Sperm DNA damage in potentially fertile homozygous ß-thalassaemia patients with iron overload

Doreen Perera1, Arnold Pizzey2, Alastair Campbell3, Maurice Katz1, John Porter2, Mary Petrou1, D.S. Irvine3 and Ratna Chatterjee1,4

1 Departments of Obstetrics and Gynaecology and 2 Haematology, UCL London and 3 MRC Reproductive Biology Unit, Edinburgh, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: To test the hypothesis that human sperm DNA could sustain iron-induced oxidative damage and reduce its fertilizing ability, we studied patients with homozygous ß-thalassaemia major (HbTh) as a model of iron overload. METHODS: Sperm from six thalassaemic patients and five age-matched controls were assessed by the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Semen parameters, endocrine markers of testicular function, iron profiles and the presence of organ dysfunction were also determined. RESULTS: All patients with HbTh were iron overloaded (median ferritin: 2251 µg/l) and had evidence of spontaneous spermatogenesis. Thalassaemic patients had more sperm DNA damage than the controls (P < 0.01). The sperm DNA damage by SCSA and TUNEL were positively correlated (P < 0.05). Sperm motility and TUNEL results were negatively correlated (P < 0.05), while the age of onset of chelation and sperm DNA damage were positively associated with both SCSA (R2 = 0.80, P = 0.016) and TUNEL data (R2 = 0.67, P < 0.044). No other biochemical or clinical data were associated with sperm DNA damage. CONCLUSIONS: The increase in sperm DNA damage and the negative correlation between sperm motility and DNA damage suggest that iron overload in HbTh predisposes sperm to oxidative injury. This finding has important implications in assisted reproductive procedures such as ICSI where there is increased risk of transmitting defective DNA to the offspring.

Key words: ß-thalassaemia/DNA damage/iron overload/oxidative stress/sperm


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The role of iron in mediating the formation of reactive oxygen species (ROS) in biological systems is well known. The detrimental effects of ROS on the sperm membrane, its structural components and its nucleus are also well documented (Aitken et al., 1998Go). Following the advent of ICSI, male reproductive success is no longer limited to those with conventionally assessed normozoospermic indices. Since sperm with damaged DNA structure are still able to fertilize oocytes and because the outcome of such a fertilization is unknown (Sakkas and Tomlinson, 2000Go), it is important to assess the integrity of the sperm nuclear material in males with high levels of circulating iron.

Homozygous ß-thalassaemia is a hereditary haemoglobinopathy that affects >1.5 million people worldwide (Chatterjee and Katz, 2000Go). Improvement of the basic management protocol of high red cell transfusion therapy has improved the longevity of the patients at least to their fourth decade. However, these patients have variable degrees of organ damage and endocrinopathies that can affect their quality of life. Hypogonadotrophic hypogonadism is the commonest endocrinopathy, affecting 80–90% of patients worldwide, for which homozygous ß-thalassaemia major (HbTh) patients have disturbance of growth, sexual maturation and impaired fertility (Chatterjee et al., 1993aGo,bGo; De Sanctis et al., 1995Go). This is largely due to iron overload, and from a 10 year prospective study we have also established that iron overload affects the hypothalamic–pituitary (H–P) axis in a dose-dependent way (Chatterjee et al., 1993bGo, 2000Go). Poorly compliant HbTh patients have sexual infantilism and severe damage to the H–P axis and even structural loss of the anterior pituitary gland (Chatterjee et al., 1998Go). On the other hand, some patients with better compliance may have spontaneous sexual maturation with intact fertility potential. There are only two studies, which describe spontaneous spermatogenesis and semen parameters in HbTh patients. A study of sperm concentrations and quality in eight British ß-thalassaemia major patients by undertaking routine semen analysis (Jensen et al., 1996Go) reported severe oligoasthenospermia in all but the youngest. Another study (Papadimus et al., 1996Go) evaluated the pituitary–testicular axis in 30 Greek thalassaemic men aged 17–35 years who were regularly transfused and who underwent chelation therapy. The investigations included endocrinological examination, evaluation of serum basal levels of FSH, LH, free testosterone and GnRH as well as a standard semen analysis. According to the results, patients were divided into three groups: group A, which included 18 eugonadal patients with moderately elevated serum ferritin; group B which included six patients who had hypogonadotrophic hypogonadism and excessive elevation of serum ferritin; and group C, which included six patients characterized as intermediate, with regard to sexual maturation and serum ferritin levels. In agreement with our previous findings (Chatterjee et al., 1998Go, 2000Go), this study also concluded that HbTh patients have variable degrees of pituitary iron overload and are therefore prone to hypophyseal damage.

Because these patients have potentially functional testes, spermatogenesis may be induced with exogenous gonadotrophins (De Sanctis et al., 1988Go). This is the only study in the literature which relates to induction of spermatogenesis in HbTh patients. In this collaborative study undertaken with the Italian group (De Sanctis et al., 1988Go) we attempted to induce or augment pubertal development and achieve spermatogenesis in 10 gonadotrophin-deficient thalassaemic patients aged 15–23 years (mean 18.9). HbTh patients were treated with exogenous gonadotrophins for 1–4 years (mean 2.1). Seven patients produced sperm during HCG treatment given for 6–14 months. However, full spermatogenesis was achieved only when HMG was added to HCG regimen. In one patient, despite cessation of gonadotrophin treatment, sexual potency, libido and spermatogenetic capacity were maintained during the past 21/2 years. However, paternity was not achieved in these patients.

Whereas pregnancy is reported in thalassaemic females (Aessopos et al., 1999Go), paternity is less common in thalassaemic men, even those with normal or near normal spermatogenesis. The exact cause for this discrepancy is unclear. Given the milieu of iron overload in which spermatogenesis is taking place, this may be due to sperm dysfunction associated with variable degrees of hypogonadotrophic hypogonadism (Chatterjee and Katz, 2000Go) and/or ROS-inflicted damage on sperm nuclear DNA (Aitken et al., 1998Go).

Advances in assisted reproductive techniques such as ICSI in which a single spermatozoon is injected into the cytoplasm of an oocyte, have improved the prospects of childbearing in oligozoospermic thalassaemic patients (Pisarska et al., 1999Go). However, little is known about the integrity of sperm DNA in patients with spontaneous or induced spermatogenesis. One study has shown evidence of oxidative DNA damage in rat sperm cells in the presence of iron overload, in vivo and in vitro (Wellejus et al., 2000Go). The degree of sperm damage correlates well with poor fertilization in natural and assisted conception (Lopes et al., 1998Go; Spano et al., 2000Go). This information is particularly important in ICSI, as during fertilization damaged sperm DNA may be transmitted to the fertilized oocyte, resulting in genotoxic effects for the embryo.

We hypothesize that the sperm of the iron-overloaded HbTh patients are more likely to have sperm DNA damage due to ROS than the non-thalassaemic controls. To test our hypothesis, we investigated the degree of sperm DNA damage in a cohort of six thalassaemic patients with spontaneous spermatogenesis and five non-thalassaemic, normozoospermic controls.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We studied spermatogenesis by assessing routine semen parameters (count, morphology, motility), endocrine markers of testicular function (FSH, LH testosterone) and sperm DNA damage in a cohort of six thalassaemia major patients aged 22–39 years (median 24). Five age-matched controls aged 25–38 years (median 26) were studied for comparative purposes. The controls were healthy fertile males of proven paternity. Sperm DNA damage was assessed by two methods: the sperm chromatin structure assay (SCSA) (Evenson et al., 1999Go) and terminal deoxynucleotidyl transferase biotin-dUTP (TUNEL) (Sun et al., 1997Go). The transfusion and chelation management plan of the thalassaemic patients has been previously published (Chatterjee and Katz, 2000Go). The thalassaemic patients received 2–3 units of blood every 4–5 weeks and were treated with 2–2.5 g of desferrioxamine (iron chelating agent) 5 nights per week. The overall mean haemoglobin was 11 g/dl. The degree of iron overload was assessed by measurement of serum ferritin, iron, total iron binding capacity and the presence of organ dysfunction (such as cirrhosis, hepatitis B, C, cardiac arrhythmia, impaired glucose tolerance test, diabetes mellitus, osteopoenia). To minimize confounding factors, only patients who had undergone spontaneous puberty and who had conventionally assessed normozoospermic indices were studied by using published criteria (World Health Organization, 1992Go).

The University College London Hospitals (UCLH) ethical committee approved the study and written, informed consent was obtained from all participants. A blood sample was drawn from the thalassaemia patients immediately before their monthly blood transfusion. The semen evaluation, SCSA and TUNEL were all carried out prior to transfusion. Semen samples were collected by masturbation after 2–3 days of abstinence. Samples were delivered to the fertility laboratory within 1 h of collection and semen analysis was performed according to published criteria (World Health Organization, 1992Go) and by computer-assisted sperm analysis (World Health Organization, 1992Go). The samples were then divided and prepared for SCSA and TUNEL.

Sperm chromatin structure assay
The SCSA provides an index of sperm with abnormal chromatin structure as assessed by the susceptibility of sperm DNA to acid-induced denaturation in situ (Evenson et al., 1999Go). Briefly, washed, ethanol-fixed and acid-treated sperm cells were stained with the nuclear dye acridine orange (AO) and analysed on an Epics Elite (Beckman-Coulter, High Wycombe, Bucks, UK). Under the assay conditions, AO exhibits green fluorescence when bound to native double-stranded DNA and red when bound to the acid-denatured, single-stranded form of the molecule. The data expressed as bivariate dot plots show red and green fluorescence on the x- and y-axes respectively. The degree of DNA abnormality was assessed by calculating the ratio of red to total fluorescent cells. This index is termed `alpha T' and has a numerical range of 0–1 where higher indices represent greater susceptibility to acid denaturation.

TUNEL
This method identifies DNA strand breaks in situ by using terminal deoxynucleotidyl transferase (TdT) to transfer biotin-dUTP to the free 3'-OH of the cleaved DNA. The biotin-labelled cleavage sites are then visualized by reaction with fluorescein-labelled streptavidin. Washed, formaldehyde-fixed sperm cells were sent to the Edinburgh MRC centre for analysis by previously described methods (Sun et al., 1997Go). Each sample of sperm acts as its own negative and positive control. In brief, an aliquot of each ejaculate acts as its own negative control by incubating it with TdT buffer but no TdT enzyme, and therefore there is no binding of biotinylated dUTP. The sperm are then incubated with the FITC but there is no binding to DNA strand breaks because there has been no binding of the biotin-dUTP. The peak on the flow cytometer histogram represents auto fluorescence of the sperm and is used as a baseline. The sample is treated with TdT and TdT buffer and therefore if DNA strand breaks are present, biotinylated dUTP binds to them due to the presence of the enzyme. When it is then incubated with streptavidin-FITC this then binds to the biotin-dUTP. On the flow cytometer, this is detected as the second peak and the percentage of sperm, which are TUNEL positive, can be calculated as the negative control has been used as a baseline and used to set the region for TUNEL positive sperm. The positive sperm is an aliquot of the ejaculate which is pretreated with DNase and therefore induces iatrogenic DNA in all sperm. The aliquot of sperm is then treated with TdT and buffer and the biotin-dUTP will bind to the DNA strand breaks which will be present in all sperm due to the action of the DNAse. Likewise the streptavidin-FITC will bind to the complex and, as all sperm will possess DNA strand breaks and therefore will bind the biotin-dUTP complex will fluorescence green because they are TUNEL positive. On the flow cytometry histogram, there is a complete shift in the curve because all the sperm are TUNEL positive due to the action of the DNase and therefore fall into the TUNEL positive region.

Serum iron and endocrine profiles
Standard operating procedures used routinely in the chemical pathology and endocrine laboratories at UCLH were employed (Chatterjee et al., 2000Go).

Statistical analysis
All data were analysed using the GB Stat statistical software (Dynamic Microsystems, Inc.) and Excel (Microsoft Corp.). A correlation matrix and linear regression analysis was used to determine the statistical association between biochemical and seminological parameters. Student's t-test was used to assess the statistical differences between data from donor controls and patients.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical data
The clinical, hormonal and iron profiles of the thalassaemia patients are shown in Table IGo. The serum ferritin levels were consistently elevated in this group. Table IIGo shows the seminological analysis for both the patient and the control groups; semen analysis parameters for all patients were within the ranges observed in the non-thalassaemic male population, with patient 3 being mildly oligozoospermic and patients 3 and 4 mildly asthenozoospermic, based on published seminological criteria (World Health Organization, 1992Go).


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Table I. Clinical, endocrine and iron profiles of patients with ß-thalassaemia
 

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Table II. Semen parameters, SCSA and TUNEL in subjects with and without ß-thalassaemia
 
Sperm DNA damage
Figure 1A and BGo shows representative flow cytometric dot plots after SCSA and TUNEL from a donor and a thalassaemic patient espectively. Figure 1CGo and Table IIGo show the range of sperm DNA damage results obtained for patient groups and controls. The mean SCSA level for thalassaemics was significantly higher than that of the controls (0.42 ± 0.14 versus 0.18 ± 0.02; P < 0.01). The mean TUNEL results of the patients were also significantly higher than those of the controls (22.9 ± 11.1 versus 11.4 ± 1.9; P < 0.05).



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Figure 1. (A) Representative flow cytometric dot plots and green fluorescence (y-axis) obtained after sperm chromatin structure assay (SCSA) using sperm from a donor and a thalassaemic patient. The normal sperm nuclei are shown as green fluorescence and abnormal sperm nuclei are represented as red fluorescence. (B) Flow cytometric single parameter histogram of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) fluorescence. On the flow cytometry histogram, there is a marked shift in the curve because all the sperm are TUNEL positive due to the action of the DNase and therefore fall into the TUNEL positive region. (C) Sperm DNA damage of patients and controls obtained by SCSA and TUNEL.

 
Figure 2AGo shows significant correlation between the age of onset of chelation therapy and sperm DNA damage as measured by both SCSA (R2 = 0.8001, P = 0.016) and TUNEL (R2 = 0.6751, P = 0.044). Figure 2B and CGo shows the negative relationship between percentage sperm motility and DNA damage. Sperm motility was recorded as both percentage total and rapid motility. Both assessments of motility were significantly correlated with the TUNEL data (R2 = 0.8, P = 0.016 and R2 = 0.67, P = 0.047 respectively). Although a negative trend was also observed with results obtained by SCSA, it did not reach statistical significance (not shown). No correlations were found between the sperm DNA damage and other sperm parameters, serum endocrine, iron profiles, or incidence of organ dysfunction.



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Figure 2. (A) Correlation between the age of onset of chelation therapy and sperm DNA damage as measured by both sperm chromatin structure assay (SCSA) (R2 = 0.80, P < 0.01) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) (R2 = 0.6751, P < 0.05). (B) Association between percentage sperm total motility and sperm DNAdamage by SCSA (P = not significant) and TUNEL (R2 = 0.80,P < 0.01). (C) Association between % rapid motility and sperm DNA damage by SCSA (P = not significant) and TUNEL(R2 = 0.67, P < 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This pilot study is the first to record evidence of genetically damaged sperm in thalassaemic patients. This finding was substantiated by two different techniques, SCSA and TUNEL. Three possible factors may contribute to sperm DNA damage in these patients: oxidative stress of iron overload, lower antioxidant levels and iron-chelating drugs.

First, generation of ROS from iron overload is likely to be the most logical explanation for the aetiology of sperm DNA damage. In rat studies, marked sperm DNA damage in acute iron toxicity has been demonstrated (Lucesoli et al., 1999Go; Wellejus et al., 2000Go). Similarly, our subjects with chronic iron overload also had evidence of significantly greater sperm damage compared with age-matched healthy controls. However, unlike the rat testicular cells, which showed a dose-dependent increase in DNA damage to oxidative stress (Aitken et al., 1992Go), there was no correlation between the degree of iron overload and the amount of sperm DNA damage in our thalassaemia patients. This could be due to a loss of statistical sensitivity of correlation at such excessive levels of exposure for long periods of time. However, there was a close relationship between initiation of chelation therapy and sperm damage. Patients whose onset of chelation was delayed had more DNA damaged sperm than those who started chelation at a younger age, indicating that duration of exposure to oxidative stress may be an important factor in the induction of sperm damage. This is consistent with the study (Aitken et al., 1992Go) showing that the damaging effect of ROS produced by WBC was dependent on the exposure of sperm to WBC.

Second, diminished amounts of antioxidants vitamin E and vitamin C in the serum of patients with thalassaemia syndrome have been reported by us (De Sanctis et al., 1988Go) and others (Jensen et al., 1996Go; Papadimas et al., 1996Go). One study (De Luca et al., 1999Go) showed a correlation between low vitamin E levels and non-transferrin-bound iron in a group of Italian thalassaemics. Hence it is possible that low levels of free radical scavengers may exacerbate ROS-inflicted damage in thalassaemic sperm.

Third, genotoxic drugs may cause DNA damage. Desferrioxamine (DFO) mesylate is an established chelating agent which is used for the management of transfusion-dependent HbTh for 30 years (Liu and Hider, 2002Go). It is an antiproliferative agent (Gao and Richardson, 2001Go) and is a proven teratogen at least in animals (Shilalukey et al., 1997Go). However, Turk et al. achieved successful therapy of iron intoxication in pregnancy with i.v. DFO and whole bowel irrigation in a severely iron-poisoned adult patient in week 26 of gestation with 10.2 g DFO (Turk et al., 1993Go). DFO was administered i.v. over 14 h and whole bowel irrigation (2 l/h of polyethylene glycol-electrolyte solution/nasogastric tube for 12 h) with a good maternal outcome and no adverse effects on the fetus. Another study (Vaskaridou et al., 1993Go) reported two cases of transfusion-dependent thalassaemia treated by DFO during the first trimesters of two successful pregnancies. Our patients were on the chelating agent DFO, and, as there are no human data to suggest that DFO is genotoxic, the sperm DNA damage in our study group is unlikely to result from DFO toxicity.

However, DFO has been teratogenic at least in some animal studies, and there is evidence of fetal teratogenecity in rats (Lauro et al., 1968Go). Furthermore, developmental toxicity is shown by an oral iron chelator deferriprone in mice models (Albina et al., 2000Go). However, none of the patients of the study group were on deferriprone. But iron chelators are suspected teratogens, and spermatogenesis in thalassaemics is, at least theoretically, occurring in a milieu of chelators, which is suboptimal for the generation of genetically intact sperm.

We observed an inverse relationship between sperm motility and DNA damage. Many studies have shown evidence of oligoasthenozoospermia in iron-overloaded thalassaemic patients with spontaneous and induced spermatogenesis (De Sanctis et al., 1988Go; Jensen et al., 1996Go; Papadimas et al., 1996Go). A study using a ram model (Upreti et al., (1997) reported impaired sperm motility due to use of DFO. Although the impaired motility in some of our thalassaemic patients may be partly due to DFO, it is more logical to suggest iron overload to be the main cause as reported before (De Sanctis et al., 1988Go; Jensen et al., 1996Go). De Lamirande and Gagnon showed that depletion of ATP plays an important role in sperm DNA damage with concomitant impairment of sperm motility from generation of ROS (De Lamirande and Gagnon, 1992Go). The sequence of events leading to loss of motility in damaged sperm and associated biochemical modulators remain to be investigated. Arguably teleologically, the loss of motility in genetically impaired sperm may be interpreted as a defence against such sperm from reaching the oocyte. Conventional semen tests using WHO criteria (World Health Organization, 1992Go) do not address the issue of sperm DNA measurement, while the association of subnormal sperm parameters and infertility is well established (Gorczyca et al., 1993Go; Sun et al., 1997Go; Lopes et al., 1998Go; Evenson et al., 1999Go; Spano et al., 2000Go). It is only recently that the relationship between genetically damaged sperm and infertility has been addressed. In infertile males, several groups have demonstrated that subnormal sperm parameters correlate well with DNA damage and the outcome of assisted conception (Sun et al., 1997Go; Lopes et al., 1998Go; Evenson et al., 1999Go; Irvine et al., 2000Go; Spano et al., 2000Go). These issues must be highlighted in fertility counselling of HbTh patients, especially during sperm cryopreservation.

It is interesting to note that in many other pathological conditions (Baccetti et al., 1996Go; Gandini et al., 2000Go), including cancer and recipients of cancer chemotherapy (Chatterjee et al., 2000Go) or in diseases where ROS is generated (Sharma et al., 1999Go), sperm DNA damage can occur. This study adds iron overload to the pathologies that can induce sperm DNA damage.

The biological implications of the higher proportion of damaged DNA in the ejaculates of thalassaemic patients is worrying as such sperm are able to fertilize oocytes and may transmit defective genetic material to the offspring, similar to recipients of cytotoxic chemotherapy (Chatterjee et al., 2000Go). This should be borne in mind when counselling thalassaemics, especially those requiring artificial reproductive techniques such as ICSI, where the natural barriers to penetration of sperm with defective DNA into the oocyte are bypassed.

Although novel, our data are derived from a small number of patients. So caution must be exercised in their interpretation. Our findings should, however, provide impetus for future research. A longitudinal study with a larger number of patients from puberty is needed to determine whether DNA damage exists ab initio or evolves later in adolescence or early adulthood. Such a study would address the issue of whether there is a dose-dependent effect of iron on sperm toxicity. In addition, a large double-blind study needs to be performed to determine whether supplementation of antioxidants vitamin E and/or vitamin C will reduce or prevent sperm DNA damage in thalassaemia patients.

In the light of our findings, we recommend that all thalassaemia patients have semen cryopreserved as early as possible, hopefully before toxic factors produce their effect. Fertility counselling offered to all patients prior to cryopreservation of sperm should include information about the potential of fetotoxicity and reproductive failure.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Dr H.H.McGarrigle and staff of Endocrine Laboratory and Haematology for their assistance in the endocrine assays.


    Notes
 
4 To whom correspondence should be addressed at: Reproductive Medicine Unit, Elizabeth Garret Anderson Hospital, iversity College Hospital, Huntley Street, London WC 1E 6AU, UK. E-mail: ratnach{at}globalnet.co.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 22, 2001; accepted on March 8, 2002.