Seminal Plasma Survivin in Fertile and Infertile Males Nagwa Roshdy and Taymour Mostafa* From the Medical Biochemistry and Andrology and Sexology (TM) Departments, Faculty of Medicine, Cairo University, Cairo, Egypt
Purpose: We assessed survivin (an inhibitor of apoptosis) protein in the seminal plasma of infertile and fertile males. Materials and Methods: Seminal plasma survivin was estimated by enzymelinked immunosorbent assay in 23 healthy fertile volunteers, 22 men with oligo-asthenozoospermia, 37 with nonobstructive azoospermia and 12 with obstructive azoospermia. Histopathology and testicular sperm extraction were done in testicular tissue biopsies from obstructive azoospermia and nonobstructive azoospermia cases. Results: Mean seminal survivin was highest in fertile controls, less in oligoasthenozoospermic cases and low in nonobstructive azoospermia cases with significant differences. In obstructive azoospermia cases seminal plasma survivin was absent. Seminal survivin positively correlated with sperm concentration and total sperm motility, and negatively correlated with the percent of sperm abnormal forms. Seminal survivin was detectable in nonobstructive azoospermia cases in which testicular sperm extraction was successful but absent in such cases when testicular sperm extraction was unsuccessful. Conclusions: Seminal survivin is testicular in origin. It is related to spermatogenesis and sperm motility processes. Seminal survivin was related to successful testicular sperm extraction in nonobstructive azoospermia cases.
Abbreviations and Acronyms NOA ⫽ nonobstructive azoospermia OA ⫽ obstructive azoospermia TESE ⫽ testicular sperm extraction Submitted for publication June 5, 2008. Study received institutional review board approval. * Correspondence: Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Cairo 11562, Egypt (telephone: ⫹2 0105150297; e-mail:
[email protected]).
Key Words: testis; sperm; infertility, male; BIRC5 protein, human NORMAL germ cell production depends on a precise balance of proliferation and apoptotic cell death. Molecular mechanisms underlying the regulation of germ cell homeostasis in the testis are partly understood. Genes involved in growth and proliferation as well as pro-apoptotic and anti-apoptotic factors seem to have important roles in this context. Impaired proliferative capacity and dysregulated apoptosis can contribute to human spermatogenic disorders.1 Genetic alterations, such as impaired anti-apoptotic gene expression, are thought to be involved in idiopathic fertility defects.2 Of the mammalian apoptosis regulators the apoptosis inhibiting protein
family is just beginning to be elucidated for its role in spermatogenic function. Survivin is an apoptosis inhibiting protein that regulates apoptosis at cell division. Survivin has been studied extensively in cancer cells but it has only recently received serious attention in male germ cells. In the rodent testis survivin expression has been localized to germ cells, especially mature spermatocytes.3 Relatively high survivin expression has been noted in normal human spermatogenesis and its downregulation is associated with spermatogenic failure.4 Taken together these studies suggest that survivin is a candidate regulatory gene in male germ cell production.
0022-5347/09/1813-1269/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 181, 1269-1272, March 2009 Printed in U.S.A. DOI:10.1016/j.juro.2008.10.158
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Late evidence of survivin involvement in the meiotic division of male rodent germ cells3,5 led to considering whether comparable expression patterns of these genes could be found at the mRNA level in human testes with varying spermatogenic function. We assessed survivin in the seminal plasma of infertile and fertile males.
MATERIALS AND METHODS This study included 71 white male patients 25 to 38 years old who were consecutively recruited at the University Hospital andrology department, in addition to healthy volunteers as controls, after receiving institutional review board approval. All men had been married more than 2 years. They were divided into group 1—23 healthy fertile volunteers as controls, group 2—22 with oligo-asthenozoospermia, group 3—37 with NOA and group 4 —12 with OA. Study exclusion criteria were trauma, torsion, radiation, gonadotoxin, abnormal karyotyping or immunological factors. General and genital examinations were done to exclude hypogonadism, cryptorchidism and congenital anomalies. High resolution color flow duplex Doppler ultrasonography was done using an 128XB device (Acuson, Golden, Colorado) to exclude varicocele or another scrotal cause. Serum follicle-stimulating hormone (reference range 1.5 to 14 mIU/ml) was assessed by radioimmunoassay in NOA cases. More than 1 semen sample was collected by masturbation after sexual abstinence for 4 days and samples were examined according to WHO guidelines6 by the computer assisted AutoSperm method (MedCalc, Mariakerke, Belgium). Sperm morphology was evaluated by phase contrast microscopy of the native sample. In addition, airdried smears were prepared and fixed in equal parts of ethanol and ether. Staining was performed using the simplified Papanicolaou stain. Samples with a white blood count of greater than 1 million per ml or positive bacterial culture were excluded from analysis. To estimate seminal plasma survivin7 after semen liquefaction cell-free seminal plasma was obtained after ini-
tial centrifugation at 1,000 ⫻ gravity for 10 minutes, followed by supernatant centrifugation at 4,000 ⫻ gravity for 20 minutes to remove debris. Samples were then stored frozen at ⫺20C. Seminal plasma survivin was estimated by Quantikine™ human survivin immunoassay using enzyme-linked immunosorbent assay with a sensitivity of 4 pg/ml (range 31.25 to 1,000) and less than 0.1% crossreactivity with human MEK-1, pJNK, p300, granzyme B, caspase-3 or caspase-9. The intra-assay and interassay precision percent coefficient of variation in low, medium and high samples was 7.2%, 2.8% and 7.4%, and 12%, 13.5% and 11.7%, respectively. Biopsy material was processed8 using a small testicular tissue sample obtained through a small incision in the tunica albuginea, which was divided into 2 portions. The first portion was subjected fresh to a TESE trial. The second portion was placed in Bouin’s fixative (a saturated aqueous solution of 75 ml picric acid-formalin and about a 40% aqueous solution of 25 ml formaldehyde and 5 ml glacial acetic acid) for histopathological interpretation.9 For statistical analysis data are shown as the median, range and mean ⫾ SD. The nonparametric Mann-Whitney U test was applied for comparison between groups that were not normally distributed and the Spearman rank correlation was used for correlations with significance considered at p ⬍0.05.
RESULTS The table lists mean data on the study groups, including patient age, sperm concentration, sperm motility, the percent of sperm normal forms and seminal plasma survivin. The mean history of infertility in infertile cases was 3.8 ⫾ 2.8 years (range 2 to 15). In NOA cases serum follicle-stimulating hormone was 5.2 to 62 mIU/ml. Fertile controls had the highest mean seminal plasma survivin compared with all other groups with statistically significant differences. Oligo-asthenozoospermic cases showed significantly higher mean seminal plasma survivin content than the 2 azoospermic groups. Mean seminal
Study group data Controls No. pts Mean ⫾ SD age Sperm count (106/ml): Median (range) Mean ⫾ SD % Sperm motility: Median (range) Mean ⫾ SD % Sperm abnormal forms: Median (range) Mean ⫾ SD Seminal survivin (pg/ml): Median (range) Mean ⫾ SD
23 29.66 ⫾
4.55
Oligo-Asthenozoospermia
NOA
OA
22 29.7 ⫾ 3.62
37 28.88 ⫾ 3.88
12 30.56 ⫾ 4.2
90 (38–140) 87.5 ⫾ 39.8
9 (3–18) 10.5 ⫾ 5.9*
0 0†
0 0†
60 (50–70) 59.0 ⫾ 5.6
15 (10–20) 15.0 ⫾ 5.1*
0 0†
0 0†
20 (15–25) 22.0 ⫾ 6.7
50 (40–80) 54.5 ⫾ 9.6*
0 0
0 0†
800 (200–1,200) 729.1 ⫾ 318.5
55 (20–180) 85.6 ⫾ 56.3*
5.0 (4.0–6.0) 5.0 ⫾ 0.85†
0 0‡
* Signficant difference vs control. † Signficant difference vs control and oligo-asthenozoospermia. ‡ Signficant difference vs control, oligo-asthenozoospermia and NOA.
SEMINAL PLASMA SURVIVIN IN FERTILE AND INFERTILE MALES
survivin in NOA cases was significantly higher than that in OA cases. Seminal survivin was absent in OA cases. There was significant positive correlation of seminal plasma survivin with sperm concentration (r ⫽ 0.989, p ⫽ 0.0001) and the percent of total sperm motility (r ⫽ 0.877, p ⫽ 0.0001), and a significant negative correlation with the percent of sperm abnormal forms (r ⫽ ⫺0.516, p ⫽ 0.002, see figure). The 37 NOA cases showed certain histopathological patterns, including hypospermatogenesis in 6, severe hypospermatogenesis in 11, germ cell arrest in 6, a mixed pattern in 10 and tubular hyalinization in 4. There were successful TESE trials in 4 of 10 mixed pattern cases, 3 of 6 germ cell arrest cases, all 6 hypospermatogenesis cases and all 11 severe hypospermatogenesis cases. Seminal survivin was detected in semen specimens in NOA cases with successful TESE but it was absent in NOA cases with unsuccessful TESE.
DISCUSSION The current study demonstrates that mean seminal survivin was highest in fertile controls, less in oligoasthenozoospermic cases and low in NOA cases with significant differences. Seminal survivin was absent in OA cases, indicating its testicular origin. These relations denote that seminal concentrations are associated with a sound spermatogenesis process as well as with its yield. The last point was supported by eliciting a significant positive correlation of seminal plasma survivin with sperm concentration, the percent of total sperm motility and the percent of sperm normal forms. To our knowledge survivin has not been previously estimated in seminal plasma. Spaulding et al reported that most adult tissues do not express survivin and when it is present it is
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largely restricted to a small subset of epithelial cells and cells with proliferative potential, such as those of the thymus.10 They noted that the testis is the only human adult tissue that highly expresses survivin with 60% to 70% positivity in the nuclei of spermatogonia. Weikert et al reported that survivin expression seems to be restricted to specimens containing meiotically dividing or haploid germ cells.4,5,11–13 These findings are in line with results in rodents demonstrating a preponderance of survivin protein and mRNA expression in meiotically dividing spermatocytes.3,14 Taken together these series raised the question of survivin involvement in the meiotic progression of germ cells. On the other hand, these studies could not exclude survivin expression by other cell types in the normal testis, including spermatogonia or even Sertoli’s cells. Weikert et al speculated that reverse transcriptase-polymerase chain reaction of survivin mRNA may lack the sensitivity to detect its low expression in pre-meiotic germ cells, such as spermatogonia or even Sertoli’s cells.5 Indeed, low survivin levels may be expressed in Leydig cells in the rodent testis.7 Since its introduction in 1993, intracytoplasmic sperm injection with testicular sperm has become a practical procedure in patients with azoospermia. Therefore, in men with NOA retrieving spermatozoa from the testes for assisted reproduction using intracytoplasmic sperm injection offers an opportunity for fertility despite limited sperm production.15 In the current study the relationship between seminal survivin and successful TESE trials in NOA cases was assessed as a step forward. Seminal survivin was shown to be related to TESE results, ie the presence of spermatogenic focus in testicular tissue irrespective of its histopathology, which is to our
A, sperm count and seminal plasma survivin. B, total sperm motility and seminal plasma survivin
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knowledge a point that has not been researched previously. This point should be investigated further in larger scale trials supplemented with polymerase chain reaction as well as immunohistochemistry to correlate survivin expression with cell type and morphology. Lin et al observed that low or no expression of anti-apoptotic genes such as survivin might have a role in the pathogenesis of spermatogenic disorders since increased germ cell apoptosis was noted in conjunction with impaired human spermatogenesis.1 Survivin is known to directly or indirectly bind and inhibit the terminal effector cell death protease cascades caspase 3 and 7 as well as inhibit the activation of caspase 9, which is the initiator in the
mitochondrial pathway for apoptosis.16 Determining whether altered survivin expression is a causative factor in spermatogenic disorders would shed a light on germ cells being intimately linked with survivin to inhibit apoptosis and prolong the cellular life span.
CONCLUSIONS We report that survivin in human seminal plasma positively correlates with sperm concentration, sperm motility and sperm normal forms. Also, seminal survivin was noted to be related to successful TESE in NOA cases.
REFERENCES 1. Lin WW, Lamb DJ, Wheeler TM, Lipshultz LI and Kim ED: In situ end-labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis. Fertil Steril 1997; 68: 1065. 2. Zhang XD, Yin LL, Zheng Y, Lu L, Zhou ZM and Sha JH: Expression of a novel beta adaptin subunit mRNA splice variant in human testes. Asian J Androl 2005; 7: 179. 3. Wang Y, Suominen JS, Hakovirta H, Parvinen M, Martinand-Mari C, Toppari J et al: Survivin expression in rat testis is upregulated by stem-cell factor. Mol Cell Endocrinol 2004; 218: 165. 4. Weikert S, Schrader M, Muller M, Krause H and Miller K: Expression of the apoptosis inhibitor survivin in testicular tissue of infertile patients. Int J Androl 2004; 27: 161. 5. Weikert S, Christoph F, Schulze W, Krause H, Kempkensteffen C, Schostak M et al: Testicular expression of survivin and human telomerase reverse transcriptase (hTERT) associated with spermatogenic function in infertile patients. Asian J Androl 2006; 8: 95. 6. World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-
Cervical Mucus Interaction, 4th ed. New York: Cambridge University Press 1999. 7. Zaffaroni N, Pennati M and Daidone MG: Survivin as a target for new anticancer interventions. J Cell Mol Med 2005; 9: 630. 8. Schulze W, Thoms F and Knuth UA: Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men. Hum Reprod, suppl., 1999; 14: 82. 9. McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM and Skakkebaek NE: Histological evaluation of the human testis—approaches to optimizing the clinical value of the assessment: mini review. Hum Reprod 2007; 22: 2. 10. Spaulding B, Pan D, Ghadersohi A, Nielsen G, Jensen S, Gellert F et al: Characterization of the 12C4 survivin monoclonal antibody and insight into the expression of survivin in human adult tissues. Histopathology 2006; 49: 622. 11. Weikert S, Schrader M, Christoph F, Schulze W, Krause H, Müller M et al: Quantification of survivin mRNA in testes of infertile patients and in testicular germ cell tumours: high levels of expression asso-
ciated with normal spermatogenesis. Int J Androl 2005; 28: 224. 12. Weikert S, Schrader M, Krause H, Schulze W, Müller M and Miller K: The inhibitor of apoptosis (IAP) survivin is expressed in human testicular germ cell tumors and normal testes. Cancer Lett 2005; 223: 331. 13. Weikert S, Schrader M, Müller M, Schulze W, Krause H and Miller K: Expression levels of the inhibitor of apoptosis survivin in testes of patients with normal spermatogenesis and spermatogenic failure. Fertil Steril, suppl., 2005; 83: 1100. 14. Print CG and Loveland KL: Germ cell suicide: new insights into apoptosis during spermatogenesis. Bioessays 2000; 22: 423. 15. Pasqualotto FF, Rossi-Ferragut LM, Rocha CC, Iaconelli A Jr and Borges E Jr: Outcome of in vitro fertilization and intracytoplasmic injection of epididymal and testicular sperm obtained from patients with obstructive and nonobstructive azoospermia. J Urol 2002; 167: 1753. 16. Song Z, Yao X and Wu M: Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis. J Biol Chem 2003; 278: 23130.
