1 Department of Obstetrics and Gynaecology, IVF Laboratory, University of Saarland, 66421 Homburg/Saar and 2 Department of Obstetrics and Gynaecology, Medical college of Luebeck, Germany
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Abstract |
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Key words: chromatin condensation/ICSI/morphology/testis extracted spermatozoa
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Introduction |
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The development of spermatids into mature spermatozoa is accomplished by a series of structural and chemical modifications including a gradual replacement of virtually lysine-rich histones by transition proteins and then by protamines which bind more tightly to DNA than do histones and this results in compaction of chromatin in the sperm nucleus, a process which is termed `sperm chromatin condensation'. Mammalian sperm DNA is the most tightly packed eukaryotic DNA, being at least six times more highly condensed than DNA in mitotic chromosomes, which allows the DNA to be compacted into a small volume (Ward and Coffey, 1991).
A number of studies have shown that spermatozoa with abnormal chromatin organization are more frequent in infertile men than in fertile men (Evenson et al., 1980; Monaco and Rasch, 1982
). A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been reported. Furthermore, many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility (Bach et al., 1990
; Foresta et al., 1992
). Recently, intracytoplasmic sperm injection (ICSI) has led to a high success rate in fertilization and pregnancy in which the husband had undergone testicular sperm extraction procedure to recover immature spermatozoa for injection into the oocyte (Silber et al., 1995
). Moreover several pregnancies have already been obtained after ICSI with testicular spermatozoa from patients with obstructive (Schoysman et al., 1993
; Devroey et al., 1994
) and non-obstructive azoospermia (Devroey et al., 1995
; Nagy et al., 1995
; Tournaye et al., 1996
; Kahraman et al., 1997
; Schlegel and Girardi, 1997
).
Edwards et al. (1994) had already suggested that spermatids could be used as substitutes for spermatozoa to achieve fertilization of human oocytes. The round spermatid, in which meiosis has been completed and the histoneprotamine transition has hardly begun, might offer the best means of transferring haploid male DNA to an oocyte. Spermatids can be collected from testicular aspirates (Blanchard et al., 1990). In humans, apparently healthy babies have been born after ICSI of spermatid nuclei (Tesarik et al., 1995
).
However, in humans normal fertilization and cleavage up to 4-cell stage (Vanderzwalmen et al., 1995) and a pregnancy (Fishel et al., 1995) were reported after ICSI of an elongated spermatid retrieved from testicular tissue. Tesarik et al. (1995) and Tesarik (1996) reported the first two pregnancies followed by delivery of healthy babies after injection of round spermatids (ROS), alternating with very severe oligozoospermia. Four other pregnancies were reported by Hannay (1995) after injection of round spermatid nuclei (ROSNI) but spontaneous miscarriages occurred in all of them.
The degree of sperm chromatin condensation (nuclear stability) can be evaluated by toluidine or aniline blue staining (Krzanowska, 1982) or by the uptake of an intercalating compound (acridine orange, ethidium bromide and propidium iodide) followed by analysis using either fluorescence microscopy (Gledhill, 1983
) or flow cytometry (Spano et al., 1984
; Evenson, 1989
; Engh et al., 1992
; Golan et al., 1997
).
Terquem and Dadoune (1983) demonstrated that the acidic aniline blue staining technique selectively stains persisting lysine-rich histones and thus allows the visualization of spermatozoa with defective chromatin condensation. Due to the shift from somatic histones to specific protamines during spermatid development (Meistrich et al., 1978) and nuclear condensation, the elongated spermatids lose their ability to be stained with acidic aniline blue dye. While normal mature spermatozoa appear unstained, remaining histones in spermatozoa with insufficient chromatin condensation and in immature elongated spermatids are stained (Dadoune and Alfonsi, 1986). Dadoune et al. (1988) observed pathological aniline blue staining in 20% of morphologically-normal spermatozoa in normozoospermia specimens; these findings were confirmed by Hofmann et al. (1990).
The aim of this study was to compare the chromatin condensation and morphology of testis biopsy extracted and ejaculated spermatozoa and to determine the relationship between the chromatin condensed spermatozoa, sperm morphology and ICSI outcome.
In addition it was also to find out whether nuclear maturity (chromatin condensation) could be a useful parameter for predicting the fertilizing capability of spermatozoa after ICSI.
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Materials and methods |
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Semen samples from the first group were produced by masturbation after 34 days abstinence. The semen was allowed to liquefy for 2030 min at room temperature. Thereafter the semen samples were analysed according to WHO guidelines (1992), many smears were made before and after sperm selection for chromatin condensation (aniline blue staining) and morphology (strict criteria) evaluation later. The semen was mixed with Ham's F10 supplemented with 10% human albumin (1:1). The spermatozoa were centrifuged at 250 g for 10 min, the supernatant was discarded and the pellet was layered with 0.5 ml Ham's F10 medium supplemented with 10% human albumin and then incubated at 37°C until ICSI was accomplished. On the other hand the testis biopsy was dissected with scissors into very small pieces under the inverted microscope in Petri dishes containing Ham's F10 medium. The Petri dishes were incubated at 37°C for 23 h to give time for the spermatozoa to be released from the seminiferous tubules. Then the medium which contained the spermatozoa was centrifuged for 10 min at 250 g and prepared like the semen samples of the first group.
Aniline blue staining
In both groups chromatin condensation was assessed by aniline blue staining as previously described (Hammadeh et al., 1996). Briefly, after sperm preparation, 5 µl of the prepared spermatozoa were spread onto a glass slide, and allowed to dry. The smears were fixed in 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30 min. The slides were then stained with 5% aqueous aniline blue mixed with 4% acetic acid (pH 3.5) for 5 min. A total of 100200 sperm cells were evaluated and the percentage of stained sperm heads were calculated. Two classes of staining intensities were distinguished: unstained and completely or partially stained.
The other smears were stained using a modified Papanicolaou method (WHO, 1992). The morphology was assessed by one observer based on strict criteria (Kruger et al., 1986, 1988
). A total of 100 spermatozoa from each slide was evaluated under oil immersion at a magnification of x1000 using bright field illumination.
Statistical analyses
Data storage and analyses were done at the Department of Biomathematics and Informatics Institute, University of Saarland.
The statistical analysis was performed using the MannWhitney U-test and the 2 test. The possible relationship between the percentage of chromatin condensation, morphology and fertilization, cleavage and pregnancy was analysed with the Spearman rank correlation test.
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Results |
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Discussion |
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Notes |
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References |
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Submitted on June 1, 1998; accepted on October 30, 1998.