Evidence of reduced single-stranded testicular sperm DNA from obstructive azoospermic men after 3 days of in-vitro culture

Serena Emiliani1,2,3, Marc Van den Bergh1,2, Anne-Sophie Vannin1, Jamila Biramane1, Miranda Verdoodt1 and Yvon Englert1,2

1 Fertility Clinic and 2 Laboratory of Biology and Psychology of Human Fertility, Erasmus Hospital French Speaking Free University of Brussels, 808, Route de Lennik, B1070 Brussels, Belgium


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to verify whether culturing testicular tissue, to obtain a higher percentage of motile spermatozoa and a better post-thaw recovery rate, affected the ratio between single/double-stranded sperm DNA and, consequently, DNA sensitivity to damage. Testicular biopsy samples from men with obstructive and secretory azoospermia, candidates for assisted reproductive treatment, were cultured for 72 h. The percentage of motile spermatozoa and the single/double stranded DNA ratio were assessed on the day of retrieval (day 0) and again on day 3. The single/double stranded DNA ratio was measured by the acridine orange (AO) staining method. Spermatozoa were classified as green (double-stranded chromatin) or red fluorescing (single-stranded chromatin). In obstructive azoospermia, median motility was 22% (range 10–44%) on day 0 and 50% (range 38–63%) on day 3 (P < 0.01). The median percentage of red stained spermatozoa was 53.5% (range 0.1–88%) on day 0 and 20% (range 2.7–99.9%) on day 3 (P < 0.05). No changes were observed in secretory azoospermia. The culture procedure from obstructive azoospermia not only increased the post-thaw recovery rate, as previously observed, but also reduced the portion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use.

Key words: acridine orange/azoospermia/in-vitro culture/sperm DNA/testicular sperm extraction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The improvement in motility of testicular spermatozoa by in-vitro culture has been reported by several authors (Craft et al., 1995Go; Zhu et al., 1996Go; Liu et al., 1997Go) and the practical implications of performing ICSI with in-vitro matured testicular spermatozoa described (Urmann et al., 1998Go; Balaban et al., 1999Go; Hu et al., 1999Go). In a previous study, we observed that the culture of testicular tissue for 48–72 h at 37°C in men suffering from obstructive azoospermia not only resulted in an increased percentage of motile spermatozoa but also in a better post-thaw recovery rate (Emiliani et al., 2000Go). A recent study showed that testicular germ cell culture for 48 h facilitates the selection of TUNEL negative (non-apoptotic) spermatids (Tesarik et al., 1999Go). We wondered whether testicular sperm culture could affect mature sperm DNA. The negative effects of oxidative stress mediated by reactive oxygen species (ROS) on spermatozoa, on their motility, on oocyte membrane fusion and on DNA integrity have been extensively described (Aitken et al., 1998Go; Lopes et al., 1998Go; Twigg et al., 1998aGo). There are many sources of ROS when spermatozoa are cultured in vitro: leukocytes (Aitken et al., 1989Go), necrotic or abnormal spermatozoa (Aitken et al., 1989Go), transition metals present in culture medium (Lloyd et al., 1997Go), and the preparation technique itself (Twigg et al., 1998bGo). Single-stranded sperm DNA is more sensitive to oxidative and denaturing stress (Gorczyka et al., 1993Go; Manicardi et al., 1995Go; Sailer et al., 1995Go). The aim of this study was to verify if the procedure for culturing testicular tissue to obtain spermatozoa with a higher motility and better post-thaw recovery rate affected the ratio of single/double-stranded sperm DNA. For this purpose, we exploited the methacromatic properties of acridine orange, a dye that binds externally to single-stranded DNA, fluorescing red and binds in an intercalate position to double-stranded DNA, fluorescing green (Evenson et al., 1986Go). As previously stated, the ratio between red/green stained spermatozoa is strictly correlated with DNA packaging (Evenson et al., 1986Go; Evenson, 1990Go). The single stranded DNA level in testicular spermatozoa was compared between the moment of retrieval (day 0) and after 72 h of culture (day 3).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Testicular biopsies were obtained, between January and November 1999, during open surgery under full anaesthesia from 13 patients suffering from obstructive azoospermia and from three patients suffering from secretory azoospermia.

Testicular biopsies: preparation and culture
The protocol for testicular specimen preparation and in-vitro culture is described elsewhere (Emiliani et al., 2000Go). Briefly, the biopsies were placed in a Petri dish and first washed in 20 mmol/l Hepes buffered modified Earl's salt solution (mHEBSS), supplemented with 0.5% human albumin (Belgian Red Cross), 0.43 mmol/l sodium pyruvate (Sigma Chemical Company, St Louis, USA) and 44 mmol/l sodium lactate (Sigma). The biopsies were mechanically dissociated with two sterile scalpels. The dissociated tissue was emptied into a 40 µm Falcon (Beckton Dickinson and Co. Meylan Cedex, France) cell strainer, placed onto a 50 ml conical Falcon tube and centrifuged at 500 g for 10 min, at room temperature, in mHEBSS with 0.5% human albumin. The pellet was then resuspended in 1 ml of mEBSS buffered with 28 mmol/l sodium bicarbonate, supplemented with sodium pyruvate, lactate and 20% of inactivated serum (35 min at 56°C), obtained from the same patient. The pellet was filtered through a 35 µm Falcon cell strainer and centrifuged again. The pellet was resuspended to a volume of 100 µl. Before ICSI or before sperm count, the pellet was further disintegrated by aspirating through a 20 µm diameter pipette to obtain nearly complete disintegration of cell clusters. The percentage of motile spermatozoa was assessed at retrieval (day 0) and after 72 h of culture (day 3), by counting at least 100 spermatozoa. Only spermatozoa displaying mature morphology and free from cell clusters were included in the count. Only motile spermatozoa were used for ICSI.

Acridine orange staining
One part of each tissue was fixed overnight on slides in Carnoy fixative (methanol:glacial acetic acid 3:1), on day 0 as well as on day 3. The staining protocol with AO is described elsewhere (Tejada et al., 1984Go). Briefly, air-dried slides were stained for 10 min with freshly prepared AO (0.19 mg/ml), washed in distilled water and then a coverslip was applied on the slide. Slides were evaluated on the same day using a fluorescent microscope (490/530 nm excitation/barrier filter; Olympus, Tokyo, Japan). In all preparations, at least 100 spermatozoa were evaluated at x40 magnification. Spermatozoa with green fluorescence were considered to have native double-stranded (DS-DNA) DNA, spermatozoa with yellow fluorescence were considered as having partially denatured single-stranded DNA and with red fluorescence as completely denatured single-stranded-DNA (SS-DNA). Percentages of green spermatozoa were assessed and compared between day 0 and day 3. Only spermatozoa displaying mature morphology were included in the count.

Statistic
Statistical analysis was conducted using a SPSS software package. Statistical significance was defined at P < 0.05. Mann–Whitney test was employed to compare median and {chi}2 test to compare percentages. Correlations were made using Pearson's product moment test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Ten patients with obstructive azoospermia had an ICSI cycle, from which eight resulted in a pregnancy (two biochemical and six clinical). All three patients with secretory azoospermia had an ICSI cycle, from which one resulted in a clinical pregnancy. Only motile spermatozoa were used for ICSI.

Obstructive azoospermia
Motility of testicular spermatozoa
Figure 1Go illustrates the percentages of motile spermatozoa at retrieval and after 3 days of in-vitro culture at 37°C. Median values of 22% (range 10–44%) on day 0 and 50% (range 38–63%) on day 3, were significantly different (P < 0.01, Mann–Whitney test). The differences in percentage motile spermatozoa were statistically significant in each sample (P < 0.01, {chi}2 test).



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Figure 1. Percentages of motile testicular spermatozoa in obstructive azoospermia: at retrieval (day 0) and after culture (day 3). Median values at day 0 (22%) and day 3 (50%) were significantly different (P < 0.01; Mann–Whitney test).

 
Single-stranded sperm DNA level
Figure 2Go illustrates the percentages of red stained spermatozoa at retrieval and after 3 days of culture. Median values of 54% (range 0.1–88%) at day 0 and 20% (range 2.7–99.99%) at day 3 were significantly different (P = 0.039, Mann–Whitney test). The differences in percentage of red stained spermatozoa were statistically significant in all samples (P < 0.05, {chi}2 test).



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Figure 2. Percentages of red fluorescing testicular spermatozoa in case of obstructive azoospermia: at retrieval (day 0) and after culture (day 3). Median values at day 0 (53.5%) and at day 3 (20%) were significantly different (P = 0.039; Mann–Whitney test).

 
Correlation test
No correlation was found between motility and percentages of red stained spermatozoa, either at retrieval (P = 0.551, r = –0.182; Pearson test), or at day 3 (P = 0.339, r = –0.289). The increases of the two parameters, observed after 72 h of culture, were also not correlated (P = 0.241, r = 0.350).

Secretory azoospermia
No differences were observed in percentages of motile spermatozoa for the three patients before and after culture (day 0: 15, 24 and 28%; day 3: 6, 16 and 34% respectively). No differences were observed in percentages of red stained spermatozoa for the three patients before and after culture (day 0: 2.1, 63 and 39%; day 3: 3.7, 55.7 and 51% respectively).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The percentage of spermatozoa containing SS-DNA decreased after 3 days of culture in the case of obstructive azoospermia but remained unchanged in the case of secretory azoospermia. Transformation of spermatozoa nuclei from red fluorescing to green fluorescing was previously observed during passage from the testis to the epididymis, as well as during epididymal transit (Kosower et al., 1992Go). A strong correlation was demonstrated previously between fluorochrome staining, chromatin packaging and DNA susceptibility to oxidative stress and damage (Bianchi et al., 1993Go; Manicardi et al., 1995Go; Sailer et al., 1995Go). DNA damage could be the result of the action of ROS (Aitken et al., 1998Go; Lopes et al., 1998c; Twigg et al., 1998aGo; Barroso et al., 2000Go). Low levels of ROS are physiologically produced by the spermatozoa and used as electron acceptors by the chromatin-associated enzyme phospholipid glutathione peroxidase (PHGPx), that promotes, during the process of compaction of DNA, the oxidation of the SH group of protamines to disulphide bound (Aitken et al., 1998Go). Higher levels of ROS, produced by impaired spermatozoa, leukocytes, sperm preparation and the culture medium, can affect various elements, including sperm membrane, sperm fusion capability and motility (Aitken et al., 1998Go), and can attack DNA, inducing strand breaks (Twigg et al., 1998aGo; Twigg et al., 1998bGo).

Our study found no correlation between the percentage of spermatozoa containing SS-DNA and motility, either at retrieval or after culture, or between the increase of the two parameters during the culture period, suggesting that the observed improvements in the two parameters are probably independent of one another. It is difficult to postulate that an enzymatic repair mechanism is involved in SS-DNA reduction, in view of the extremely compacted nature of sperm DNA. A consistent reduction of apoptosis-related DNA damage in human spermatids from testicular biopsies after 48 h of culture was demonstrated previously (Tesarik et al., 1999Go). The observed phenomenon was explained by the disintegration of apoptotic cells at the beginning of the culture, in parallel with the ongoing differentiation of healthy cells. A similar process of degeneration of SS-DNA-containing spermatozoa and the parallel development of immature DS-DNA spermatids in mature spermatozoa, during the culture, could be postulated in our case. Furthermore, the latter mechanism might explain the lack of reduction of SS-DNA in samples from patients with non-obstructive azoospermia who are known to be more likely to have higher rates of DNA damage among immature germ cells (Tesarik et al., 1998aGo). Finally, the effects of sample processing before culture on the changes observed should not be underestimated. For example, we wonder what effect temperature change may have; from the physiological value in the testis (about 30°C: Tesarik et al., 1998bGo,cGo) to 37°C in the culture. In a previous study similar observed motility pattern of cultured spermatozoa induced by two different temperatures (32 and 37°C) (Emiliani et al., 2000Go) suggested that this is not the principal factor implicated in the motility improvement of cultured spermatozoa. On the other hand, in this study the observed lack of correlation between the changes in motility and the proportino of spermatozoa containing SS-DNA proportion suggest independent control of the two processes. The process of DNA packaging is produced by two factors: (i) the gradual substitution of lysine-rich histones by protamines, which seems to be completed in late spermatid (Poccia, 1986Go); (ii) the process of oxidation of SH groups, present in protamine residual of cysteine to SS groups (Balhorn, 1989Go; Bianchi et al., 1993Go), that, on the contrary, is not completed in the testis [between 4 and 44% of DS-DNA spermatozoa was reported in the testis: (Kosower et al., 1992Go; Golan et al., 1996Go)]. A role of the temperature increase or of the changes in the physico-chemical environment, induced by sample processing and in-vitro culture, could simulate some physiological changes in vivo, during sperm transit through the testicular–epididymal–deferential compartments, thereby activating such oxidative enzymes (Balhorn, 1989Go; Bianchi et al., 1993Go) and inducing similar DNA structural modifications.

In conclusion, we present evidence that the in-vitro culture of testicular spermatozoa does not increase their susceptibility to DNA damage. For patients with obstructive azoospermia, the proportion of spermatozoa containing SS-DNA decreased after 3 days of culture, whilst this parameter remained unchanged in spermatozoa retrieved from patients with secretory azoospermia. Several previous studies have demonstrated the negative effects of SS-DNA spermatozoa on several clinical parameters. Lower semen quality, fertilization rate, embryo cleavage rate and pregnancy rate after IVF or ICSI are closely associated with a high SS-DNA-containing sperm concentration (Peluso et al., 1992Go; Hoshi et al., 1996Go; Duran et al., 1998Go; Evenson et al., 1999Go). Furthermore, a relationship was found between oxidative DNA damage and childhood cancer (Fraga et al., 1996Go; Ji et al., 1997Go) and it was stated that the percentage of SS-DNA spermatozoa is highly predictive of pregnancy failure (Evenson et al., 1999Go). There are no commercially available vital sperm dyes that can be used to select directly DS-DNA-containing spermatozoa for ICSI. However, the increased percentage of these spermatozoa after culture, in obstructive azoospermia, increases the probability of selecting one of these (more safe) spermatozoa for ICSI.


    Notes
 
3 To whom correspondence should be addressed at: Fertility Clinic, Department of Obstetrics and Gynaecology, Erasmus Hospital, Route de Lennik 808, 1070 Brussels, Belgium. E-mail: semilian{at}ulb.ac.be Back


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 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 21, 2000; accepted on February 26, 2001.