TUNEL analysis of sperm DNA fragmentation in kidney transplant patients

1 Introduction

The male genome abnormalities depicted by DNA damage may indicate male-subfertility regardless of the routinely used semen properties [1–3]. Increased proportions of fragmented sperm DNA are associated with poor semen quality [2, 4], but the origin of this damage is still not fully elucidated. Previously published papers have demonstrated nucleus anomalies of spermatozoa [4]. Three factors have been suggested as the cause of DNA damage in the germline: chromatin packaging deficiencies, apoptosis (abortive), and oxidative stress.

Infertility is a common complication in chronic kidney disease (CKD) patients, however, successful kidney transplantation usually enables fertility [5]. Besides, endocrine abnormalities are common in CKD patients and lead to sexual dysfunction, altered mineral metabolism, hyperparathyroidism, and anemia [6, 7]. Erectile dysfunction in men decreased libido, and infertility in both sexes are common clinical problems [8]. Moreover, the male reproductive function may be affected by prescribed immunosuppressive therapy to prevent rejection after transplantation. In this context, it is important to evaluate the influential factors such as medication procedures and the pre-treatment period. Male infertility is also related to impaired spermatogenesis, as indicated by decreased total sperm counts and sperm motility along with elevated levels of sperm abnormality, following the treatment. There is a need for studies on the evaluation of DNA fragmentation in renal transplant patients. Therefore, this study was aimed to investigate the sperm parameters of kidney transplant patients, including sperm DNA defects.

2 Material and Method

2.4 DNA fragmentation analysis

the TUNEL assay was performed to determine sperm DNA fragmentation. In this context, an In-Situ Cell Death Detection Kit (Roche Diagnostics) was used as instructed by the manufacturer. Shortly, the spermatozoa were immediately fixed in Carnoy’s solution (2 : 1 methanol/acetic acid) after trypsinization (Trypsin-EDTA) and stored at –20°C for at least 1 h. Sperm pellets were washed with phosphate-buffered saline (PBS), were afterward permeabilized for 20 min with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO) in sodium citrate dehydrate (VWR, Fontenay sous Bois, France; 3.4 mmol l^–1, pH 6.8), and washed again with PBS. The cells were later incubated with FITC-labelled dUTP and a terminal deoxyribonucleotidyl transferase enzyme (TUNEL solution). The positive control sample was treated with 100μl of DNase (1 U μl^–1) (Fermentas, Maryland, USA) at 37°C before incubation with the TUNEL solution for 1 h, and in the negative control samples, TUNEL solution was omitted. The cells were next washed twice in PBS and spread over glass slides. In a dark environment at room temperature, slides were dried and DAPI solution (VECTASHIELD Mounting Medium with DAPI, 1.5μg ml^–1; Vector Laboratories, Burlingame, CA) was added to the spermatozoa. The slides were examined by fluorescence microscopy. 200 spermatozoa (at least) were assessed, and the total DNA fragmentation rate was calculated as the number of FITC-positive cells divided by the total number of sperm nuclei (labeled with DAPI). Figure 1 shows a sample area with DNA fragmented and unfragmented spermatozoa.

OPEN IN VIEWER

Fig. 1

Sperm cell TUNEL test. Dark green painted cells show sperm cell without DNA fragmentation and light green glittering cells show DNA fragmented sperm cells.

3 Results

This study included 56 kidney transplant male patients (34 married and 22 single) aged 22 to 53 (mean 35.7) years and 25 controls (aged 22–50 years, mean 35.5) (P = 0.8). As shown in Table 1, eight (14%) out of 56 men with kidney transplantation had azoospermia and were excluded from sperm DNA fragmentation analysis. In the kidney transplantation group, 16 patients had normozoospermia and the remaining 32 patients had oligozoospermia (n = 1), asthenozoospermia (n = 4), teratozoospermia (n = 6) or a combination of these abnormalities (n = 21). Table 2 shows the seminal parameters of the patients and the control individuals.

The sperm DNA fragmentation in kidney transplant patients was slightly higher compared to controls (P = 0.09) (Fig. 2). Spermatozoa motility and morphology of the kidney transplant patients were significantly lower than those of the controls (P < 0.05) (Table 2).

OPEN IN VIEWER

Fig. 2

Box plots showing the distribution of sperm DNA fragmentation among the controls and patients using the TUNEL assay.

The post-transplantation duration, which was defined as the duration of the exposure period to immunosuppressive treatment, was less than 12 months for 18 patients after transplantation surgery (four patients, 22.2%, had normozoospermia in this group), 12–24 months for 6 patients after surgery (two patients, 33.3%, had normozoospermia in this group), and 24 months in 24 patients after surgery (ten patients, 41.6%, had normozoospermia in this group). Differences in the sperm DNA fragmentation index did not reach statistically significant levels among these groups (P = 0.835) (Fig. 3).

OPEN IN VIEWER

Fig. 3

Box plots showing the distribution of sperm fragmentation among patients according to duration after renal transplantation.

Regarding sirolimus treatment, three patients had severe oligoasthenoteratozoospermia (OAT) and they experienced infertility. The mean of DNA fragmentation index was slightly higher than the mean of those in the remainder of the kidney transplantation group (P < 0.1). In the kidney transplantation group, the sperm DNA fragmentation index was lower in the patients who had normozoospermia (15.6±10.58) than those whose spermogram results showed oligo-, astheno- or teratozoospermia or a combination of these parameters (23.7±13.1) (P = 0.038). The number of patients in the immunosuppression treatment protocol is reported in Table 3.

Testicular volumes (P = 0.13), LH levels (P = 0.97), and total testosterone levels (P = 0.31) were not statistically significant among groups but significantly elevated levels of FSH were observed in the group treated with cyclosporin A + mycophenolate mofetil+steroid (P = 0.028).

Loss of libido, impotence, and infertility due to impaired spermatogenesis and testicular damage are frequently seen in kidney failure [10]. Moreover, hypothalamic-pituitary-gonadal dysfunction characterized by waned sperm maturation is common in these patients [11]. Semen analyses typically show low-volume ejaculate, oligoazoospermia or azoospermia (complete), and decreased percentages of motile sperms. A low spermatogenic activity has been demonstrated by testicular histology. It is characterized by mature spermatocyte insufficiency or germinal element aplasia. The factors associated with the testicular damage in uremia remain unclear. Plasticizers in dialysis-tubing (such as phthalate) may be evaluated as a possible factor in patients with hemodialysis. Moreover, gonadal steroidogenesis might be impaired by uremia. Typically, a decrease is observed in the serum total and free testosterone concentrations. Institution of maintenance dialysis may improve these problems, but normalization often cannot be achieved and quality of life is decreased [12, 13]. Normally, a kidney transplant likely restores sexual activity by six months after successful kidney transplantation; however, the reproductive function might partially remain impaired [11]. Some improvements have been reported in the three main parameters of semen quality (spermatozoa number, motility, and morphology) after kidney transplantation [14] and the sex hormone profile tends to normalize [15]. Prolonged peritoneal dialysis, elevated levels of FSH in pre-transplant duration, and poor graft function may impair recovery of sexual/reproductive activity in these patients [14]. Sperm DNA integrity is a prerequisite for the accurate flow of genetic information. In this respect, sperm DNA damage may cause male infertility regardless of spermatozoa number, morphology, and motility [16]. Although the importance of sperm DNA integrity testing has been adequately demonstrated, there are many questions still to be answered concerning the origins of spermatozoa’s DNA damage.

In this study, the deficiencies in semen parameters of kidney transplant patients were shown. In these patients, the DNA damage amount of the spermatozoa can be interpreted as a considerable factor for unsuccessful reproductive treatment or infertility even if the sperm DNA fragmentation of the kidney transplant patients was not significantly higher compared to the controls (P = 0.09) [17]. For our patient population, the DNA fragmentation percentage was 17.8% ±14.1 in the transplantation group and 16.5% ±6.5 in the control group (P = 0.1), which was lower than the threshold value for infertility (20.05% by TUNEL assay) reported elsewhere [18, 19].

It has been suggested that there is no significant association between immunosuppressive drugs commonly used in kidney transplant patients and adverse effects on patient spermatogenesis or with teratogenic effects on their offspring [18]. Nevertheless, several studies have reported that some of these agents are potentially gonadotoxic with respect to their negative effects on testicular function and fertility [20, 21].

CsA is an immunosuppression enhancer for recipients of kidney transplants [22] and a potential gonadotoxic drug. CsA’s negative influences on reproductive capability have been demonstrated in vivo experiments. In rats, the CsA administration leads to seminiferous tubule degeneration and spermatogenesis impairment, which are unlikely to be secondary to the potential hepatic or nephrotoxic effects of the drug [23]. Moreover, CsA was associated with stimulation of oxidative stress and apoptosis mediation via reactive oxygen species in various cells [24].

Concerning Tac, sperm counts and motility dose-dependently decreased without observation of histopathological testicular injury [21] even though Tac is 50–100 times more potent than CsA [25]. When the drug treatment is stopped, the number of sperms together with motility can return to control levels [21]. In rats, however, spermatogenic damage and Sertoli cell-reduction exist in prolonged subcutaneous administration of Tac (30 to 60 days) [26].

Sirolimus may block spermatogenesis by interrupting the crucial stem cell factor/c-kit system through the rapamycin-sensitive PI3K/Akt/P70S6K/cyclinD3 pathway-inhibition [27]. Therefore, azoospermia/oligozoospermia and infertility may be observed in male kidney transplant recipients with sirolimus treatment [28, 29]. The abnormalities caused by sirolimus administration are similar to CsA but are more severe than those observed with the administration of Tac. Nevertheless, testicular toxicity and infertility by the sirolimus are potentially reversible [20]. In this respect, treatment changing from sirolimus to Tac significantly provides spermatogenesis improvement and the restoration of fertility in kidney transplant recipients after [28, 29].

AZA has been suggested to be genotoxic. It is frequently used in combination with CsA. Information on the effects of AZA on human reproduction is limited. Previously published papers suggest that prednisone might not be associated with sperm cell damage [30]. In addition, the incidence of neonatal malformations in pregnancies fathered by transplant recipients does not increase [15].

Combination therapy used in kidney transplant patients made it difficult to conclude which drug had a more detrimental effect on human spermatogenesis. In our group of patients, 34 patients were on the Tac+MMF+steroid treatment (12 had normozoospermia), five were on the Tac+AZA+steroid treatment (no patient had normozoospermia), 14 on the CsA+MMF+steroid treatment (four had normozoospermia), and three were on the MMF+sirolimus+steroid treatment (none had normozoospermia) (Table 3). There were no statistical differences between the groups regarding the effect of the drugs on spermatogenesis (P = 0.264).

5 Conclusion

In the present study, undesirable consequences of semen parameters in kidney transplant patients were observed. In this context, sperm motility and morphology were significantly lower in the patients group. Sperm DNA fragmentation of renal transplant patients was slightly higher than controls. Sirolimus impairs testicular function in male recipients of an organ transplant and three patients on sirolimus treatment had severe OAT in their ejaculate. Nevertheless, sperm DNA fragmentation rates were not significantly higher than the rest of the transplant group. FSH levels were significantly higher in the group treated with cyclosporin A + mycophenolate mo-fetil+steroid. Kidney transplantation is the best option for patients with severe kidney damage. Although it provides promising possibilities for the patients to restore their general health, it is worth noting that, some features of reproductive function might remain impaired. Although the difference between the renal transplant patients and control group was not statistically significant regarding sperm DNA fragmentation rate, infertility or unsuccessful assisted reproductive treatments may be partially explained by the DNA damage of spermatozoa. Furthermore, experimental designs with a large number of individuals may be required to confirm the present results and to obtain novel clinical aspects.