Sperm DNA fragmentation in men with malignancy

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1 Sperm DNA fragmentation in men with malignancy Simon McDowell, a Keith Harrison, M.Sc., b Ben Kroon, a,c Emily Ford, M.P.H., a,c and Anusch Yazdani, C.R.E.I. a,c a Queensland Fertility Group Research Foundation; b Queensland Fertility Group; and c University of Queensland, Brisbane, Queensland, Australia Objective: To determine if men with malignancy have increased sperm DNA fragmentation compared with men presenting for sperm donation. Design: Retrospective observational study. Setting: Tertiary-level fertility center. Patient(s): Eighty-nine men with cancer presenting for prophylactic semen cryopreservation and 35 men presenting for sperm donation. Intervention(s): None. Main Outcome Measure(s): Sperm DNA fragmentation index (DFI) measured by sperm chromatin assay. Result(s): The mean sperm DFI in men with a diagnosis of cancer, 9.88% (95% confidence interval [CI] 7.84% 12.44%), did not differ from that found in men presenting for sperm donation 10.46% (95% CI 8.68% 11.80%). There were no significant differences in mean sperm DFI within cancer subgroups or when comparing testicular and nontesticular cancers. Subgroup analysis lacked statistical power. Men with testicular cancer have significantly reduced sperm concentration compared with both control subjects and men with nontesticular cancer. Conclusion(s): In our study population there was no difference in sperm DFI between men undergoing prophylactic semen cryopreservation and men presenting for sperm donation. Sperm DFI assessment has limited utility in the routine evaluation of men presenting for semen cryopreservation. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Sperm DNA fragmentation, SCSA, DNA damage, cancer, sperm cryopreservation Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/mcdowellsj-sperm-dna-fragmentation-cancer/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Men diagnosed with cancer are commonly offered semen cryopreservation as a means of fertility preservation (1). Chemotherapy and radiotherapy may detrimentally affect gonadal function and spermatogenesis, resulting in impaired fertility. Such treatment may leave men azoospermic or with varying degrees of spermatogenic compromise. Cryopreservation of spermatozoa is a viable fertility preservation option for men and couples before gonadotoxic chemo- and/or radiotherapy. Received October 5, 2012; revised February 7, 2013; accepted February 8, 2013; published online March 5, S.M. has nothing to disclose. K.H. has nothing to disclose. B.K. has nothing to disclose. E.F. has nothing to disclose. A.Y. has nothing to disclose. Reprint requests: Simon McDowell, Queensland Fertility Group Research Foundation, 1st Floor, 225 Wickham Terrace, Brisbane, Queensland 4000, Australia ( simon.mcdowell@ hotmail.co.uk). Fertility and Sterility Vol. 99, No. 7, June /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. Malignancy alone may be associated with male infertility. A Dutch study in 2010 identified 764 male cancer patients presenting for semen cryopreservation before chemotherapy and radiotherapy. Almost two-thirds of all semen samples were abnormal, and men with testicular germ cell cancer (n ¼ 292) had significantly lower sperm concentrations (2). High levels of sperm DNA fragmentation have been implicated in delayed assisted conception, higher miscarriage rates, increased pregnancy loss, and adverse effects on the short- and long-term health of children born from assisted reproductive technologies (3). Currently there is insufficient evidence regarding the impact of malignancy on sperm DNA, but there are concerns that those with cancer may have a higher level of sperm DNA damage (4). Routine testing for sperm DNA damage in men with cancer is thought to assist practitioners in conveying prognostic information regarding likelihood of success with cryopreserved sperm, andtoplanwhatmodalityofassisted reproduction would be required in the future. In the present study, we measured standard semen parameters and the sperm DNA fragmentation index (DFI) in men presenting for semen cryopreservation before antineoplastic treatment. The type of cancer was noted 1862 VOL. 99 NO. 7 / JUNE 2013

2 Fertility and Sterility and analysis performed on cancer subgroups as well as the broad subgroups of testicular versus nontesticular cancers. The findings were compared with a control population of men presenting to the same fertility clinic for altruistic sperm donation. MATERIALS AND METHODS A retrospective analysis was performed of all men with cancer who elected to have semen cryopreservation before the initiation of oncologic treatment at a single sperm storage facility from 2007 to The results of sperm concentration, total motility, semen volume, and sperm chromatin assay (SCSA) were analyzed. Semen parameters were assessed according to World Health Organization (WHO) guidelines 1999 (5). SCSA was performed according to Evenson et al. (6). The assay uses the metachromatic properties of the stain acridine orange (AO), a DNA probe, in combination with flow cytometry. The extent of DNA fragmentation was quantified with the use of a FACScalibur (Beckton-Dickinson) flow cytometer measuring the amount of red (single-stranded DNA) and green (double-stranded DNA) fluorescence emitted from individual sperm cells. Tris-NACl-EDT buffer diluted semen samples were exposed to acid detergent and the reaction stopped by the addition of AO. Five thousand cells were evaluated in each sample. The primary outcome measure was the mean sperm DFI. Secondary outcome measures included sperm concentration, total motility and semen volume. Sperm morphology results were not available for analysis. A subgroup analysis was performed for cancer subtype, including testicular versus nontesticular cancer. Statistical analysis was performed with the use of SPSS 20.0 software. DNA fragmentation indices and sperm concentration were subjected to a logarithmic transformation for parametric analysis and then back transformed. An independent-samples t test was performed to compare the sperm DNA fragmentation index in men with cancer and men presenting for sperm donation. For the cancer subgroups, analysis of variance (ANOVA) was performed. Scheffe test was used for post hoc comparison owing to unequal sample sizes. Statistical significance was assumed when P<.5. The study was sufficiently powered only to demonstrate a 6.0% difference in sperm DFI between the men with cancer and control groups. The study had Institutional Review Board approval. RESULTS In total, 89 men with cancer were identified in the period from 2007 to The data set was complete on all patients. The mean age in men with cancer (31.1 years) was similar to that of the control population (32.3 years); 41.6% (n ¼ 37) had a histologic diagnosis of testicular cancer, 9.0% (n ¼ 8) Hodgkin lymphoma, 4.5% (n ¼ 4) non-hodgkin lymphoma, 12.4% (n ¼ 11) other lymphoma, 7.9% (n ¼ 7) leukemia, and 24.7% (n ¼ 22) other neoplasm; 35 men were included in the control population (Table 1). Both men with cancer and control subjects were sequentially included in the study during the time period specified. The mean sperm DFI in men with cancer was 10.46% (95% confidence interval [CI] 8.68% 11.80%), and in men presenting for altruistic sperm donation 9.88% (95% CI 7.84% 12.44%). There was no significant difference in mean DFI between the two study groups: P¼.851; t ¼ with df 122 (Fig. 1). A one-way ANOVA showed that there were no significant differences in mean sperm DFI according to cancer subgroup: F(6, 117) ¼ 0.864; P¼.524 (Fig. 2). There were also no significant differences in sperm DFI between men presenting with testicular cancer and nontesticular cancer: F(2, 121) ¼ 1.306; P¼.275. Analysis of standard semen parameters showed that there was no difference in sperm concentration between men with cancer and control subjects (P¼.06). Men with testicular germ cell cancer, however, had reduced sperm concentration: F(2, 120) ¼ 9.181; P<.001. A Scheffe post hoc comparison of the mean concentration in three groups (testicular germ cell cancer, nontesticular germ cell cancer, and control population) indicated that the control population had significantly higher concentration (56.63 million/ml, 95% CI ), compared with the testicular cancer group (22.9 million/ml, 95% CI ; P¼.001). Men with nontesticular cancer also had significantly higher mean sperm concentration (52.98 million/ml) than those in the TABLE 1 Sperm DNA fragmentation index and semen parameters in men with cancer compared to men presenting for sperm donation categorized into cancer subtype. Diagnosis Patients (n) Age (y), mean (range) SCSA (DFI%), mean (CI) Volume (ml), mean (CI) Concentration (10 6 /ml), mean (CI) Total motility (% motile), mean (CI) All cancers (15 62) ( ) 3.1 ( ) ( ) ( ) Testicular germ cell cancer ( ) 8.73 ( ) 3.0 ( ) ( ) ( ) Hodgkin lymphoma 8 30 (20 44) 8.66 ( ) 3.6 ( ) ( ) ( ) Non-Hodgkin lymphoma 4 32 (29 38) 8.31 ( ) 2.2 ( ) ( ) ( ) Other lymphoma (15 48) ( ) 2.4 ( ) ( ) ( ) Leukemia 7 28 (17 37) ( ) 3.8 ( ) ( ) ( ) Other neoplasm (17 62) ( ) 3.3 ( ) ( ) ( ) Control (18 44) 9.88 ( ) 2.9 ( ) ( ) ( ) Note: CI ¼ confidence interval; DFI% ¼ DNA fragmentation index percent; SCSA ¼ sperm chromatin assay. VOL. 99 NO. 7 / JUNE

3 ORIGINAL ARTICLE: ANDROLOGY FIGURE 1 Sperm DNA fragmentation (sperm chromatin assay [SCSA]) in men with cancer compared with men presenting for sperm donation. *Outliers. testicular cancer group: 95% CI ; P¼.001. There were no significant differences between the control population and men with nontesticular cancer (P¼.954). There were no significant differences in sperm motility and seminal volume between case and control subjects and subgroups. FIGURE 2 Sperm DNA fragmentation (sperm chromatin assay [SCSA]) in men with cancer according to type of cancer and men presenting for sperm donation. TGCC ¼ testicular germ cell cancer. *Outliers VOL. 99 NO. 7 / JUNE 2013

4 Fertility and Sterility DISCUSSION This study failed to demonstrate a significant difference in sperm DFI in men presenting for semen cryopreservation after a diagnosis of cancer compared with men presenting for altruistic sperm donation. An a priori analysis of cancer subgroups did not detect a difference in sperm DNA damage, even in those with testicular cancer compared with nontesticular cancer. Many male patients with malignancy have poor sperm quality before treatment for malignancy (7).Semen parametersmay be adversely affected through an increased catabolic rate, subsequent malnutrition, and hypothalamic dysfunction. Malignancy may suppress the release of GnRH via increased production of stress hormones, which alter the balance of catecholamines and endogenous opiates (8). It is unclear whether malignancy can adversely affect the genomic integrity of the sperm and thisstudyhelpsclarifyexistingpublishedliterature. There is limited and conflicting evidence on the effect of cancer on sperm DNA fragmentation rates. Smit et al. studied a population of 127 patients diagnosed with various malignancies. Only patients with Non-Hodgkin lymphoma (n ¼ 15) had increased DNA fragmentation of 25.1% (95% CI 8.7% 66.7%). In a similar study of 121 patients, those authors found that sperm DNA fragmentation was significantly higher in cancer patients than in healthy fertile control subjects, but only in patients with testicular germ cell cancer and Hodgkin lymphoma (9). Said et al. compared 39 patients with testicular malignancy and 50 with systemic malignancy with 20 proven fertile sperm donors (10). In both groups, men with cancer had significantly higher sperm DNA fragmentation. Sperm donor SCSA values in the study by Said et al. were similar to those of the donor population in our study; however, men with testicular cancer had increased SCSA at 17.8% and men with systemic malignancy 21.0%. O'Flaherty et al. found that sperm DFI was increased in men with testicular cancer and Hodgkin lymphoma compared with fertile control subjects, but was less than that of infertile men (11). A follow-up study in 2010 after chemotherapy showed that sperm chromatin damage persisted (12). A smaller study of 37 men with cancer found a significantly higher rate of DNA damage in men with cancer compared with control subjects (n ¼ 12) (13). Mesequer et al. also found that cancer patients had higher DNA fragmentation than control subjects, with an average of 34.3% compared with 10.8% in fertile sperm donors (14). Those with testicular cancer were not statistically different from other cancer subgroups. Riberio et al. noted that 48 men with testicular cancer did not have increased sperm DNA fragmentation compared with men with proven fertility (15). Stahl et al. investigated DNA fragmentation in 74 patients with testicular cancer and 2,778 military conscripts who served as a control population (16). Interestingly, DNA fragmentation increased to 18% in those who had radiotherapy treatment and decreased to 9.1% in those who had chemotherapy. Those who had higher doses of chemotherapy had an even greater reduction in DNA fragmentation to 7.3%. In our study cohort, the only difference in standard semen parameters was reduced sperm concentration in men with testicular cancer compared with control subjects or men with nontesticular cancer. The difference in sperm concentration in all men with cancer and control subjects was not significant (P¼.06). The mean sperm concentration in men with testicular cancer (22.9 million/ml) was within normal semen parameters according to WHO guidelines. Several studies have similarly found significant reductions in sperm concentration in men with testicular cancer (17, 18). Testicular cancer may have a local effect on testicular function. Also, it has been hypothesized that a common defect is implicated in the causation of testicular cancer and hypospermatogenesis (19). The present study has several limitations. The overall number of patients in individual cancer subgroups was small and the study thus not powered to detect a small difference within cancer subgroups. Moreover, our assessment was limited by the lack of sperm morphology, limited patient demographics, and posttreatment sperm parameters. Within the limitations of our data, there appears to be limited utility of sperm DNA fragmentation testing in males with cancer. This study adds to the limited body of evidence regarding the impact of malignancy on male fertility. Future studies evaluating pregnancy rates, live birth rates, and miscarriage rates will shed further light on the long-term consequences of the impact of malignancy on a man's reproductive potential. REFERENCES 1. Stahl P, Stember D, Mulhall J. Options for fertility preservation in men and boys with cancer. Adv in Exp Med Biol 2012;732: van Castern N, Boellaaard W, Tomijn J, Dohle G. Gonadal dysfunction in male cancer patients before cytotoxic treatment. Int J Androl 2010;33: Lewis S, Simon L. Clinical implications of sperm DNA damage. Hum Fertil 2010;13: Smit M, van Casteren N, Wildhagen M, Romijn J, Dohle G. Sperm DNA integrity in cancer patients before and after cytotoxic treatment. Hum Reprod 2010;25: World Health Organization. WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press; Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22: Hendry W, Stedronska J, Jones C, Blackmore C, Barret A, Peckham M. Semen analysis in testicular cancer and Hodgkin's disease: pre- and post-treatment findings and implications for cryopreservation. Br J Urol 1983;55: Schenker J, Meirow D, Schender E. Stress and human reproduction. Eur J Obstet Gynecol Reprod Biol 1992;45: Stahl O, Eberhard J, Cavallin-Stahl E, Jepson K, Friberg B, Tingsmark C, et al. Sperm DNA integrity in cancer patients: the effect of disease and treatment. Int J Androl 2008;32: Said T, Tellez S, Evenson D, del Valle A. Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies. Andrologia 2009;41: O'Flaherty C, Vaisheva F, Chan P, Robaire B. Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy. Hum Reprod 2008;23: O'Flaherty C, Hales B, Chan P, Robaire B. Impact of chemotherapeutics and advanced testicular cancer or Hodgkin lymphoma on sperm deoxyribonucleic acid integrity. Fertil Steril 2010;94: Kobayashi H, Larson K, Sharma R, Nelson D, Evenson D, Toma H, et al. DNA damage in patients with untreated cancer as measured by the sperm chromatin structure assay. Fertil Steril 2001;75: VOL. 99 NO. 7 / JUNE

5 ORIGINAL ARTICLE: ANDROLOGY 14. Meseguer M, Santisa R, Garrido N, Fernandex J. The effect of cancer on sperm DNA fragmentation as measured by the sperm chromatin dispersion test. Fertil Steril 2008;90: Riberio T, Bertolla R, Spaine D, Fraietta R, Ortiz V, Cedenho A. Sperm nuclear apoptotic DNA fragmentation in men with testicular cancer. Fertil Steril 2008;90: Stahl O, Eberhard J, Jepson K, Spano M, Cwikiel M, Cavallin-Stahl E, et al. The impact of testicular carcinoma and its treatment on sperm DNA integrity. Cancer 2004;100: van Casteren N, van Santbrink E, van Inzen W, Romjin J, Dohle G. Use rate and assisted reproduction technologies outcome of cryopreserved semen from 629 cancer patients. Fertil Steril 2008;90: Gandini L, Lombardo F, Salacone P, Paoli D, Anselmo AP, Culasso F, et al. Testicular cancer and Hodgkin's disease: evaluation of semen quality. Hum Reprod 2003;18: Jacobsen R, Bostofte E, Engholm G, Hansen J, Olsen J, Skakkebaek N, et al. Risk of testicular cancer in men with abnormal semen characteristics: cohort study. BMJ 2000;321: VOL. 99 NO. 7 / JUNE 2013