 30 tubules were scored in each biopsy by counting in each tubule, the number of elongating spermatids (SD) and the number of nuclei of Sertoli cells (SE). The total number of spermatids was divided by the total number of Sertoli cell nuclei, in order to obtain the number of elongating spermatids per 100 Sertoli cell nuclei (SD/SE). A minimum of 200 Sertoli cell nuclei were counted in each biopsy. The seminiferous epithelium in cases of deranged spermatogenesis contains germ cells that are undergoing apoptotic degeneration (Lin et al., 1999Go; Francavilla et al., 2000Go), and the microscopic analysis of Epon-embedded toluidine blue staining sections allows an accurate identification of cellular changes associated with apoptosis (Blanco-Rodriguez and Martinez-Garcia, 1997Go). The frequency of apoptotic cells in the seminiferous epithelium was therefore assessed in the same specimens submitted to the count of elongating spermatids, by using the same counting criteria. A germ cell degeneration index was determined by dividing the number of degenerated germ cells per 100 Sertoli cell nuclei (DG/SE).

Ultrastructure
The ultrastructural analysis of elongating spermatids by transmission electron microscopy (TEM) was applied to all testicular specimens submitted to the count of elongated spermatids and degenerated germ cells by light microscopy. Ultrathin sections (800–1200 Å) were contrasted with uranyl acetate and lead hydroxyde (AGAR Scientific Ltd) and evaluated in a Philips CM100 TEM (Philips Electronics, Eindhoven, Holland). In each biopsy, at least 20 heads of elongated spermatids in the stage Sd2 according to De Kretser (De Kretser, 1969Go) were scored for the condensation of chromatin, presence of an acrosome with homogeneous electron-dense matrix, and continuity of cell and nuclear membranes. Sd2 indicates fully elongated spermatids with chromatin granules coalesced to form homogeneous electron-dense masses. To compare spermatids at the same advanced stage of spermiogenesis in different testicular specimens we included only observations made on stage I of the seminiferous epithelium which contains round spermatids along with mature elongated spermatids (Clermont, 1963Go). To reach the number of spermatids required for the quantitative evaluation in biopsies affected by LMA, it was necessary to collect numerous sections of the same biopsy, separated by at least 5 µm, to avoid studying the same cell twice. Only nine biopsies from 19 patients affected by LMA could be evaluated appropriately according to the inclusion criteria of TEM analysis. Results obtained from this group were compared with those obtained from a group of 10 normal histology biopsies, to allow an accurate comparative analysis of the percentage of elongating spermatids with normal chromatin condensation.

Statistical analysis
Due to the small sample size, non-parametric tests were used for data analysis. Comparison between groups was assessed using the Mann–Whitney U-test, while correlations were performed using the Spearman rank correlation test. Statistical analysis was performed by use of the Complete Statistical System for personal computers (CSS/pc), release 2.1, version B 640, 1988 (Stat Soft Inc., New York, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Table IGo gives an overview of the histological findings of the 50 biopsies grouped according to the results of sperm recovery after wet preparation, and the presence of some spermatozoa in at least one ejaculate in the past semen analysis. Numerous spermatozoa after wet preparation were obtained in 20 cases, few spermatozoa in 18 cases, while no spermatozoa were obtained in 12 cases (24% of total). Twenty biopsies showed normal histology and 18 of these (90%) were in the group with numerous spermatozoa retrieved; in one of these 20 cases of obstructive azoospermia, ejaculated spermatozoa were reported in a previous semen analysis. Nineteen biopsies were classified as LMA and 14 of these were in the group in which few spermatozoa were retrieved, two in the group with numerous spermatozoa and three (15%) in the group with no spermatozoa retrieved. Five biopsies were classified as EMA and in no case were spermatozoa retrieved, although two patients reported ejaculated spermatozoa in one previous semen analysis. Complete SCOS was found in two cases associated with failed sperm retrieval. A mixed atrophy was finally found in four cases, two of which had spermatozoa retrieved and two no spermatozoa retrieved. The data show that the retrieval of spermatozoa from wet preparations of testicular tissue was mostly associated with normal histology or with LMA. Twelve out of 30 biopsies with an altered histology were associated with failed sperm recovery (40%). It was interesting to note that the presence of ejaculated spermatozoa in the past semen analysis was observed in only 1/20 cases of obstructive azoospermia (5%), while this was reported in 8/30 cases of abnormal histology (26%). This suggests that occasional ejaculated spermatozoa were more commonly a feature of an altered testicular function, and not of obstruction of the genital tract.


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Table I. Histological findings, retrieval of spermatozoa after wet preparation, presence of spermatozoa in a previous ejaculate and delivery after ICSI, in 50 patients submitted to testicular biopsy
 
Table IIGo shows the outcome of ICSI after grouping the cycles according to the number of spermatozoa retrieved by wet preparation, according to the use of fresh or frozen–thawed spermatozoa, and finally after grouping cycles on the basis of testicular histology. There was no difference between groups in term of percentage of oocytes fertilized. One pregnancy loss was reported in a case of scattered spermatozoa recovered, and deliveries were obtained mostly in cases with numerous spermatozoa retrieved from biopsy (20.8% of cycles), while only one delivery was obtained in the group with scattered spermatozoa retrieved (4.7% of cycles). ICSI with fresh spermatozoa resulted in a higher number of deliveries compared with ICSI with frozen–thawed spermatozoa (15.3 and 10.5% of cycles respectively), although the rather small number of cycles does not allow any conclusion. Finally, the delivery rate was influenced by the testicular histology. Four deliveries/cycle (16.6%) were obtained from biopsies with normal histology, while only two were obtained (9.5%) from cases of LMA. This suggests that testicular spermatozoa retrieved in cases of LMA fertilize oocytes after ICSI, but do show a decreased ability to support a pregnancy compared with cases with normal histology, although the difference was not statistically significant due to the overall low number of deliveries.


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Table II. Testicular volume, serum concentration of FSH and ICSI outcome after grouping cycles according to the number of spermatozoa retrieved after testis biopsy, use of fresh or frozen–thawed spermatozoa, and testicular histology
 
In this section, we comparatively evaluated testes with normal histology and with LMA, for the ultrastructure of spermatids during the late phase of nuclear maturation and for the occurrence of degenerating germ cells in the seminiferous epithelium, as a marker of efficiency of spermatogenesis. Analysis by light microscopy documented the frequent occurrence of degenerating germ cells in cases of LMA (Figure 1Go). These were easily recognized by the heavy toluidine blue staining of the nucleus due to chromatin condensation, and by cytoplasm vesiculation. The localization of degenerating cells in the adluminal compartment of the seminiferous epithelium suggested that they might be primary spermatocytes or spermatids. The ultrastructural analysis showed diffuse altered chromatin of elongated spermatids in LMA compared with normal testes. The ultrastructural analysis was performed by selecting stage I of the seminiferous epithelium, in order to analyse elongated spermatids in the same stage of advanced maturation in all specimens (Figure 2aGo). Mature normal spermatids in stage I showed a compact aggregation of chromatin and an acrosome which tightly surrounded the rostral portion of the nucleus (Figure 2bGo). Two types of defects were observed in LMA. The first defect involved shrinkage or collapse of chromatin, which appeared unravelled into filamentous threads in a wide almost empty perinuclear area, and contained numerous small and large rarefied areas (Figure 3aGo). This was associated to a pronounced separation of the acrosome from the nucleus, and to a continuity of the nuclear and cell membranes, all findings indicating apoptotic degeneration. The second type of defect involved altered chromatin maturation in an otherwise normal cell or in a cell with multiple aberrations such as multiple nuclei joined together by commonly shared acrosomes or acrosomal defects. The chromatin kept a finely or coarsely granular immature pattern and did not progress through coalescence into a homogeneous mass (Figure 3bGo). The first type of defect was seldom encountered and involved isolated cells; the second type of chromatin defect was very common and involved all adjacent elongating spermatids observed in a single stage of the seminiferous epithelium (Figure 3cGo).



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Figure 1. Light micrograph of a segment of the seminiferous tubule in a case of incomplete late maturation arrest. Two degenerating cells are present in the adluminal compartment of the seminiferous epithelium. The two cells are deeply stained by toluidine blue and show cell shrinkage (asterisk), and chromatin condensation (arrow). The localization and the cellular size, suggest that degenerating cells are primary spermatocytes. L = lumen; P = primary spermatocyte (bar = 10 µm).

 


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Figure 2. Electron micrographs of spermatids in a stage I of the seminiferous epithelium in a case of normal testicular histology. (a) Overview with round spermatids and adjacent fully elongated spermatids with condensed chromatin (bar = 5 µm); (b) head of an elongated spermatid; the rostral aspect of the head is homogeneously covered by the acrosome and the nucleus contains electron-dense, condensed chromatin and scattered small irregular empty vacuoles (bar = 1 µm).

 


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Figure 3. Electron micrographs of spermatids in incomplete late maturation arrest of spermatogenesis. (a) Degenerating elongated spermatid. Chromatin contains numerous large rarefied vacuoles and appears unravelled into filamentous threads in an empty perinuclear area, due to shrinkage of the nuclear content. The integrity of nuclear membrane, as well as of the inner and outer acrosome membranes, suggests that the cell is undergoing apoptotic degeneration (bar = 0.5 µm). (b) The head of an elongated spermatid in stage I of the seminiferous epithelium shows a normal acrosome and a coarse granular pattern of chromatin (bar = 1 µm). (c) The incomplete chromatin coalescence is present in all elongated spermatids (arrow) observed in an overview of stage I of the seminiferous epithelium; compare with Figure 2aGo (bar = 2 µm).

 
Table IIIGo reports the number of degenerating germ cells and of the elongating spermatids referred to 100 Sertoli cell nuclei, evaluated with light microscopy, as well as the percentage of elongated spermatids with normal chromatin evaluated with electron microscopy in nine cases of LMA and in 10 cases with normal histology. Besides the reduced number of spermatids, LMA showed a significantly increased number of degenerating germ cells compared with normal testes, and a significantly reduced number of spermatids with normal chromatin condensation (P < 0.0005, Mann–Whitney U-test). Six out of nine patients with LMA showed no normal spermatids, while all specimens with normal histology showed >40% of elongated spermatids with normal chromatin. Patients with LMA showed also an increased level of FSH and a decreased testicular volume (P < 0.005) compared with patients with normal histology. The percentage of elongated spermatids with normal chromatin was strongly negatively correlated with the level of FSH (r = –0.86; P < 0.0001) and the number of degenerated germ cells (r = –0.68; P = 0.001) and resulted positively correlated with the number of elongated spermatids per 100 Sertoli cells (r = 0.81; P < 0.0001). This suggests that the altered chromatin in elongated spermatids was related to an abnormal spermatogenesis. The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was lower, but not statistically different, in cases of LMA compared with cases of normal histology (Tables I and IIGoGo).


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Table III. Testicular volume, FSH serum level, number of degenerating germ cells (Deg.Gc) and of elongating spermatids (Sd) referred to 100 Sertoli cell nuclei, evaluated at light microscopy, and the percentage of elongated spermatids with normal chromatin, evaluated at electron microscopy
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This preliminary study investigated the ultrastructure of elongated spermatids in a limited number of testicular biopsies of azoospermic men and its relation with the efficiency of spermatogenesis, as well as with the clinical outcome of ICSI. A diffuse and almost ubiquitous defect of chromatin condensation in mature spermatids was observed in cases of incomplete late maturation arrest but not in testes with normal histology. The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization. The delivery rate after ICSI performed with testicular spermatozoa was lower in cases of LMA compared with normal testicular histology, although larger studies are required to confirm this observation.

The reported pregnancies obtained with a few spermatozoa extracted from testicles affected by severe spermatogenic failure and then microinjected into the oocyte, has generated widespread use of TESE–ICSI in almost any form of azoospermia (Devroey et al., 1996Go; Silber et al., 1996Go; Palermo et al., 1999Go). Different methods to extract testicular spermatozoa were proposed to increase the chance of sperm retrieval in cases of deranged spermatogenesis, and significant differences among studies are reported in the relationship between sperm retrieval and testicular histology. In this study, spermatozoa were recovered from all 20 specimens with normal histology (obstructive azoospermia) and from 18 out of 30 specimens with deranged spermatogenesis (60%). The histological pattern associated with successful sperm retrieval in cases of deranged spermatogenesis was an incomplete late maturation arrest (75%) and mixed pattern (50%), while no spermatozoa were obtained in cases of complete SCOS or complete EMA. This reinforces the opinion that the presence of elongated spermatids in testicular biopsy is highly predictive of sperm retrieval (Tournaye et al., 1996Go, 1997Go; Muhall at al., 1997; Silber et al., 1997Go; Ezeh et al. 1998Go; Amer et al., 1999Go; Su et al., 1999Go). In our study, tissue was recovered from a single testicular site, to minimize the possible devascularization reported after multiple biopsies in the same testis (Schlegel and Su, 1997Go). This simple procedure allowed us to retrieve spermatozoa in 75% of cases of LMA, compared with a retrieval rate ranging between 79 and 100% obtained with multiple biopsies (Tournaye et al., 1997Go; Ezeh et al., 1998Go; Amer et al., 1999Go; Su et al., 1999Go). The lack of sperm retrieval in cases of complete SCOS or complete EMA, as reported here, could be influenced by the low number of cases of SCOS and EMA evaluated, as well as by the single biopsy site. Multiple biopsies are indeed associated with sperm retrieval in cases of SCOS ranging between 19 and 24% of cases, (Tournaye et al., 1997Go; Ezeh et al., 1998Go; Amer et al., 1999Go; Su et al., 1999Go) and this favours the hypothesis of a patchy distribution of tubules with normal spermatogenesis in cases of severe testicular failure (Tournaye et al., 1996Go). A possible explanation for the reduced sperm recovery in this study compared with others, in cases of deranged spermatogenesis could be also related to the testicular region selected for biopsy. The lateral or medial region of the upper pole of the testis was selected in our study for sperm biopsy, since this is a region with very few sub-albugineal end-arteries compared with other testicular areas (Jarow, 1990Go). This region was therefore selected to reduce the risk of regional devascularization that is well documented after testicular biopsy (Harrington et al., 1996Go; Schlegel and Su, 1997Go). It is possible that the spermatogenesis in this relatively less vascular testicular region is not as well preserved compared with more vascular area as recently reported (Foresta et al., 1998Go).

This study suggests that viable testicular spermatozoa obtained from testes with incomplete late maturation arrest do not show the same ability to support a normal pregnancy, if microinjected in the oocyte, as compared with spermatozoa obtained from testes with normal spermatogenesis. The ultrastructural study performed in mature spermatids showed diffuse, almost ubiquitous, alteration of chromatin condensation, in cases of LMA. This did not seem to be due to chromatin disorganization, as a feature of spermatid degeneration, since chromatin defects associated with the evidence of cell degeneration were rarely observed. Most mature spermatids showed altered chromatin condensation associated with a normal appearance of other cell structures such as mitochondria, cytoplasm vesicles, and membranes. This suggests that a primary abnormal structural organization of the nucleus is a common, if not ubiquitous feature of testicular spermatozoa extracted in cases of LMA.

During spermiogenesis, transition proteins 1 and 2 replace many of the somatic and testicular forms of nuclear histones. These contribute to convert the histone-containing nucleosomes into condensed chromatin fibres. Concomitant with the substitution of histones, transcription terminates, and at the end of spermiogenesis the transition proteins are replaced by protamines, testis-specific arginine-rich proteins (Hecht, 2000Go). The protamines tightly compact the sperm nucleus by forming chromatin-stabilizing disulphide bonds (Bedford and Calvin, 1974Go). Altered chromatin condensation represents one of the most common defect encountered in ejaculated spermatozoa from infertile men affected by oligoasthenoteratozoospermia (Zamboni, 1987Go; Francavilla et al., 1996Go), and this is associated with an abnormal chromatin protein complement (Chevaillier et al., 1987Go). The diffuse or almost ubiquitous defect in chromatin condensation in mature spermatids in testes affected by LMA supports the hypothesis that in this condition, the complex mechanism which underlines chromatin changes during spermiogenesis, and which requires an ordered activation of gene expression, modulated by transcriptional and post-transcriptional regulation (Hecht, 2000Go), is deranged. The ultrastructural defect of chromatin condensation is in keeping with recent findings on altered expression of genes involved in spermatid differentiation reported in cases of incomplete late maturation arrest of spermatogenesis. The gene encoding transition protein 1 (TP1) is not expressed at both mRNA and protein level in round spermatids of men affected by maturation arrest (Steger et al., 1999Go). Protamine genes, TNP1 gene and other testis-specific genes, products which are required for structuring spermatozoa, are modulated by cAMP-responsive element modulator (CREM) (Tamai et al., 1997Go). The activation isoform of CREM transcript is not expressed in cases of spermatid arrest in human testes (Peri et al., 1998Go; Weinbauer et al., 1998Go; Steger et al., 1999Go), and the same defect is also observed in ejaculates of men affected by oligoasthenoteratozoospermia (Peri et al., 1998Go). This suggests that the same gene defect might ensue in different testicular phenotypes ranging from an almost total block of late spermatid differentiation to the production of a reduced number of mature testicular spermatozoa with diffuse structural defects. Testes with LMA, besides a reduced number of spermatids, and a diffuse defect of spermatid maturation, also demonstrated a high rate of apoptotic loss of meiotic and post-meiotic germ cells, compared with testes with normal spermatogenesis, as well as increased expression of apoptosis-inducer FAS receptor (Francavilla et al., 2000Go). Apoptosis of germ cells is a strictly regulated mechanism (Blanco-Rodriguez, 1998Go), resulting in the possible elimination of defective cells. Therefore increased disposal of meiotic and post-meiotic germ cells may contribute to a reduced number of spermatids and this is associated with diffuse defective differentiation of the few maturing spermatids, in cases of incomplete late maturation arrest. Taken together, data suggest that both meiotic and post-meiotic germ cells undergo altered maturation in cases of LMA of spermatogenesis, and this may have a negative effect on delivery rate after ICSI performed with testicular spermatozoa. A very high rate of abnormal testicular spermatozoa has already been reported in a few selected cases of incomplete spermatogenic arrest, and this was associated with a lack of pregnancies and with a very high rate of autosomal (1,17) and sex chromosome non-disjunction in testicular spermatids (Bernardini et al., 2000Go). Most of these non-disjunction errors seem to occur at meiosis, and seem to be strictly linked to altered spermatogenic parameters (Huang et al., 1999Go; Vendrell et al., 1999Go), including the degree of maturation of spermatids (Bernardini et al., 2000Go).

All data discussed suggest that incomplete late maturation arrest, according to our results, represents the most frequent testicular phenotype successfully submitted to TESI for ICSI in non-obstructed azoospermic men. This testicular defect may be associated with a variety of chromosomal and gene defects which hinder normal meiotic and post-meiotic germ cell maturation. Germ cells harbouring chromosomal and gene defects and which escaped from a block of maturation or from elimination through apoptosis during meiosis I, probably show a poor reproductive ability due to their diffuse structural and chromatin defects. Abnormal chromatin packaging in ejaculated spermatozoa is indeed associated with infertility and early miscarriages (Evenson et al., 1999Go). Assisted reproduction by ICSI does not seem to restore the reproductive competence of ejaculated or testicular spermatozoa carrying diffuse chromatin defects; it is associated indeed with a low oocyte fertilization (Sakkas et al., 1996Go) and with a low ability to support embryonic development (Evenson et al, 1999Go; present study). These observations expand and help to explain the results of previous studies which have shown a low fertilization rate and an impaired potential for embryo development after ooplasmic injection of immature ejaculated or testicular spermatids in non-obstructed azoospermic men (Fishel et al., 1995Go; Tesarik et al., 1996Go; Sofikitis et al., 1998Go). Large studies are warranted to explore more fully the relationship between the occurrence of chromosome and gene defects in maturing germ cells, the outcome of spermatogenesis by sound morphological techniques and the outcome of ICSI with testicular spermatozoa and elongating spermatids.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The technical assistance of Dr Maria Giammatteo, at Centre of Microscopy, University of L'Aquila, is deeply appreciated.


    Notes
 
3 To whom correspondence should be addressed. E-mail: sandrof{at}univaq.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 21, 2000; accepted on March 22, 2001.