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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: chromatin/intracytoplasmic sperm injection/spermatogenesis/TESE/ultrastructure
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A major conclusion of results obtained with TESEICSI 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., 1996). 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., 2000
). Incomplete chromatin condensation has been observed in testicular spermatozoa of men affected by non-obstructive azoospermia (Hammadeh et al., 1999
), and an abnormal chromatin packaging in ejaculated spermatozoa is associated with infertility or with early miscarriage (Evenson et al., 1999
) and with low oocyte fertilization after ICSI (Sakkas et al., 1996
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 24 ml of erythrocyte lysing buffer for 10 min at room temperature (Nagy et al., 1997), 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 2542 years) of azoospermic men, using a GnRH agonist to achieve pituitary suppression, and recombinant FSH. Ovulation was induced with 500010 000 IU of human chorionic gonadotrophin (HCG) (Profasi, Serono, Rome, Italy). Oocytes were harvested 3436 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 1618 h by the presence of 2 polar bodies and 2 pronuclei (2PN). Embryo cleavage and quality was assessed ~4044 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, 1998).
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, 1971). 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., 1999
; Francavilla et al., 2000
), 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, 1997
). 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 (8001200 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, 1969) 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, 1963
). 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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 TESEICSI in almost any form of azoospermia (Devroey et al., 1996; Silber et al., 1996
; Palermo et al., 1999
). 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., 1996
, 1997
; Muhall at al., 1997; Silber et al., 1997
; Ezeh et al. 1998
; Amer et al., 1999
; Su et al., 1999
). 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, 1997
). 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., 1997
; Ezeh et al., 1998
; Amer et al., 1999
; Su et al., 1999
). 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., 1997
; Ezeh et al., 1998
; Amer et al., 1999
; Su et al., 1999
) and this favours the hypothesis of a patchy distribution of tubules with normal spermatogenesis in cases of severe testicular failure (Tournaye et al., 1996
). 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, 1990
). This region was therefore selected to reduce the risk of regional devascularization that is well documented after testicular biopsy (Harrington et al., 1996
; Schlegel and Su, 1997
). 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., 1998
).
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, 2000). The protamines tightly compact the sperm nucleus by forming chromatin-stabilizing disulphide bonds (Bedford and Calvin, 1974
). Altered chromatin condensation represents one of the most common defect encountered in ejaculated spermatozoa from infertile men affected by oligoasthenoteratozoospermia (Zamboni, 1987
; Francavilla et al., 1996
), and this is associated with an abnormal chromatin protein complement (Chevaillier et al., 1987
). 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, 2000
), 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., 1999
). 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., 1997
). The activation isoform of CREM transcript is not expressed in cases of spermatid arrest in human testes (Peri et al., 1998
; Weinbauer et al., 1998
; Steger et al., 1999
), and the same defect is also observed in ejaculates of men affected by oligoasthenoteratozoospermia (Peri et al., 1998
). 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., 2000
). Apoptosis of germ cells is a strictly regulated mechanism (Blanco-Rodriguez, 1998
), 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., 2000
). Most of these non-disjunction errors seem to occur at meiosis, and seem to be strictly linked to altered spermatogenic parameters (Huang et al., 1999
; Vendrell et al., 1999
), including the degree of maturation of spermatids (Bernardini et al., 2000
).
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., 1999). 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., 1996
) and with a low ability to support embryonic development (Evenson et al, 1999
; 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., 1995
; Tesarik et al., 1996
; Sofikitis et al., 1998
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amer, M., El Haggar, S., Moustafa, T. et al. (1999) Testicular sperm extraction: impact of testicular histology on outcome, number of biopsies to be performed and optimal time of repetition. Hum. Reprod., 14, 30303034.
Bedford, J.M. and Calvin, H. (1974) The occurrence and possible functional significance of S-S cross links in sperm heads, with particular reference to eutherian mammals. J. Exp. Zool., 188, 137156.[ISI][Medline]
Bernardini, L., Gianaroli, L., Fortini D. et al. (2000) Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile men. Hum. Reprod., 15, 21652172.
Blanco-Rodriguez, J. (1998) A matter of death and life: the significance of germ cell death during spermatogenesis. Int. J. Androl., 21, 236248.[ISI][Medline]
Blanco-Rodriguez, J. and Martinez-Garcia, C. (1997) Apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium of the adult rat. J. Reprod. Fertil., 110, 6170.[Abstract]
Chevaillier, P., Mauro, N., Feneux, D. et al. (1987) Anomalous protein complement of sperm nuclei in some infertile men. Lancet, ii, 806807.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 3551.[ISI]
De Kretser, D. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch., 98, 477505.[ISI][Medline]
Devroey, P., Nagy, P., Tournaye, H. et al. (1996) Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 10151018.[Abstract]
Egozcue, S., Blanco, J., Vendrell, J.M. et al. (2000) Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum. Reprod. Update, 6, 93105.
Evenson, D.P., Jost, L.K., Marshall, D. et al. (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod., 14, 10391049.
Ezeh, U.I.O., Moore, H.D.M. and Cooke, I.D. (1998) Correlation of testicular sperm extraction with morphological, biophysical and endocrine profiles in men with azoospermia due to primary gonadal failure. Hum. Reprod., 13, 30663074.[Abstract]
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatids. Lancet, 345, 16411642.
Foresta, C., Garolla, A., Ferlin, A. et al. (1998) Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function. Hum. Reprod., 13, 30903093.[Abstract]
Francavilla, S., Cordeschi, G., Gabriele, A. et al. (1996) Chromatin defects in normal and malformed human ejaculated and epididymal spermatozoa: a cytochemical ultrastructural study. J. Reprod. Fertil., 106, 259268.[Abstract]
Francavilla, S., D'Abrizio, P., Rucci, N. et al. (2000) Fas and Fas Ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J. Clin. Endocrinol. Metab., 85, 26922700.
Friedler, S., Raziel, A., Strassburger, D. et al. (1997a) Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy with non-obstructive azoospermia. Hum. Reprod., 12, 14881493.[Abstract]
Friedler, S., Raziel, A., Soffler, Y. et al. (1997b) Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with non-obstructive azoospermia a comparative study. Fertil. Steril., 68, 892897.[ISI][Medline]
Ghazzawi, I.M., Sarraf, G., Taher, R. et al. (1998) Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection. Hum. Reprod., 13, 348352.[Medline]
Habermann, H., Seo, R., Cieslak, J. et al. (2000) In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozenthawed testicular spermatozoa. Fertil. Steril., 73, 955960.[ISI][Medline]
Hammadeh, M.E., Al-Hasani, S., Doerr, S. et al. (1999) Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome. Hum. Reprod., 14, 363367.
Harrington, T.G., Schauer, D. and Gilbert, B.R. (1996) Percutaneous testis biopsy: an alternative to open testicular biopsy in the evaluation of the sub fertile man. J. Urol., 156, 16471651.[ISI][Medline]
Hecht, N.B. (2000) Molecular mechanisms of male germ cell differentiation. In Francavilla, F., Francavilla, S. and Forti, G. (eds), Andrology, 2000, Proceedings of the First European Congress of Andrology, Collana di Studi Abruzzesi, Italy, pp. 1328.
Huang, W.J., Lamb, D.J., Kim, E.D. et al. (1999) Germ cell nondisjunction in testes biopsies of men with idiopathic infertility. Am. J. Hum. Genet., 64, 16381645.[ISI][Medline]
Jarow, J.P. (1990) Intratesticular arterial anatomy. J. Androl., 11, 255259
Kupker, W., Schlegel, P.N., Al-Hasani, S. et al. (2000) Use of frozenthawed testicular sperm for intracytoplasmic sperm injection. Fertil. Steril., 73, 453458.[ISI][Medline]
Lin, W.W., Lamb, D.J., Wheeler, T.M. et al. (1999) In situ-end-labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis. Fertil. Steril., 68, 10651069.
Mulhall, J.P., Burgess, C.M., Cunningham, D. et al. (1997) The presence of mature sperm in testicular parenchyma of men with non-obstructive azoospermia: prevalence and predictive factors. Urology, 49, 9195.[ISI][Medline]
Nagy, Z.P., Verheyen, G., Tournaye, H. et al. (1997) An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil. Steril., 68, 376379.[ISI][Medline]
Palermo, G.D., Schlegel, P.N., Hariprashad, J. et al. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum. Reprod., 14, 741748.
Peri, A., Krausz, C., Cioppi, F. et al. (1998) Cyclic adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men. J. Clin. Endocrinol. Metab., 83, 37223726.
Rowley, M.J. and Heller, C.H. (1971) Quantitation of the cells of the seminiferous epithelium of the human employing the Sertoli cell as a constant. Z. Zellforsch., 115, 461472.[ISI][Medline]
Sakkas, D., Urner, F., Bianchi, P.G. et al. (1996) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum. Reprod., 11, 837843.[Abstract]
Schlegel, P.N. and Su, L. (1997) Physiological consequences of testicular sperm extraction. Hum. Reprod., 12, 16881692.[Abstract]
Schlegel, P.N, Palermo, G.D., Goldstein, M. et al. (1997) Testicular sperm extraction with intracytoplasmic sperm injection for non-obstructive azoospermia. Urology, 49, 435440.[ISI][Medline]
Silber, S.J. and Johnson, L. (1998) Are spermatid injections of any clinical value? Hum. Reprod., 13, 509515.
Silber S.J., Van Steirteghem A., Liu J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, 148152.[Abstract]
Silber S.J., Van Steirteghem A., Nagy Z. et al. (1996) Normal pregnancies resulting from testicular sperm extraction and intracytoplasmic sperm injection for azoospermia due to maturation arrest. Fertil. Steril., 66, 110117.[ISI][Medline]
Silber S.J., Nagy Z., Doevrey, P. et al. (1997) Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum. Reprod., 12, 24222428.[Abstract]
Sofikitis, N.V., Yamamoto, Y., Miyagawa, I. et al. (1998) Ooplasmic injection of elongating spermatids for the treatment of non-obstructive azoospermia. Hum. Reprod., 13, 709714.[Abstract]
Steger, K., Klonisch, T., Gavenis, K. et al. (1999) Round spermatids show normal testis-specific H1t but reduced camp-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest. J. Androl., 20, 747754.
Su, L., Palermo, G.D., Goldstein, M. et al. (1999) Testicular sperm extraction with intracytoplasmic sperm injection for non-obstructive azoospermia: testicular histology can predict success of sperm retrieval. J. Urol., 161, 112116.[ISI][Medline]
Tamai, K.T., Monaco, L., Nantel, F. et al. (1997) Coupling signaling pathways to transcriptional control: nuclear factors responsive to cAMP. In Conn, P.M. (ed.), Recent Progress in Hormone Research. Endocrine Society, Bethesda, 2, 121140.
Tesarik, J., Rolet, F., Brami, C. et al. (1996) Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum. Reprod., 11, 780783.[Abstract]
Tournaye, H., Liu, J., Nagy, P.Z. et al. (1996) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 11, 127132.[Abstract]
Tournaye, H., Verheyen, G., Nagy, P.Z. et al. (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum. Reprod., 12, 8086.[ISI][Medline]
Vendrell, J.M., Garcia, F., Veiga, A. et al. (1999) Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum. Reprod., 14, 375378.
Weinbauer, G.F., Behr, R., Bergmann, M. et al. (1998) Testicular cAMP-response element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest. Mol. Hum. Reprod., 4, 915.[Abstract]
Zamboni, L. (1987) The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil. Steril., 48, 711734.[ISI][Medline]
Submitted on November 21, 2000; accepted on March 22, 2001.