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The Biochemical and Pathological Correlates of Successful Semen Cryopreservation From Patients With Testicular Cancer: A Single Tertiary Center Experience
Infertility The Biochemical and Pathological Correlates of Successful Semen Cryopreservation From Patients With Testicular Cancer: A Single Tertiary Center Experience Yuan-Hung Pong, Ming-Hong Kao, Yu-Chuan Lu, Ivy Chung-Yung Lee, Ju-Ton Hsieh, Vincent F. S. Tsai, and Hong-Chiang Chang OBJECTIVE MATERIALS AND METHODS
RESULTS
CONCLUSION
To determine the predictors for success with regard to semen cryopreservation and good semen quality of patients with testicular cancer. A total of 50 men (aged 16-36 years) with testicular cancer, referred for semen cryopreservation prior to gonadotoxic treatment, were included. Age, alpha fetal protein (α-FP), beta human chorionic gonadotropin, lactate dehydrogenase, clinical staging, tumor volume, and pathological reports were evaluated as correlates of successful semen cryopreservation and good semen quality. The overall success rate was 52%. α-FP (4113.1 ng/mL vs 81.2 ng/mL) and tumor volume (77.7 mL vs 25.5 mL) were significantly higher in the failure group as compared to the success group. The seminoma to nonseminomatous germ cell tumor ratio was lower in the failure group as compared to the success group (9/17 vs 3/21). There was nearly a significant difference (P = .066). The optimal cutoff value for α-FP > 1000 ng/mL showed the highest Youden index (0.689) and resulted in a sensitivity of 0.625 and specificity of 1.0 for predicting poor outcome. In terms of multivariate analysis, the α-FP (P = .013), tumor volume (P = .047), and α-FP > 1000 ng/mL (P = .010) were significantly associated with poor semen quality and failure to preserve semen. Sperm quality was found to be higher in the seminoma versus the nonseminomatous germ cell tumor patients in: sperm concentration (21.5 million/mL vs 11.8 million/mL, P < .027). Furthermore, tumor volume is correlated to α-FP (P = .018) and is weakly correlated to lactate dehydrogenase (P = .067) Elevated α-FP and tumor volume are independently poor factors for semen quality and semen cryopreservation. In clinical use, α-FP is a noninvasive tool to predict the success of semen cryopreservation and patients with α-FP > 1000 ng/mL should be informed of the higher risk of poor semen quality and semen cryopreservation concerns. UROLOGY 88: 76–80, 2016. © 2016 Elsevier Inc.
T
esticular cancer is relatively rare, accounting for approximately 1-1.5% of all cancers in men. Most of the patients were diagnosed before conception of the first offspring.1 Testicular cancers show excellent cure Yuan-Hung Pong and Ming-Hong Kao contributed equally. Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, National Taiwan University Hospital Hsin-Chu Branch, National Taiwan University, Taipei, Taiwan; the Department of Urology, TenChan Hospital, Taoyuan, Taiwan; and the Department of Urology, College of Medicine, National Taiwan University, Taipei, Taiwan Address correspondence to: Hong-Chiang Chang, M.D., Ph.D., Department of Urology, National Taiwan University Hospital Hsin-Chu Branch, National Taiwan University, Taipei, Taiwan. E-mail: [email protected] Submitted: April 14, 2015, accepted (with revisions): November 10, 2015
76
© 2016 Elsevier Inc. All rights reserved.
rates, mainly because of early diagnosis and its outstanding chemo- and radiosensitivity.2 Chemotherapy and radiotherapy have both been proven to be gonadotoxic.3,4 Therefore, semen cryopreservation is recommended for patients with testicular cancer preceding treatment. Currently, most of the related studies have reported that spermatogenesis might be already impaired at the time of testicular cancer being detected.5-9 It is also accepted that men with fertility problems were more likely to develop testicular cancer.10-12 Nevertheless, there have been no studies to identify the predictors for successful semen cryopreservation or semen quality in men with testicular cancer. The aim of the present study is to detect the predictors for the success of semen cryopreservation and semen quality. http://dx.doi.org/10.1016/j.urology.2015.11.015 0090-4295
MATERIALS AND METHODS A total of 53 men with testicular cancer were asked for semen cryopreservation before the initiation of surgical intervention, chemotherapy, or radiotherapy between July 2002 and July 2008 at the National Taiwan University Hospital. Three patients were excluded because of inability to ejaculate. A total of 50 patients were enrolled, of which 4 (8.0%) were adolescents. Specimens were collected by masturbation into a sterile bottle and letting it liquefy for 30 minutes before analysis. All patients were requested abstinence for 3-4 days before masturbation. Semen analysis was done manually according to World Health Organization laboratory manual for the examination of human semen and sperm–cervical mucus interaction 4th edition. Sperm cryopreservation was suggested only when ejaculated volume > 1.5 mL, sperm concentration > 5 × 106 sperm/mL, and sperm motility > 10%. Patients who were placed in the failure group had undergone semen analysis twice or more, in which their semen quality never met the cryopreservative criteria. The tumor volume was measured by pathologic report. All data were collected by retrospective review of charts. All patients were provided with detailed information about the purposes and procedures of semen cryopreservation. We also discussed fertility issues in patients with testicular cancer. All patients provided signed informed consent. Ethical committee approval (Institutional Review Board of the National Taiwan University Hospital) was obtained and all treatments applied were part of routine standard care.
STATISTICS Using SPSS 19, associations between categorical variables were calculated by Student t test, Mann-Whitney U test, and Fisher’s exact test. Most parameters were nonnormally distributed so analyses were performed by MannWhitney U test and Fisher’s exact test. A plausible biological and pathological relationship to the dependent outcome variable was subjected to nonconditional univariate and multivariate analysis to identify independent factors significantly associated with the success of semen cryopreservation and good semen quality. Variables with a P < .05 underwent receiving operating characteristic curve analysis to determine their sensitivity and specific-
ity for predicting poor outcome. The correlation between tumor volume and tumor markers was calculated with Pearson’s correlation test.
RESULTS Of the 50 eligible patients, 26 patients successfully preserved their sperm (success group) whereas 24 patients failed (failure group). The latter failed to preserve their semen because of poor semen quality. There were nine seminomas in the success group and three in the failure group. On the other hand, there were 17 nonseminomatous germ cell tumors (NSGCTs) in the success group and 21 in the failure group. The overall success rate was 52%. Table 1 shows the demographic data and clinical characteristics of the success and failure groups. The mean age of the success group was 25.5 years old (standard deviation [SD] = 5.6), whereas the mean age in the failure group was 25.8 years old (SD = 6.2). Alpha fetal protein (α-FP) was significantly higher in the failure group as compared with the success group (4113.1 ng/mL vs 81.2 ng/mL). Tumor volume was also significantly larger in the failure group than in the success group (77.7 mL vs 25.5 mL). The seminoma to NSGCT ratio was lower in the failure group as compared with the success group (9/17 vs 3/21). There was nearly a statistically significant difference observed (P = .066). Other parameters did not reach significant difference between both groups. The α-FP and tumor volume were independent variables that were significantly different between success and failure groups. The α-FP was evaluated by receiving operating characteristic analysis, and the area under the curve, sensitivity, and specificity were estimated. The optimal cutoff value for α-FP > 1000 ng/mL showed the highest Youden index (0.689), and resulted in a sensitivity of 0.625 and a specificity of 1.0 for predicting a poor outcome (Supplementary Fig. S1). Table 2 shows the results of multivariate analysis of seven independent variables in association with poor semen quality. There were no significant differences between both groups with respect to age, beta human chorionic gonadotropin,
Table 1. Demographic data and clinical characteristics of patients with testicular cancer Variable Age Stage I Stage II Stage III β-HCG (mIU/mL) α-FP (ng/mL) LDH (IU/L) Tumor volume (mL) Seminoma/NSGCT Sperm concentration (M/mL) Forward motility (%) Normal morphology (%)
Success Group (n = 26) Mean (SD)
Failure Group (n = 24) Mean (SD)
P Value
25.5 (5.6) 18 6 2 4509.9 (14,402) 81.2 (146.8) 696.19 (1444.1) 25.5 (37.8) 9/17 24.2 (17.6) 31.4 (19.3) 27.4 (15.8)
25.8 (6.2) 13 8 3 4933.4 (21,902) 4113.1 (7588.4) 841.3 (1292.4) 77.7 (119.6) 3/21 2.9 (5.0) 6.6 (13.0) 3.1 (2.3)
.858 .309 .545 .517 .936 .009* .739 .040* .066 .001* .001* .001*
α-FP, alpha fetal protein; β-HCG, beta human chorionic gonadotropin; LDH, lactate dehydrogenase; NSGCT, nonseminomatous germ cell tumor; SD, standard deviation. * Mann-Whitney U test, P < .05.
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Table 2. Multivariate analysis of predictors significant for poor semen quality Variable Age β-HCG (mIU/mL) α-FP (ng/mL) LDH (IU/L) Seminoma/NSGCT Tumor volume (mL) α-FP > 1000 ng/mL NSGCT
Success Group (n = 26) Mean (SD)
Failure Group (n = 24) Mean (SD)
25.5 (5.6) 4509.9 (14,402) 81.2 (146.8) 696.19 (1444.1) 9/17 25.5 (37.8)
25.8 (6.2) 4933.4 (21,902) 4113.1 (7588.4) 841.3 (1292.4) 3/21 77.7 (119.6)
Odds Ratio
P Value
1.61 (1.17-2.18) 2.76 (0.81-9.03)
.823 .731 .013* .739 .061 .047* .010* .062
Abbreviations as in Table 1. * Analyzed by multivariate logistic regression, P < .05.
Table 3. The comparison of semen quality between seminoma and NSGCT
Sperm concentration (million/mL) Forward motility (grade a + b, %)
Seminoma (n = 12) Mean (SD)
NSGCT (n = 38) Mean (SD)
P Value
21.5 (22.9) 26.1 (22.3)
11.8 (14.4) 16.7 (19.6)
.027* .206
Abbreviations as in Table 1. * Analyzed by Mann-Whitney U test, P < .05.
Table 4. The correlation of tumor volume and tumor markers Tumor Volume (ml)
α-FP (ng/mL)
β-HCG (mIU/mL)
LDH (IU/L)
Pearson’s correlation P value
0.842 .018*
−0.119 .417
0.275 .067
Abbreviations as in Table 1. * Analyzed by Pearson’s correlation test, P < .05.
lactate dehydrogenase (LDH), and NSGCT. The α-FP (P = .013), tumor volume (P = .047), and α-FP > 1000 ng/ mL (P = .010) were significantly associated with poor semen quality and failure to preserve semen. Otherwise, NSGCT is higher in the failure group than in the success group. It did not reach a significant difference but was at a nearly significant difference (P = .062). As for the correlation between semen quality and pathological report, the semen quality was found to be higher in the seminoma vs the NSGCT patients in sperm concentration (21.5 vs 11.8 million/mL, P < .027, Table 3). Furthermore, tumor volume is correlated to α-FP (P = .018) and is weakly correlated to LDH (P = .067) according to Pearson’s correlation test (Table 4 and Supplementary Fig. S2).
COMMENT Testicular cancer is relatively rare, accounting for approximately 1-1.5% of all cancers in men. Treatment efficiency has improved dramatically and the overall number of survivors has increased considerably. Most of the patients were diagnosed before conception of the first offspring.1 Therefore, semen cryopreservation is recommended for patients with testicular cancer preceding the start of treatment. 78
In this single-centered study, we found that the overall success rate of semen cryopreservation in patients with testicular cancer was 52%. This is relatively lower than other series. Bahadur et al’s evaluation of a total of 238 adolescent cancer patients showed a success rate of 86.1%.13 Menon et al reported that semen sampling from 156 adolescents had a success rate at 88.5%.14 Postovsky et al’s study showed a 65% (40 out of 62 patients) overall success rate.15 Van Casteren et al evaluated 80 boys with a median age of 16.6 years (aged 13.7-18.9 years) who received cryopreservation prior to gonadotoxic treatment; a 67% of overall success rate was noted in this study.16 Hagenas et al showed a 82.5% (71 out of 86 patients) success rate with regard to semen cryopreservation.17 One important reason for the discrepancy in successful collection is the strict cryopreservative criteria in our study than in other studies, which is collected as the “presence of live spermatozoa”. Moreover, all of the above series evaluated variable cancer patients, including those with leukemia, lymphoma, testicular cancer, and so on, who were prepared to receive gonadotoxic therapy. In the present study, we evaluated patients with only testicular cancer. It is widely accepted that the semen quality in testicular cancer patients is inferior to normal adolescents.18 Thus, we thought that these are two important reasons for the relatively lower success rate in our study. Otherwise, it has been reported that spermatogenesis might be already impaired at the time of testicular cancer detection.5-9,18 An explanation for this phenomenon is the majority of deoxyribonucleic acid (DNA) damage in testicular cancer patients. It is found that DNA fragmentation is higher in men with testicular cancer than in fertile groups.19,20 Other possible explanations are intratesticular factors (local inflammation, inflammatory markers, and local tissue trauma) and environmental factors (stress of the disease and treatment).21 UROLOGY 88, 2016
In the present study, the α-FP is thought to be a poor predictor for semen cryopreservation and semen quality. The optimal cutoff value was set at α-FP > 1000 ng/mL because of the highest Youden index (0.689). To the best of our knowledge, this is the first study to identify poor biochemical predictors. It is still unclear why α-FP influences the success rate of semen cryopreservation and the semen quality. We suppose that high α-FP represents a high tumor burden and advanced testicular cancer, which are associated with low normal testicular volume as well. Testicular volume has a direct correlation with semen profiles.22 Therefore, high tumor burden and advanced testicular cancer induced poor semen quality. The α-FP has a potential value as a noninvasive marker to predict the success of semen cryopreservation and semen quality for patients with testicular cancer. As noted in a previous series, Hagenas et al stated that testicular volume correlated with sperm concentration and percentage of motile spermatozoa.17 Reproductive hormones (including follicle-stimulating hormone [FSH], luteinizing hormone [LH], testosterone, and inhibin B) were not indicated in successful semen collection and good semen quality.17 However, there are some predictors for spermatogenic ability that covaries with pubertal development and age, including FSH, LH, inhibin B, and testosterone.23-25 As for pathological analysis, the NSGCT is higher in the failure group than in the success group. It did not reach a statistically significant difference but nearly was a poor predictor for semen cryopreservation and semen quality. The semen quality among seminoma and NSGCT remains controversial. Botchan et al showed that sperm quality was found to be superior in the seminoma to the NSGCT patients in sperm concentration, total motile sperm counts, post-thaw forward motile concentration, and motility percentage.26 Paduch et al stated that men with stage II or NSGCT have higher chance of azoospermia.11 Conversely, Hansen et al reported lower sperm counts in seminoma patients compared with those in NSGCT patients.27 Thachil et al and Fossa et al reported no significant difference between seminoma and NSGCT.28,29 Záková et al also declared that there is no significant difference of sperm count in relation to the histologic diagnosis of the cancer type.30 In the present study, sperm quality was found to be higher in the seminoma vs the NSGCT patients in sperm concentration (21.5 vs 11.8 million/mL, P < .027). However, the forward motile sperm did not reach significant difference between two groups. As for the utilization of cryopreserved semen in the groups, only 7 (14%) patients utilized their specimens for reproductive purposes. Three patients tried to have a baby via intrauterine insemination and the other four patients via intracytoplasmic sperm injection. The pregnancy rates of intrauterine insemination and intracytoplasmic sperm injection were 33.3% (1/3) and 50.0% (2/4). Take-home baby rates were also 33.3% (1/3) and 50.0% (2/4). The low rates of sperm utilization for fertility in cancer patients in this study suggest that there is a need for greater emphaUROLOGY 88, 2016
sis of assisted reproductive technique choice for male oncology patients. In our opinion, this is a new point regarding biochemical and pathological correlations of successful semen cryopreservation from patients with testicular cancer. Nevertheless, there are several limitations that should be highlighted with regard to our study. Firstly, this is a retrospective analysis and some biochemical data were missing, including reproductive hormones (FSH, LH, testosterone, and inhibin B) and semen parameters. Secondly, the small number of patients could account for the lack of significance of some of the factors analyzed. Thirdly, it is difficult to evaluate whether or not semen quality in our patients is normal or influenced by cancer. Based on our findings, we introduce a new idea to correlate the successful semen cryopreservation and semen quality from patients with testicular cancer. Further large-scale prospective studies are needed to identify other predictors.
CONCLUSION In our study, elevated α-FP and tumor volume are independently poor factors for semen quality and semen cryopreservation. In clinical use, α-FP is a noninvasive tool to predict the success of semen cryopreservation and patients with α-FP > 1000 ng/mL should be informed of the high risk of poor semen quality and semen cryopreservation concerns. Besides, the NSGCT is close to be a poor predictor for semen quality and semen cryopreservation. Acknowledgment. The authors acknowledge and appreciate the statistical experiment assistance provided by the Ten Chan Medical Group Calculating Center.
References 1. de Kretser D. Testicular cancer and infertility. BMJ. 2000;321:781782. 2. Kliesch S, Bergmann M, Hertle L, et al. Semen parameters and testicular pathology in men with testicular cancer and contralateral carcinoma in situ or bilateral testicular malignancies. Hum Reprod. 1997;12:2830-2835. 3. Howell SJ, Shalet SM. Testicular function following chemotherapy. Hum Reprod Update. 2001;7:363-369. 4. Lass A, Akagbosu F, Brinsden P. Sperm banking and assisted reproduction treatment for couples following cancer treatment of the male partner. Hum Reprod Update. 2001;7:370-377. 5. Agarwal A, Allamaneni SS. Disruption of spermatogenesis by the cancer disease process. J Natl Cancer Inst Monogr. 2005;9-12. 6. Dohle GR. Male infertility in cancer patients: review of the literature. Int J Urol. 2010;17:327-331. 7. Gandini L, Lombardo F, Salacone P, et al. Testicular cancer and Hodgkin’s disease: evaluation of semen quality. Hum Reprod. 2003;18:796801. 8. Martin RH, Chernos JE, Rademaker AW. Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm. Mol Reprod Dev. 1991;30:159-163. 9. Rives N, Perdrix A, Hennebicq S, et al. The semen quality of 1158 men with testicular cancer at the time of cryopreservation: results of the French National CECOS Network. J Androl. 2012;33:13941401.
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10. Jacobsen R, Bostofte E, Engholm G, et al. Risk of testicular cancer in men with abnormal semen characteristics: cohort study. BMJ. 2000;321:789-792. 11. Paduch DA. Testicular cancer and male infertility. Curr Opin Urol. 2006;16:419-427. 12. Raman JD, Nobert CF, Goldstein M. Increased incidence of testicular cancer in men presenting with infertility and abnormal semen analysis. Journal of Urology. 2005;174:1819-1822, discussion 1822. 13. Bahadur G, Ling KL, Hart R, et al. Semen quality and cryopreservation in adolescent cancer patients. Hum Reprod. 2002;17:3157-3161. 14. Menon S, Rives N, Mousset-Simeon N, et al. Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital. Hum Reprod. 2009;24:37-44. 15. Postovsky S, Lightman A, Aminpour D, et al. Sperm cryopreservation in adolescents with newly diagnosed cancer. Med Pediatr Oncol. 2003;40:355-359. 16. van Casteren NJ, Dohle GR, Romijn JC, et al. Semen cryopreservation in pubertal boys before gonadotoxic treatment and the role of endocrinologic evaluation in predicting sperm yield. Fertil Steril. 2008;90:1119-1125. 17. Hagenas I, Jorgensen N, Rechnitzer C, et al. Clinical and biochemical correlates of successful semen collection for cryopreservation from 12-18-year-old patients: a single-center study of 86 adolescents. Hum Reprod. 2010;25:2031-2038. 18. McDowell S, Harrison K, Kroon B, et al. Sperm DNA fragmentation in men with malignancy. Fertil Steril. 2013;99:1862-1866. 19. O’Flaherty C, Vaisheva F, Hales BF, et al. Characterization of sperm chromatin quality in testicular cancer and Hodgkin’s lymphoma patients prior to chemotherapy. Hum Reprod. 2008;23:1044-1052. 20. Said TM, Tellez S, Evenson DP, et al. Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies. Andrologia. 2009;41:377382. 21. Molnar Z, Mokanszki A, Kassai Bazsane Z, et al. Sperm concentration, hyaluronic acid-binding capacity, aneuploidy and persistent histones in testicular cancer. Hum Reprod. 2014;29:1866-1874.
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22. Arai T, Kitahara S, Horiuchi S, et al. Relationship of testicular volume to semen profiles and serum hormone concentrations in infertile Japanese males. Int J Fertil Womens Med. 1998;43:40-47. 23. Andersson AM, Juul A, Petersen JH, et al. Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metab. 1997;82:39763981. 24. Kulin HE, Frontera MA, Demers LM, et al. The onset of sperm production in pubertal boys. Relationship to gonadotropin excretion. Am J Dis Child. 1989;143:190-193. 25. Nielsen CT, Skakkebaek NE, Darling JA, et al. Longitudinal study of testosterone and luteinizing hormone (LH) in relation to spermarche, pubic hair, height and sitting height in normal boys. Acta Endocrinol Suppl (Copenh). 1986;279:98-106. 26. Botchan A, Hauser R, Yogev L, et al. Testicular cancer and spermatogenesis. Hum Reprod. 1997;12:755-758. 27. Hansen PV, Trykker H, Andersen J, et al. Germ cell function and hormonal status in patients with testicular cancer. Cancer. 1989;64:956961. 28. Fossa SD, Abyholm T, Aakvaag A. Spermatogenesis and hormonal status after orchiectomy for cancer and before supplementary treatment. Eur Urol. 1984;10:173-177. 29. Thachil JV, Jewett MA, Rider WD. The effects of cancer and cancer therapy on male fertility. J Urol. 1981;126:141-145. 30. Záková J, Lousova E, Ventruba P, et al. Sperm cryopreservation before testicular cancer treatment and its subsequent utilization for the treatment of infertility. Scientificworldjournal. 2014;2014: 575978.
APPENDIX SUPPLEMENTARY DATA
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.urology .2015.11.015.
UROLOGY 88, 2016
RESULTS
CONCLUSION
To determine the predictors for success with regard to semen cryopreservation and good semen quality of patients with testicular cancer. A total of 50 men (aged 16-36 years) with testicular cancer, referred for semen cryopreservation prior to gonadotoxic treatment, were included. Age, alpha fetal protein (α-FP), beta human chorionic gonadotropin, lactate dehydrogenase, clinical staging, tumor volume, and pathological reports were evaluated as correlates of successful semen cryopreservation and good semen quality. The overall success rate was 52%. α-FP (4113.1 ng/mL vs 81.2 ng/mL) and tumor volume (77.7 mL vs 25.5 mL) were significantly higher in the failure group as compared to the success group. The seminoma to nonseminomatous germ cell tumor ratio was lower in the failure group as compared to the success group (9/17 vs 3/21). There was nearly a significant difference (P = .066). The optimal cutoff value for α-FP > 1000 ng/mL showed the highest Youden index (0.689) and resulted in a sensitivity of 0.625 and specificity of 1.0 for predicting poor outcome. In terms of multivariate analysis, the α-FP (P = .013), tumor volume (P = .047), and α-FP > 1000 ng/mL (P = .010) were significantly associated with poor semen quality and failure to preserve semen. Sperm quality was found to be higher in the seminoma versus the nonseminomatous germ cell tumor patients in: sperm concentration (21.5 million/mL vs 11.8 million/mL, P < .027). Furthermore, tumor volume is correlated to α-FP (P = .018) and is weakly correlated to lactate dehydrogenase (P = .067) Elevated α-FP and tumor volume are independently poor factors for semen quality and semen cryopreservation. In clinical use, α-FP is a noninvasive tool to predict the success of semen cryopreservation and patients with α-FP > 1000 ng/mL should be informed of the higher risk of poor semen quality and semen cryopreservation concerns. UROLOGY 88: 76–80, 2016. © 2016 Elsevier Inc.
T
esticular cancer is relatively rare, accounting for approximately 1-1.5% of all cancers in men. Most of the patients were diagnosed before conception of the first offspring.1 Testicular cancers show excellent cure Yuan-Hung Pong and Ming-Hong Kao contributed equally. Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, National Taiwan University Hospital Hsin-Chu Branch, National Taiwan University, Taipei, Taiwan; the Department of Urology, TenChan Hospital, Taoyuan, Taiwan; and the Department of Urology, College of Medicine, National Taiwan University, Taipei, Taiwan Address correspondence to: Hong-Chiang Chang, M.D., Ph.D., Department of Urology, National Taiwan University Hospital Hsin-Chu Branch, National Taiwan University, Taipei, Taiwan. E-mail: [email protected] Submitted: April 14, 2015, accepted (with revisions): November 10, 2015
76
© 2016 Elsevier Inc. All rights reserved.
rates, mainly because of early diagnosis and its outstanding chemo- and radiosensitivity.2 Chemotherapy and radiotherapy have both been proven to be gonadotoxic.3,4 Therefore, semen cryopreservation is recommended for patients with testicular cancer preceding treatment. Currently, most of the related studies have reported that spermatogenesis might be already impaired at the time of testicular cancer being detected.5-9 It is also accepted that men with fertility problems were more likely to develop testicular cancer.10-12 Nevertheless, there have been no studies to identify the predictors for successful semen cryopreservation or semen quality in men with testicular cancer. The aim of the present study is to detect the predictors for the success of semen cryopreservation and semen quality. http://dx.doi.org/10.1016/j.urology.2015.11.015 0090-4295
MATERIALS AND METHODS A total of 53 men with testicular cancer were asked for semen cryopreservation before the initiation of surgical intervention, chemotherapy, or radiotherapy between July 2002 and July 2008 at the National Taiwan University Hospital. Three patients were excluded because of inability to ejaculate. A total of 50 patients were enrolled, of which 4 (8.0%) were adolescents. Specimens were collected by masturbation into a sterile bottle and letting it liquefy for 30 minutes before analysis. All patients were requested abstinence for 3-4 days before masturbation. Semen analysis was done manually according to World Health Organization laboratory manual for the examination of human semen and sperm–cervical mucus interaction 4th edition. Sperm cryopreservation was suggested only when ejaculated volume > 1.5 mL, sperm concentration > 5 × 106 sperm/mL, and sperm motility > 10%. Patients who were placed in the failure group had undergone semen analysis twice or more, in which their semen quality never met the cryopreservative criteria. The tumor volume was measured by pathologic report. All data were collected by retrospective review of charts. All patients were provided with detailed information about the purposes and procedures of semen cryopreservation. We also discussed fertility issues in patients with testicular cancer. All patients provided signed informed consent. Ethical committee approval (Institutional Review Board of the National Taiwan University Hospital) was obtained and all treatments applied were part of routine standard care.
STATISTICS Using SPSS 19, associations between categorical variables were calculated by Student t test, Mann-Whitney U test, and Fisher’s exact test. Most parameters were nonnormally distributed so analyses were performed by MannWhitney U test and Fisher’s exact test. A plausible biological and pathological relationship to the dependent outcome variable was subjected to nonconditional univariate and multivariate analysis to identify independent factors significantly associated with the success of semen cryopreservation and good semen quality. Variables with a P < .05 underwent receiving operating characteristic curve analysis to determine their sensitivity and specific-
ity for predicting poor outcome. The correlation between tumor volume and tumor markers was calculated with Pearson’s correlation test.
RESULTS Of the 50 eligible patients, 26 patients successfully preserved their sperm (success group) whereas 24 patients failed (failure group). The latter failed to preserve their semen because of poor semen quality. There were nine seminomas in the success group and three in the failure group. On the other hand, there were 17 nonseminomatous germ cell tumors (NSGCTs) in the success group and 21 in the failure group. The overall success rate was 52%. Table 1 shows the demographic data and clinical characteristics of the success and failure groups. The mean age of the success group was 25.5 years old (standard deviation [SD] = 5.6), whereas the mean age in the failure group was 25.8 years old (SD = 6.2). Alpha fetal protein (α-FP) was significantly higher in the failure group as compared with the success group (4113.1 ng/mL vs 81.2 ng/mL). Tumor volume was also significantly larger in the failure group than in the success group (77.7 mL vs 25.5 mL). The seminoma to NSGCT ratio was lower in the failure group as compared with the success group (9/17 vs 3/21). There was nearly a statistically significant difference observed (P = .066). Other parameters did not reach significant difference between both groups. The α-FP and tumor volume were independent variables that were significantly different between success and failure groups. The α-FP was evaluated by receiving operating characteristic analysis, and the area under the curve, sensitivity, and specificity were estimated. The optimal cutoff value for α-FP > 1000 ng/mL showed the highest Youden index (0.689), and resulted in a sensitivity of 0.625 and a specificity of 1.0 for predicting a poor outcome (Supplementary Fig. S1). Table 2 shows the results of multivariate analysis of seven independent variables in association with poor semen quality. There were no significant differences between both groups with respect to age, beta human chorionic gonadotropin,
Table 1. Demographic data and clinical characteristics of patients with testicular cancer Variable Age Stage I Stage II Stage III β-HCG (mIU/mL) α-FP (ng/mL) LDH (IU/L) Tumor volume (mL) Seminoma/NSGCT Sperm concentration (M/mL) Forward motility (%) Normal morphology (%)
Success Group (n = 26) Mean (SD)
Failure Group (n = 24) Mean (SD)
P Value
25.5 (5.6) 18 6 2 4509.9 (14,402) 81.2 (146.8) 696.19 (1444.1) 25.5 (37.8) 9/17 24.2 (17.6) 31.4 (19.3) 27.4 (15.8)
25.8 (6.2) 13 8 3 4933.4 (21,902) 4113.1 (7588.4) 841.3 (1292.4) 77.7 (119.6) 3/21 2.9 (5.0) 6.6 (13.0) 3.1 (2.3)
.858 .309 .545 .517 .936 .009* .739 .040* .066 .001* .001* .001*
α-FP, alpha fetal protein; β-HCG, beta human chorionic gonadotropin; LDH, lactate dehydrogenase; NSGCT, nonseminomatous germ cell tumor; SD, standard deviation. * Mann-Whitney U test, P < .05.
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Table 2. Multivariate analysis of predictors significant for poor semen quality Variable Age β-HCG (mIU/mL) α-FP (ng/mL) LDH (IU/L) Seminoma/NSGCT Tumor volume (mL) α-FP > 1000 ng/mL NSGCT
Success Group (n = 26) Mean (SD)
Failure Group (n = 24) Mean (SD)
25.5 (5.6) 4509.9 (14,402) 81.2 (146.8) 696.19 (1444.1) 9/17 25.5 (37.8)
25.8 (6.2) 4933.4 (21,902) 4113.1 (7588.4) 841.3 (1292.4) 3/21 77.7 (119.6)
Odds Ratio
P Value
1.61 (1.17-2.18) 2.76 (0.81-9.03)
.823 .731 .013* .739 .061 .047* .010* .062
Abbreviations as in Table 1. * Analyzed by multivariate logistic regression, P < .05.
Table 3. The comparison of semen quality between seminoma and NSGCT
Sperm concentration (million/mL) Forward motility (grade a + b, %)
Seminoma (n = 12) Mean (SD)
NSGCT (n = 38) Mean (SD)
P Value
21.5 (22.9) 26.1 (22.3)
11.8 (14.4) 16.7 (19.6)
.027* .206
Abbreviations as in Table 1. * Analyzed by Mann-Whitney U test, P < .05.
Table 4. The correlation of tumor volume and tumor markers Tumor Volume (ml)
α-FP (ng/mL)
β-HCG (mIU/mL)
LDH (IU/L)
Pearson’s correlation P value
0.842 .018*
−0.119 .417
0.275 .067
Abbreviations as in Table 1. * Analyzed by Pearson’s correlation test, P < .05.
lactate dehydrogenase (LDH), and NSGCT. The α-FP (P = .013), tumor volume (P = .047), and α-FP > 1000 ng/ mL (P = .010) were significantly associated with poor semen quality and failure to preserve semen. Otherwise, NSGCT is higher in the failure group than in the success group. It did not reach a significant difference but was at a nearly significant difference (P = .062). As for the correlation between semen quality and pathological report, the semen quality was found to be higher in the seminoma vs the NSGCT patients in sperm concentration (21.5 vs 11.8 million/mL, P < .027, Table 3). Furthermore, tumor volume is correlated to α-FP (P = .018) and is weakly correlated to LDH (P = .067) according to Pearson’s correlation test (Table 4 and Supplementary Fig. S2).
COMMENT Testicular cancer is relatively rare, accounting for approximately 1-1.5% of all cancers in men. Treatment efficiency has improved dramatically and the overall number of survivors has increased considerably. Most of the patients were diagnosed before conception of the first offspring.1 Therefore, semen cryopreservation is recommended for patients with testicular cancer preceding the start of treatment. 78
In this single-centered study, we found that the overall success rate of semen cryopreservation in patients with testicular cancer was 52%. This is relatively lower than other series. Bahadur et al’s evaluation of a total of 238 adolescent cancer patients showed a success rate of 86.1%.13 Menon et al reported that semen sampling from 156 adolescents had a success rate at 88.5%.14 Postovsky et al’s study showed a 65% (40 out of 62 patients) overall success rate.15 Van Casteren et al evaluated 80 boys with a median age of 16.6 years (aged 13.7-18.9 years) who received cryopreservation prior to gonadotoxic treatment; a 67% of overall success rate was noted in this study.16 Hagenas et al showed a 82.5% (71 out of 86 patients) success rate with regard to semen cryopreservation.17 One important reason for the discrepancy in successful collection is the strict cryopreservative criteria in our study than in other studies, which is collected as the “presence of live spermatozoa”. Moreover, all of the above series evaluated variable cancer patients, including those with leukemia, lymphoma, testicular cancer, and so on, who were prepared to receive gonadotoxic therapy. In the present study, we evaluated patients with only testicular cancer. It is widely accepted that the semen quality in testicular cancer patients is inferior to normal adolescents.18 Thus, we thought that these are two important reasons for the relatively lower success rate in our study. Otherwise, it has been reported that spermatogenesis might be already impaired at the time of testicular cancer detection.5-9,18 An explanation for this phenomenon is the majority of deoxyribonucleic acid (DNA) damage in testicular cancer patients. It is found that DNA fragmentation is higher in men with testicular cancer than in fertile groups.19,20 Other possible explanations are intratesticular factors (local inflammation, inflammatory markers, and local tissue trauma) and environmental factors (stress of the disease and treatment).21 UROLOGY 88, 2016
In the present study, the α-FP is thought to be a poor predictor for semen cryopreservation and semen quality. The optimal cutoff value was set at α-FP > 1000 ng/mL because of the highest Youden index (0.689). To the best of our knowledge, this is the first study to identify poor biochemical predictors. It is still unclear why α-FP influences the success rate of semen cryopreservation and the semen quality. We suppose that high α-FP represents a high tumor burden and advanced testicular cancer, which are associated with low normal testicular volume as well. Testicular volume has a direct correlation with semen profiles.22 Therefore, high tumor burden and advanced testicular cancer induced poor semen quality. The α-FP has a potential value as a noninvasive marker to predict the success of semen cryopreservation and semen quality for patients with testicular cancer. As noted in a previous series, Hagenas et al stated that testicular volume correlated with sperm concentration and percentage of motile spermatozoa.17 Reproductive hormones (including follicle-stimulating hormone [FSH], luteinizing hormone [LH], testosterone, and inhibin B) were not indicated in successful semen collection and good semen quality.17 However, there are some predictors for spermatogenic ability that covaries with pubertal development and age, including FSH, LH, inhibin B, and testosterone.23-25 As for pathological analysis, the NSGCT is higher in the failure group than in the success group. It did not reach a statistically significant difference but nearly was a poor predictor for semen cryopreservation and semen quality. The semen quality among seminoma and NSGCT remains controversial. Botchan et al showed that sperm quality was found to be superior in the seminoma to the NSGCT patients in sperm concentration, total motile sperm counts, post-thaw forward motile concentration, and motility percentage.26 Paduch et al stated that men with stage II or NSGCT have higher chance of azoospermia.11 Conversely, Hansen et al reported lower sperm counts in seminoma patients compared with those in NSGCT patients.27 Thachil et al and Fossa et al reported no significant difference between seminoma and NSGCT.28,29 Záková et al also declared that there is no significant difference of sperm count in relation to the histologic diagnosis of the cancer type.30 In the present study, sperm quality was found to be higher in the seminoma vs the NSGCT patients in sperm concentration (21.5 vs 11.8 million/mL, P < .027). However, the forward motile sperm did not reach significant difference between two groups. As for the utilization of cryopreserved semen in the groups, only 7 (14%) patients utilized their specimens for reproductive purposes. Three patients tried to have a baby via intrauterine insemination and the other four patients via intracytoplasmic sperm injection. The pregnancy rates of intrauterine insemination and intracytoplasmic sperm injection were 33.3% (1/3) and 50.0% (2/4). Take-home baby rates were also 33.3% (1/3) and 50.0% (2/4). The low rates of sperm utilization for fertility in cancer patients in this study suggest that there is a need for greater emphaUROLOGY 88, 2016
sis of assisted reproductive technique choice for male oncology patients. In our opinion, this is a new point regarding biochemical and pathological correlations of successful semen cryopreservation from patients with testicular cancer. Nevertheless, there are several limitations that should be highlighted with regard to our study. Firstly, this is a retrospective analysis and some biochemical data were missing, including reproductive hormones (FSH, LH, testosterone, and inhibin B) and semen parameters. Secondly, the small number of patients could account for the lack of significance of some of the factors analyzed. Thirdly, it is difficult to evaluate whether or not semen quality in our patients is normal or influenced by cancer. Based on our findings, we introduce a new idea to correlate the successful semen cryopreservation and semen quality from patients with testicular cancer. Further large-scale prospective studies are needed to identify other predictors.
CONCLUSION In our study, elevated α-FP and tumor volume are independently poor factors for semen quality and semen cryopreservation. In clinical use, α-FP is a noninvasive tool to predict the success of semen cryopreservation and patients with α-FP > 1000 ng/mL should be informed of the high risk of poor semen quality and semen cryopreservation concerns. Besides, the NSGCT is close to be a poor predictor for semen quality and semen cryopreservation. Acknowledgment. The authors acknowledge and appreciate the statistical experiment assistance provided by the Ten Chan Medical Group Calculating Center.
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APPENDIX SUPPLEMENTARY DATA
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.urology .2015.11.015.
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