RECENT ADVANCES OF THE SEMEN ANTIOXIDATIVE RESERVE AND SPERM PROTEOMICS EVALUATIONS TO ASSESS MALE REPRODUCTIVE POTENTIAL
Semir A. S. Al Samarrai
Approximately 10% of couples are infertile with male factors contributing to as many as 50% of these cases (1). Men with abnormal semen parameters frequently achieve spontaneous pregnancies, and only half of infertile men have a recognizable cause detected by conventional semen analysis (2).
Over the past 50 years, semen analysis has represented the cornerstone of the laboratory evaluation for male infertility with little substantive change over time, serving as surrogate measure of male reproductive potential. Although this semen analysis test has profound influence of male infertility, its use is plagued by significant limitations.
The limited ability of this important tool to predict productivity risks misclassifying a subject’s true fertility status and compromises its value as a basic screening test.
Fortunately, the advent of more sophisticated assays including measurements of seminal oxidative stress, antioxidant capacity, and sperm deoxyribonucleic acid (DNA) fragmentation rate, as well as recent advances in the field of sperm proteomics has provided promising alternative tests to better assess male reproductive potential.
A growing body of evidence suggests that excessive production of reactive oxygen species (ROS) can overwhelm the total antioxidant reserve in semen leading to abnormal semen parameters and DNA damage in the sperm nuclear and mitochondrial genomes (2, 3). When used in conjunction with conventional semen analysis, measurements of oxidative stress can help. First is to distinguish between fertile and infertile men, second to identify subfertile men who are most likely to conceive over time. Third, to identify the subgroup of infertile men who may benefit the most from antioxidant supplementation (2). Physiological levels of ROS are of paramount importance for containing sperm functions (e.g. capacitation); however, excessive ROS pose a threat to sperm plasma membrane integrity (4). For this reason, spermatozoa and seminal plasma contain a variety of enzymatic and nonenzymatic antioxidant systems capable of counteracting the negative effects of ROS (5, 6). However, a number of lifestyle (e.g. smoking), environmental (e.g. pesticides, air pollution, electromagnetic radiation), and health (e.g. chemotherapy exposure, urogenital tract infections, neutrophil or macrophage infiltration) factors significantly raise production of ROS, overwhelming the total antioxidant capacity (TAC) of sperm (5, 7). This imbalance termed oxidative stress, disrupts sperm plasma membrane fluidity, impairs sperm motility, and interferes with membrane fusion events (5).
Multiple assays for measuring seminal ROS levels have been developed, however, the chemiluminescence method is the most common technique used to measure the ROS generated by spermatozoa (2). The ROS level for men with normal semen parameters is 1.5 x 104 cpm/20 million sperm/mL, and infertile men may be classified as being oxidative stress positive if they exhibit a ROS level higher than this level (2). The seminal TAC, representing the total antioxidant protection in seminal plasma, may also be measured using chemiluminescence assays that quantify the ability of seminal antioxidants to block oxidation of specific reagents (8).
There is growing body of evidence that infertile men possess lower levels of individual antioxidants, a reduced TAC, and increased ROS levels relative to fertile men (13). Elevated ROS levels are detected in the semen of 25%-40% of infertile men (4), and infertile men have been shown to have TAC levels that are 30-43% lower than those measured in fertile controls (9).
Thus, infertile men as a whole are prone to imbalance between the harmful effects of excess ROS and the protective functions of seminal antioxidants (4). When the generation of ROS overwhelms the ROS scavenging system, the resulting oxidation stress compromises sperm function and viability and leads to sperm DNA fragmentation.
Men who exhibit excessive ROS demonstrate higher rate of sperm abnormal morphology and head defects (10). Conversely, TAC level has been positively associated with each of the spermiogram parameters, including sperm concentration, motility, and morphology (9). Oxidative stress also produces DNA strand breaks (14); infact, every 25% increase in seminal ROS levels is associated with a 10% increase in sperm DNA fragmentation rate (8).
Cumulatively, these structural, functional, and DNA defects impair sperm fertilization potential. Not surprisingly, men with elevated ROS have been shown to be less likely to achieve spontaneous pregnancy. Moreover infertile men with favorable ROS-TAC scores are more likely to ultimately initiate successful pregnancy then infertile men with unfavorable scores (4).
The aforementioned relationship between ROS levels, TAC, and sperm fertilization potential has formed basis for antioxidant therapy of infertile men, the goal being to restore the balance between ROS-generating and antioxidant defense systems in patients. A number of studies have evaluated the efficacy of various oral antioxidants (e.g. vitamins C and E, Zinc, Selenium, Folate and Carnitine) on sperm quality, pregnancy rate, and live birth rate in infertile men (11). (Figure 1 & 2 )
In fact, it has been reported that men taking oral antioxidants have more than 4 times the pregnancy rate and an almost 5-fold higher live birth rate when undergoing assisted reproductive technique (ART) (12).
DNA Fragmentation Rate:
Sperm chromatin integrity is vital for both transmission of paternal genetic information and proper embryo development. Spermatozoal chromatin is highly compacted by nuclear proteins called protamines, and this high degree of chromatin condensation protects the DNA from stress and breakage (2). Despite this protection, sperm DNA damage can still occur in the form of single or double-stranded DNA breaks, inter or intra-strand cross linkage, or base deletions or modifications (3).
A number of different factors have been shown to produce these DNA effects, including tobacco use, chemotherapy, malignancy, leukospermia and high measured levels of oxidate stress (2).
Many tests of sperm DNA damage are available, including both direct measures of DNA fragmentation and indirect tests of sperm chromatin compaction (2).
Clinically, high rates of DNA fragmentation are negatively correlated with bulk semen parameters and have been associated with several infertility phenotypes:
- Longer time for natural conception
- Idiopathic infertility
- Recurrent intrauterine insemination (IUI) and in vitro fertilization (IVF) failure (7)
- Spontaneous miscarriage (3)
A DNA fragmentation index (DFI) cut off 19.25% can distinguish infertile men with DNA damage from healthy controls with sensitivity with 65%. Moreover, high DNA fragmentation rates have been associated with lower biochemical pregnancy, clinical pregnancy and delivery rates after IUI (13). Bungum et al (13) demonstrated that clinical pregnancy rates were tenfold higher for men with DFI 30% compared with men with DFI >30% have been associated with approximately half the likelihood of success with conventional IVF methods (2, 7).
In addition to lower pregnancy rates, high levels of DNA fragmentation are associated with greater rates of pregnancy loss. For example, a meta-analysis of couples conceiving spontaneously or via assisted conception in the form of IUI, IVF, or intracytoplasmic sperm injection (ICSI) has demonstrated that men with high DNA damage show a greater than 2-fold increase in miscarriage rate compared with patients with normal levels of DNA-damage (14). Thus, although men with a high DFI experience a modest reduction in conception rates with conventional IVF and little effect with ICSI, they are 2-3 times more likely to experience a pregnancy loss than men with normal DFI (24). DNA fragmentation testing may be used in combination with the conventional semen analysis to help explain idiopathic male factor infertility, to confirm adverse effects of oxidative stress, and to establish the DNA integrity of sperm before undertaking more expensive ART procedures.
Proteomics refer to the comprehensive analysis of all the proteins expressed by a cell, or organism, the field of sperm proteomics specifically characterizes the entire human sperm protein repertoire (15).
There are 6,000 different sperm proteins. Sperm proteomic assessment begins with isolation and purification of sperm. Sperm proteins are then separated. One of these methods is the gel electrophoresis (SDS-PAGE).
Spermatozoa may also be divided into subcellular fractions such as heads or tails by differential centrifugation, and each of these fractions may be analyzed independently. This approach, dubbed �subcellular proteomics�, gives us more thorough assessment of spermatozoal content and helps pinpoint which cellular compartments the various sperm proteins localize to (17). For example, assessment of isolated tail fractions has led to the identification of numerous metabolic enzymes essential for motility and analysis of sperm membrane fractions has uncovered various membrane integral proteins that play a role in spermatozoa-oocyte interaction (16).
A number of studies have subsequently characterized proteomics anomalies in infertile men with asthenospermia. Hosseinifar et al (18) compared semen samples from normospermic men without varicoceles with oligospermic men with varicoceles and noted 15 consistent differences in protein expression between the groups. More recently, Zylbertztein et al (19) found that apoptosis regulation proteins were overexpressed in semen from adolescents with varicocele and abnormal semen quality whereas spermatogenesis proteins whereas overexpressed in adolescents with varicocele and normal semen quality.
Proteomics analysis have also revealed that the nuclear protamine content differs between fertile and infertile men, suggesting that sperm from the latter may have reduced DNA integrity and be more succeptible to oxidative damage (20). Moreover, a recent proteomic analysis demonstrated that the expression profile of proteins present in human spermatozoa differs in men with high and low levels of ROS (21). Various other studies have highlighted major groups of sperm proteins implicated in cases of ART failure (22). Already, researchers have successfully identified novel protein markers that can discriminate between men with obstructive azoospermia (OA) and nonobstructive azoospermia without need for surgery. Although conventional semen analysis remains a valuable tool in the evaluation of male factor infertility but this basic screening test may not accurately reflect all reproductive potentials. So that measurements of sperm DNA-damage, oxidative stress and TAC can enable clinicians to predict pregnancy and reproductive outcome more accurately. Moreover, we can now differ between fertile men and infertile men and in men with obstructive azoospermia and nonobstructive azoospermia through the investigations of the differential expression of sperm and semen plasma proteins. This field of the semen antioxidative reserve and sperm proteins evaluations holds the key to the development of more novel diagnostic and prognostic markers for the evaluation of infertile men.
Thacker S, Yadav SP, Sharma RK, et al. Evaluation of sperm proteins in infertile men: a proteomic approach. Fertil Steril. 2011; 95: 2745-2748.
Sabanegh E Jr, Agarwal Ashok. Chapter 21: Male Infertility. In: Wein AJ, Kavoussi LR, Novick AC, et al., eds. Campbell-Walsh Urology. 10th ed. Philadelphia: Saunders, An Imprint of Elsevier; 2011.
Feij? CM, Esteves SC. Diagnostic accuracy of sperm chromatin dispersion test to evaluate sperm deoxyribonucleic acid damage in men with unexplained infertility. Fertil Steril. 2014 Jan; 101: 58-63. e3.
Sharma RK, Pasqualotto FF, Nelson DR, et al. The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999; 14: 2801-2807.
Benedetti S, Tagliamonte MC, Catalani S, et al. Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality. Reprod Biomed Online. 2012; 25: 300-306.
Chen SJ, Allam JP, Duan YG, et al. Influence of reactive oxygen species on human sperm functions and fertilizing capacity including therapeutical approaches. Arch Gynecol Obstet. 2013; 288: 191-199.
Novotny J, Aziz N, Rybar R, et al. Relationship between reactive oxygen species production in human semen and sperm DNA damage assessed by Sperm Chromatin Structure Assay. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013; 157: 383-386.
Mahfouz R, Sharma R, Thiyagarajan A, et al. Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril. 2010; 94: 2141-2146.
Pahune PP, Choudhari AR, Muley PA. The total antioxidant power of semen and its correlation with the fertility potential of human male subjects. J Clin Diagn Res. 2013; 7: 991-995.
Chen H, Zhao HX, Huang XF, et al. Does high load of oxidants in human semen contribute to male factor infertility? Antioxid Redox Signal. 2012; 16: 754-759.
Ross C, Morriss A, Khalaf Y, et al. A systematic review of the effect of oral antioxidants on male infertility. Reprod Biomed Online. 2010; 20: 711-723.
Showell MG, Brown J, Yazdani A, et al. Antioxidants for male subfertility. Cochrane Database Syst Rev. 2011; 19: CD007411.
Bungum M, Humaidan P, Axmon A, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod. 2007; 22: 174-179.
Robinson L, Gallos ID, Conner SJ, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum Reprod. 2012; 27: 2908-2917.
Rahman MS, Lee JS, Kwon WS, et al. Sperm proteomics: road to male infertility and contraception. Int J Endocrinol. 2013; 2013: 360986.
Amaral A, Castillo J, Ramalho-Santos J, et al. The combined human sperm proteome: cellular pathways and implications for basic and clinical science. Hum Reprod Update. 2014; 20: 40-62.
Nowicka-Bauer K, Kurpisz M. Current knowledge of the human sperm proteome. Expert Rev Proteomics. 2013; 10: 591-605.
Hosseinifar H, Gourabi H, Salekdeh GH, et al. Study of sperm protein profile in men with and without varicocele using two-di
mensional gel electrophoresis. Urology. 2013; 81: 293-300.
Zylbersztejn DS, Andreoni C, Del Giudice PT, et al. Proteomic analysis of seminal plasma in adolescents with and without varicocele. Fertil Steril. 2013; 99: 92-98.
De Yebra L, Ballesc JL, Vanrell JA, et al. Complete selective absence of protamine P2 in humans. J Biol Chem. 1993; 268: 10553-10557.
Sharma R, Agarwal A, Mohanty G, et al. Proteomic analysis of human spermatozoa proteins with oxidative stress. Reprod Biol Endocrinol. 2013; 11: 48.
Zhu Y, Wu Y, Jin K, et al. Differential proteomic profiling in human spermatozoa that did or did not result in pregnancy via IVF and AID. Proteomics Clin Appl. Doi: 10.1002/prca.201200078, accessed February 2014.
Prof. Dr. SEMIR AHMED SALIM AL SAMARRAI
Professor Doctor of Medicine-Urosurgery, Andrology, and Male Infertility
Dubai Healthcare City, Dubai, United Arab Emirates.
Mailing Address: Dubai Healthcare City, Bldg. No. 64, Al Razi building, Block D,
2nd floor, Dubai, United Arab Emirates, PO box 13576