clinical consequences of oxidative stress in male infertility · 2014. 1. 31. · culture media...

535A. Agarwal et al. (eds.), Studies on Men’s Health and Fertility, Oxidative Stress in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-776-7_24, © Springer Science+Business Media, LLC 2012

Abstract Male infertility affects 40% of infertile couples in the USA and may be attributed to conditions such as varicocele, leukocytospermia, infection, and idio-pathic infertility. Such conditions may be associated with elevated levels of reactive oxygen species (ROS), decreased antioxidants, and oxidative stress (OS). OS can lead to male infertility in both an in vitro and in vivo setting. The negative effects of ROS on male fertility present as sperm DNA damage, decreasing motility, apopto-sis, and lipid peroxidation. ROS and antioxidant levels can be measured and quanti-fi ed in order to detect OS in semen samples. Both oral antioxidant therapy and culture media supplementation have proven to be effective in reducing OS. Future research is still needed in order to better understand the mechanisms involved in oxidative damage in the context of male infertility and to improve the treatments available for patients with OS-mediated male factor infertility.

Keywords Male infertility • Oxidative stress • Reactive oxygen species • Varicocele • Infection • Leukocytospermia • Idiopathic infertility • DNA damage • Antioxidants

Chapter 24 Clinical Consequences of Oxidative Stress in Male Infertility*

Tamer M. Said , Sheila R. Gokul , and Ashok Agarwal

T. M. Said MD, PhD, HCLD (ABB) Andrology Laboratory and Reproductive Tissue Bank , The Toronto Institute for Reproductive Medicine , 56 Aberfoyle Crescent , Toronto , ON , Canada M8X2W4

S. R. Gokul Center for Reproductive Medicine , Cleveland Clinic , 9500 Euclid Avenue , Cleveland , OH 44195 , USA

A. Agarwal, PhD (*) Center for Reproductive Medicine , Cleveland Clinic, Lerner College of Medicine , 9500 Euclid Avenue , Cleveland , OH 44195 , USA e-mail: [email protected]

* This research was conducted at the Cleveland Clinic’s Center for Reproductive Medicine, Cleveland, OH.

536 T.M. Said et al.

24.1 Introduction

Male factor infertility is a contributing factor in roughly 40% of infertile couples in the USA [ 1 ] . It is also thought to be the sole cause in about half of these cases [ 2 ] . Several conditions have been described as potential causes for male infertility including varicocele, leukocytospermia, genital infection, and idiopathic infertility, as well as exposure to environmental factors such as smoking and pollutants. These clinical conditions have been linked to oxidative stress (OS), which occurs when there is an imbalance between reactive oxygen species (ROS) and antioxidants in the body.

ROS are reactive molecules that contain oxygen, which includes oxidizing free radicals that are necessary for normal biological functions. When produced in excess, ROS can cause sperm damage and may lead to selective cell death (apopto-sis) [ 3 ] . Spermatozoa are particularly susceptible to oxidative damage because of their lack of an antioxidant defense system outside of the cytoplasm, which is mostly lost during the maturation process [ 4 ] . When the sperm cytoplasm is retained as cytoplasmic droplets, higher OS is to be expected due to ROS-producing enzymes in the cytoplasm [ 5 ] . Another source of ROS is leukocytes in the seminal fl uid. Oxidative damage can affect the lipids of the sperm plasma membrane, as well as the sperm DNA. This type of damage does not only affect sperm function but also lead to negative consequences with the developing embryo and pregnancy rates [ 6 ] .

In this chapter, we review recent literature and research to assess the current knowledge regarding the impact of OS on male infertility. Also discussed are the mechanisms behind oxidative damage and its contribution to the pathogenesis of male infertility.

24.2 Manifestations of Oxidative Stress

OS can lead to sperm damage and infertility through several pathways. ROS can react with DNA, proteins, carbohydrates, and lipids to cause sperm dysfunction and cell death. These detrimental effects have consequences both in vivo and in vitro, and may ultimately lead to infertility in males. The extent of oxidative damage depends on the nature of the ROS as well as the amount of exposure to OS.

24.2.1 Lipid Peroxidation

Lipids are found in the sperm plasma membrane in form of polyunsaturated fatty acids (PUFA). These fatty acids contain double bonds that make them susceptible to attack by free radicals. The initial attack by the hydroxyl radical leads to a series of chemical reactions referred to as lipid peroxidation [ 7 ] . Lipid peroxidation results

53724 Clinical Consequences of Oxidative Stress in Male Infertility

in damage of the axonemal structure, which has consequences related to sperm motility [ 8 ] . Lipid peroxidation is considered to be the fi rst step to sperm dysfunc-tion infl icted by ROS, as well as the most important pathologic process that results in the decrease of sperm parameters [ 9 ] . Oxidative damage to the sperm lipid mem-brane also leads to a loss of membrane integrity, and as a result DNA and proteins can become susceptible to damage.

24.2.2 Decreased Motility

There are several hypotheses showing how ROS and OS can lead to decreased sperm motility. One of these hypotheses postulates that hydrogen peroxide dif-fuses across the plasma membrane into the spermatozoa and affects enzymes such as glucose-6-phosphate dehydrogenase and NADPH oxidase that are vital to nor-mal spermatozoa function [ 10 ] . Another hypothesis suggests that ROS can lead to a decrease in protein phosphorylation and mitochondrial activity, consequently leading to sperm immobilization by reducing membrane fl uidity [ 11 ] . Damage to the mitochondrial membrane by ROS can further potentiate the effects of OS on motility by causing a loss of intracellular ATP, leading to axonemal damage [ 12 ] . Finally, it was also reported that oxidative stress impacts the sperm motility pat-tern and motion kinetics. While low levels of nitric oxide are needed for hyperac-tivation, excess levels of NO have been shown to inhibit motility by impairing sperm respiration [ 13 ] .

24.2.3 DNA Damage

DNA is usually protected from oxidative damage by its condensed form and the antioxidants present in the cytoplasm. After lipid peroxidation, DNA in both the mitochondria and nucleus becomes more susceptible to react with ROS. Sperm DNA damage has been associated with poor sperm parameters, infertility, and poor in vitro fertilization (IVF) outcomes [ 14 ] . DNA damage may present as fragmenta-tion, deletions, frameshifts, point mutation, and DNA cross-links, as well as other genetic modifi cations and rearrangements. OS can also lead to single-strand DNA and double-strand DNA breaks [ 15 ] . If the amount of oxidative DNA damage is considerable, then apoptosis and embryo fragmentation may also occur.

24.2.4 Apoptosis

Apoptosis is an ongoing physiological phenomenon that leads to the elimination of abnormal spermatozoa in order to limit the number of male germ cells that can be


maintained by Sertoli cells in the testes. Apoptosis is initiated in spermatozoa when high levels of ROS damage the mitochondrial membranes, and cytochrome-c proteins are released [ 1 ] . This activates caspases 9 and 3, which play essential roles in apoptosis. High levels of cytochrome-c and caspases have been correlated with increased levels of sperm DNA damage such as single and double-stranded DNA strand breaks [ 16 ] . Caspases have been also implicated in the decrease of sperm motility [ 17 ] .

24.2.5 Sperm Morphology

Caspase-mediated apoptosis and increased OS have a positive relationship with increased sperm damage and abnormal sperm morphology [ 3 ] . There is also a higher incidence of abnormal sperm morphology in conditions related to OS [ 8 ] . DNA damage and ROS production has also been found to correlate with abnormal head morphology and cytoplasmic retention in immature sperm, but not in mature sperm [ 4 ] . This may be a result of OS affecting the regulation of spermiogenesis, the fi nal stage of spermatogenesis where immature spermatids develop into mature sperma-tozoa [ 4 ] . Morphologically abnormal and immature sperm can lead to even higher levels of ROS production during sperm migration, and consequently lead to OS-related damage in mature sperm [ 4 ] . Abnormal morphology related to OS is not limited to immature spermatids and can extend to mature spermatozoa [ 14 ] .

24.3 Clinical Conditions Associated with OS

24.3.1 Varicocele

The incidence of varicoceles in the general male population is 20% [ 18 ] , while vari-coceles are thought to be present in 40% of infertile males [ 19 ] . In males with sec-ondary infertility, varicoceles are seen in 70–80% of patients [ 20 ] . Although the exact mechanisms through which varicocele damages spermatogenesis and sperm quality are not well understood, varicoceles have been associated with increased NO production [ 21 ] , intratesticular temperatures, and low antioxidant capacity [ 22 ] . NO is released by phagocytes and endothelial cells in the male reproductive tract [ 23 ] . In patients with varicocele, excessive NO is released by the dilated veins, which can lead to the dysfunction of spermatozoa [ 24 ] . Also, NO reacts with the superoxide anion and produce peroxynitrate, a strong oxidant that can negatively impact sperm function [ 25 ] .

While the exact mechanisms that lead to infertility and lower sperm parameters in men with varicocele are yet to be confi rmed, reactive oxygen and nitrogen species


may play a role [ 26 ] . OS and other forms of damage associated with increased reactive oxygen and nitrogen species leads to infertility in 15% of males with vari-cocele [ 27 ] . Patients with varicocele have higher ROS levels regardless of whether or not they are infertile [ 28 ] . A study by Allamaneni et al. found a positive correlation between the severity of varicocele and higher ROS production [ 29 ] . Other studies have reported lower antioxidant concentrations along with OS in patients with vari-cocele [ 30, 31 ] . On the other hand, some studies report no association between levels of OS and varicocele grade or fertility status in males [ 28, 32 ] . In support of the association of OS and varicoceles, varicocelectomy and antioxidant treatments have been shown to improve male fertility in patients with varicocele [ 33 ] .

24.3.2 Leukocytospermia

Leukocytes should only make up 5% of the round cell population in semen in nor-mal males [ 34 ] . Leukocytospermia is defi ned as the presence of peroxidase-positive leukocytes in concentrations of greater than 1 × 10 6 per mL of semen. Increased leukocyte infi ltration in semen is common in conditions that involve infl ammation such as genitourinary tract infection. Leukocytes exhibit increased ROS production in an early defense mechanism to kill infecting bacteria [ 35 ] . Therefore, high amounts of seminal leukocytes may even be used to diagnose a genitourinary tract infection [ 36 ] . However, leukocytospermia may be seen in situations unrelated to infection, such as smoking or heavy alcohol consumption [ 37 ] .

Although ROS production by leukocytes is necessary for normal sperm func-tion, leukocytes are considered to be one of the main sources of excessive ROS and OS in the male reproductive tract. Activated leukocytes have been shown to pro-duce up to 100 times the amount of ROS produced by nonactivated leukocytes [ 38 ] . Leukocytes may also lead to oxidative damage in levels below the 1 × 10 6 per mL threshold for diagnosis of leukocytospermia [ 39 ] . Increased seminal leuko-cytes result in high levels of lipid peroxidation and DNA damage through ROS production. Nonsteroidal anti-infl ammatory drugs and antioxidants have been proven to be effective treatments for male patients with leukocytospermia [ 40 ] . Sperm preparation may also be used to decrease the effects of ROS and OS on mature spermatozoa by separating leukocytes and immature sperm from the mature fraction [ 39 ] .

A decline in sperm parameters has been correlated with leukocytospermia; a decrease that may be dependent on the concentration of leukocytes in the seminal fl uid [ 41 ] . Parameters that were shown to be impaired by leukocytospermia include decreased motility, acrosomal damage, abnormal morphology [ 42, 43 ] , decreased sperm concentration [ 44 ] , decreased hyperactivation, sperm DNA damage, and impaired oocyte penetration [ 45 ] . However, other contradictory studies reported no signifi cant relationship between leukocytospermia and decreased sperm function or parameters [ 46, 47 ] .


24.3.3 Genitourinary Tract Infection

Genitourinary tract infections may be caused by a number of bacteria, including Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis, Chlamydia tra-chomatis, and Ureaplasma urealyticum [ 36 ] . Genitourinary tract infection may originate in the kidney, bladder, epididymis, prostate, or urethra, and includes diagnoses such as prostatitis, epididymitis, orchitis, pyelonephritis, bacterial cysti-tis, and urethritis. These types of infections are associated with infl ammation and increased leukocytes in the seminal fl uid, which may lead to increased levels of ROS and OS [ 8 ] .

High levels of ROS production by leukocytes occurs as a defense mechanism designed to kill microbes [ 35 ] . ROS generation may continue due to stimulation mediated by cytokines such as interleukin-6 and -8, which are associated with infec-tion and infl ammation [ 48 ] . Leukocytospermia and DNA damage have been found in patients with genitourinary tract infections [ 49 ] . Chronic infections may lead to increased damage over time. Patients with both varicocele and some type of genito-urinary tract infection have been found to have higher levels of ROS than patients with just one of the conditions [ 44 ] . The most common method of treatment is with antibiotics such as tetracyclines, although antioxidant treatments involving carniti-nes have been also proven to be effective [ 50 ] .

In male accessory gland infection, there is a high prevalence of abnormal semen quality and infertility [ 51 ] . Infection of the epididymis can cause asthenozoo-spermia, while impairment of the seminal vesicles can lead to obstruction, decreased semen volumes and fructose levels [ 52 ] . Infection of the epididymis is also a source of obstructive azoospermia [ 2 ] . Infection of the prostate can cause semen hypervis-cosity due to ineffi cient secretion of proteolytic enzymes and antigens [ 53 ] , which can affect the sperm motility and progression through the female reproductive tract. Semen samples of men with an accessory gland infection have been shown to have lower concentrations of certain key antioxidants such as citric acid and zinc, which are involved in maintaining seminal pH levels as well as DNA condensation and chromatin stability [ 54– 57 ] .

24.3.4 Idiopathic Infertility

Infertile men are diagnosed with idiopathic infertility when normal sperm parame-ters and no other clinical condition that may result in infertility are seen. Oxidative damage may be a contributing factor to infertility in these normozoospermic males. While the exact cause of infertility may be unknown, idiopathic infertility has been correlated with higher levels of ROS and lower antioxidant levels than in fertile men [ 58 ] . A study found that men with idiopathic infertility had the second highest level of OS among the clinical diagnoses studied [ 44 ] . Therefore, it would be expected that infertile men diagnosed with idiopathic infertility would benefi t from antioxi-dant treatments. Idiopathic infertility has also been associated with increased sperm


DNA damage. A study by Wang et al. found a relationship between increased sperm damage by ROS and higher levels of cytochrome c and caspases 9 and 3 [ 3 ] . This may also indicate elevated apoptosis in males with idiopathic infertility.

Studies have reported that anywhere from 25 to 40% of males with idiopathic infertility have high ROS levels [ 59 ] . Idiopathic infertility may be explained by OS in infertile men with normal sperm and semen parameters [ 39 ] . Although idiopathic infertility may only be a temporary diagnosis, an estimated 64% of diagnosed males will remain infertile after 1 year [ 3 ] . The sperm mitochondria have also been found to be damaged by ROS production in men with idiopathic infertility. This mitochon-drial damage can lead to the release of proteins such as cytochrome c, and result in apoptosis and DNA damage [ 3 ] . However, apoptosis, DNA damage, and reduced sperm quality do not always present together [ 50 ] .

24.3.5 Environmental and Lifestyle Factors

Saleh et al. found a correlation between smoking and increased seminal leukocyte levels, as well as increased ROS production and lower antioxidant levels [ 60, 61 ] . Similarly, another study by Close et al. found a correlation between increased semi-nal leukocyte infi ltration in cigarette-smoking men [ 37 ] . Additionally, high levels of sperm DNA damage have been reported in male smokers [ 60, 62 ] . This may be due to the mutagens and carcinogens associated with cigarette smoke [ 62 ] .Cigarettes contain nicotine, cotinine, hydroxycotinine, alkaloids, and nitrosamines, which are all sources of free radicals in the body [ 63 ] . Smoking has been associated with up to a 48% increase in leukocyte levels in semen, as well as up to a 107% increase in seminal ROS levels [ 60 ] .

Environmental pollutants are a major source of ROS production that have been implicated in the pathogenesis of poor quality sperm [ 64 ] . A study by De Rosa et al. found that NO and lead in air pollution can negatively affect semen quality [ 65 ] . OS has been suggested to play a role in the development of these negative effects of pollution [ 66 ] . The accumulation of pollutants that may act as endocrine disruptors has been shown to have a negative effect on male reproductive function [ 67 ] . Air pollution from motor vehicles may have negative impact on male fertility through the release of NO [ 65 ] . Pollution may be also a contributing factor to the overall decline in male fecundity that has been seen in the past few decades [ 68, 69 ] .

24.4 OS and In Vitro Infertility

Reactive oxygen and nitrogen species that lead to OS have a negative impact not only on male fertility in natural conception but also on assisted reproductive technologies (ART) in an in vitro setting. In any ART procedure, there is a risk of oxidative damage from sources such as exposure to ambient air [ 70 ] . Other sources


of OS in an ART setting include the oocyte, embryo, cumulus cells, and immature sperm cells [ 6 ] . In addition, spermatozoa used in any ART procedure originate from an environment conducive to OS, which can lead to DNA damage [ 71 ] .

Studies have shown that the ROS levels in the seminal fl uid correlate with fertil-izing potential and the IVF rate in procedures such as intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) [ 71, 72 ] . Also, high ROS levels in day 1 embryo culture media have been related to decreased blastocyst and cleavage rate, low fertilization rate, and increased embryo fragmentation in ICSI cycles. Consistently, high day 1 ROS levels in culture media have been correlated with decreased pregnancy rates in ICSI and IVF [ 73 ] .

ROS production and sperm DNA damage are associated with apoptosis [ 3 ] , which has been shown to be associated with a decrease in fertilization rate [ 74 ] . It is of importance to note that sperm with DNA damage has the potential to lead to poor embryo development and carries the risk of birth defects [ 14 ] . Miscarriage rates were found to be higher in ICSI than in IVF, which may be explained by the fact that in the ICSI procedure, there is a greater chance of DNA damaged sperm being injected into the oocyte [ 75 ] . DNA damaged sperm is less likely to be used to fertilize an oocyte in IVF or IUI because of associated damage to the sperm plasma membrane, which is necessary for fertilization [ 1 ] .

The general sources, mechanisms, and consequences of OS on male fertility are summarized in Fig. 24.1 . Clinical conditions related to OS include idiopathic infer-tility, leukocytospermia, varicocele, genitourinary tract infection, environmental and lifestyle factors. OS acts through several mechanisms that lead to subfertility, such as lipid peroxidation, DNA damage, and apoptosis. OS can lead to several consequences related to male fertility, both in an in vivo and in vitro setting.

24.5 Management of Oxidative Stress

OS in a clinical setting can be diagnosed through a variety of tests that measure levels of ROS and antioxidant capacities in semen. OS and the conditions that lead to OS can be treated through various means, including oral and surgical treatments as well as laboratory techniques.

24.5.1 Diagnosis of OS and ROS Levels

The chemiluminescence assay is a direct method of quantifying extracellular ROS levels. This assay uses lucigenin and luminol to assess the generation of the super-oxide anion and hydrogen peroxide [ 76 ] . Lucigenin detects superoxide anion, while luminol detects hydrogen peroxide. These agents are oxidizable substrates that react with certain oxygen species in order to measure levels of ROS. The chemilumines-cence assay or calorimetric assay can also be used to measure the total antioxidant


capacity (TAC) of a semen sample. Subsequently, a ROS-TAC score is used to quantify if an imbalance exists between ROS and free radical scavengers. Low ROS-TAC scores are indicative of overall OS and have been identifi ed in patients with idiopathic infertility, varicocele, and male accessory gland infection [ 77, 78 ] . These scores may be useful in predicting infertility when compared to using just ROS or TAC scores [ 78 ] . Patients with varicocele and other conditions associated with male factor infertility have low TAC scores [ 77 ] , indicating an inability to scavenge ROS.

An indirect method of testing for ROS levels is through the examination of malondialdehyde (MDA) levels. MDA is a by-product of lipid peroxidation, which can be measured to detect the amount of lipid peroxidation in a semen sample. The MDA levels correlate with sperm motility and the potential for sperm–oocyte fusion [ 79 ] . This assay is useful in determining ROS levels before ART procedures and in diagnosing patients with subfertility [ 80 ] . Some studies have shown that high MDA levels correlate with other decreased sperm parameters such as concentration and motility [ 81, 82 ] . Higher MDA levels have been found in patients with varicocele when compared to controls, indicating increased peroxidative activity [ 31 ] .

Fig. 24.1 The general sources, mechanisms, and consequences of oxidative stress (OS) on male fertility are summarized. Clinical conditions related to OS include idiopathic infertility, leukocy-tospermia, varicocele, genitourinary tract infection, environmental and lifestyle factors. OS acts through several mechanisms which lead to subfertility, such as lipid peroxidation, DNA damage, and apoptosis. OS can lead to several consequences related to male fertility, both in an in vivo and in vitro setting


24.5.2 In Vivo and In Vitro Antioxidant Treatments

Oral antioxidant supplements may be used to counteract OS and treat male infertil-ity. Studies have shown that antioxidants correlate with improvement in sperm parameters; however, in excess, these oral antioxidant supplements may have detri-mental effects [ 83 ] . As an example, improvement in sperm motility and lower levels of ROS were attributed to vitamin E and selenium oral supplementation [ 84 ] . Other antioxidant supplementations that have been proven to be effective in reducing ROS levels include glutathione, l -carnitine, vitamin A, and N -acetyl cysteine [ 39, 50 ] .

Antioxidant treatments can be also added to culture media in vitro during sperm preparation techniques in order to improve in vitro sperm fertilization ability. Vitamin C supplementation alone and in conjunction with vitamin E has been shown to be effective both orally and in culture media [ 85, 86 ] . Media supplementation with vitamin C and urate can lead to protection of spermatozoa from DNA damage [ 87 ] .

24.5.3 Sperm Preparation Techniques

Sperm preparation techniques can be used to separate ROS producing agents such as leukocytes and immature spermatozoa from mature spermatozoa. The use of den-sity gradients and centrifugation separates mature and immature sperm populations based on morphology and motility [ 88 ] . Swim up techniques may also be used to separate highly motile and morphologically normal sperm from the rest of the sperm population [ 89 ] . Percoll density gradient preparation is available, but is recom-mended for research purposes only.

24.5.4 Specifi c Treatments

Effective treatments have been developed for specifi c male conditions related to OS. One of the most effective treatments for patients with varicocele is varicocelectomy, the surgical means of varicocele repair [ 90 ] . Varicocelectomy is effective in decreas-ing oxidative stress, sperm DNA damage and increasing antioxidant capacity in seminal plasma of subfertile patients with varicocele [ 91 ] . Prevention and avoid-ance are also necessary in case of environmental factors induced oxidative stress such as smoking and pollutants that contribute to OS.

24.6 Conclusions

Conditions such as varicocele, genitourinary tract infection, leukocytospermia, and idiopathic infertility are associated with OS and have the potential of causing male factor infertility. Environmental factors such as smoking and pollution can also lead


to elevated levels of ROS and subfertility in males. OS results in in vitro and in vivo clinical complications that may affect male fecundity. High ROS levels impair male fertility through lipid peroxidation, decreased motility, DNA damage, and apopto-sis. OS can be detected and treated to lower ROS levels. Various approaches are currently available to prevent and treat OS in vivo and in vitro, which may lead to improvement of male fertility. There is still a need for further investigation into the mechanisms and pathways that lead to oxidative damage, as well as the need for improved fertility treatments specifi c to OS and the related conditions.

High levels of ROS or low levels of antioxidants can lead to OS, affecting male • fertility. OS can impair male fertility through lipid peroxidation, decreased motility, DNA • damage, and apoptosis. Clinical conditions associated with OS include varicocele, leukocytospermia, • genitourinary tract infection, and idiopathic infertility. ROS levels can be diagnosed by the chemiluminescence assay, and OS can be • identifi ed using the TAC levels, and the ROS-TAC levels. Oral antioxidants and antioxidant treatment of the media culture may be helpful • in ART procedures to prevent OS and improve fertility.

References

1. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infer-tility: an update. Am J Reprod Immunol. 2008;59(1):2–11.

2. Sharlip I, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. Best practice poli-cies for male infertility. Fertil Steril. 2002;77(5):873–82.

3. Wang X, Sharma RK, Sikka SC, Thomas Jr AJ, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril. 2003;80(3):531–5.

4. Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implica-tions in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16(9):1912–21.

5. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl. 1996;17(3):276–87.

6. Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. Oxidative stress in an assisted reproductive techniques setting. Fertil Steril. 2006;86(3):503–12.

7. Aitken J, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefi t and risk. Bioessays. 1994;16(4):259–67.

8. Saleh R, Agarwal A, Kandirali E, Sharma RK, Thomas AJ, Nada EA, et al. Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa. Fertil Steril. 2002;78(6):1215–24.

9. Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril. 1979;31(5):531–7.

10. Aitken R. Molecular mechanisms regulating human sperm function. Mol Hum Reprod. 1997;3(3):169–73.


11. de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between benefi cial and detrimental effects. Hum Reprod. 1995;10 Suppl 1:15–21.

12. Sikka S. Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 2001;8(7):851–62.

13. Herrero M, de Lamirande E, Gagnon C. Nitric oxide is a signaling molecule in spermatozoa. Curr Pharm Des. 2003;9(5):419–25.

14. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999;4(1):31–7.

15. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril. 1997;68(3):519–24.

16. Said T, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update. 2004;10(1):39–51.

17. Weng S, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S. Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod. 2002;8(11):984–91.

18. Pasqualotto F, Agarwal A. Varicocele and male infertility: an evidence based review. Arch Med Sci. 2009;5(1A):S20–7.

19. Madgar I, Weissenberg R, Lunenfeld B, Karasik A, Goldwasser B. Controlled trial of high spermatic vein ligation for varicocele in infertile men. Fertil Steril. 1995;63(1):120–4.

20. Naughton C, Nangia A, Agarwal A. Varicocele and male infertility: part II: pathophysiology of varicoceles in male infertility. Hum Reprod. 2001;7(5):473–81.

21. Romeo C, Ientile R, Santoro G, Impellizzeri P, Turiaco N, Impalà P, et al. Nitric oxide produc-tion is increased in the spermatic veins of adolescents with left idiophatic varicocele. J Pediatr Surg. 2001;36(2):389–93.

22. Agarwal A, Prabakaran S, Allamaneni SS. Relationship between oxidative stress, varicocele and infertility: a meta-analysis. Reprod Biomed Online. 2006;12(5):630–3.

23. Davies M, Fulton GJ, Hagen PO. Clinical biology of nitric oxide. Br J Surg. 1995;82(12):1598–610.

24. Ozbeka E, Turkozc Y, Gokdenizb R, Davarcia M, Ozugurluc F. Increased nitric oxide produc-tion in the spermatic vein of patients with varicocele. Eur Urol. 2000;37(2):172–5.

25. Weinberg J, Doty E, Bonaventura J, Haney AF. Nitric oxide inhibition of human sperm motil-ity. Fertil Steril. 1995;64(2):408–13.

26. Agarwal A, Sharma RK, Desai N, Prabakran S, Tavares A, Sabanaegh E. Role of oxidative stress in pathogenesis of varicocele and infertility. Urology. 2009;73(3):461–9.

27. Schoor R, Elhanbly SM, Niederberger C. The pathophysiology of varicocele-associated male infertility. Curr Urol Rep. 2001;2(6):432–6.

28. Hendin B, Kolettis PN, Sharma RK, Thomas Jr AJ, Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol. 1999;161(6):1831–4.

29. Allamaneni S, Naughton CK, Sharma RK, Thomas Jr AJ, Agarwal A. Increased seminal reac-tive oxygen species levels in patients with varicoceles correlate with varicocele grade but not with testis size. Fertil Steril. 2004;82(6):1684–6.

30. Künzle R, Mueller MD, Hänggi W, Birkhäuser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril. 2003;79(2):287–91.

31. Abd-Elmoaty M, Saleh R, Sharma R, Agarwal A. Increased levels of oxidants and reduced antioxidants in semen of infertile men with varicocele. Fertil Steril. 2010;94(4):1531–4.

32. Cocuzza M, Athayde KS, Agarwal A, Pagani R, Sikka SC, Lucon AM, et al. Impact of clinical varicocele and testis size on seminal reactive oxygen species levels in a fertile population: a prospective controlled study. Fertil Steril. 2008;90(4):1103–8.

33. Unal D, Yeni E, Verit A, Karatas OF. Clomiphene citrate versus varicocelectomy in treatment of subclinical varicocele: a prospective randomized study. Int J Urol. 2001;8(5):227–30.

34. Eggert-Kruse W, Bellmann A, Rohr G, Tilgen W, Runnebaum B. Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male fertility. Fertil Steril. 1992;58(5):1046–55.


35. Saran M, Beck-Speier I, Fellerhoff B, Bauer G. Phagocytic killing of microorganisms by radical processes: consequences of the reaction of hydroxyl radicals with chloride yielding chlorine atoms. Free Radic Biol Med. 1999;26(3–4):482–90.

36. Munuce M, Bregni C, Carizza C, Mendeluk G. Semen culture, leukocytospermia, and the presence of sperm antibodies in seminal hyperviscosity. Arch Androl. 1999;42(1):21–8.

37. Close C, Roberts PL, Berger RE. Cigarettes, alcohol and marijuana are related to pyospermia in infertile men. J Urol. 1990;144(4):900–3.

38. Plante M, de Lamirande E, Gagnon C. Reactive oxygen species released by activated neutro-phils, but not by defi cient spermatozoa, are suffi cient to affect normal sperm motility. Fertil Steril. 1994;62(2):387–93.

39. Agarwal A, Said TM. Oxidative stress, DNA damage and apoptosis in male infertility: a clini-cal approach. BJU Int. 2005;95(4):503–7.

40. Vicari E, La Vignera S, Calogero AE. Antioxidant treatment with carnitines is effective in infertile patients with prostatovesiculoepididymitis and elevated seminal leukocyte concentra-tions after treatment with nonsteroidal anti-infl ammatory compounds. Fertil Steril. 2002;78(6):1203–8.

41. Sharma R, Pasqualotto AE, Nelson DR, Thomas Jr AJ, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. J Androl. 2001;22(4):575–83.

42. Aziz N, Agarwal A, Lewis-Jones I, Sharma RK, Thomas Jr AJ. Novel associations between specifi c sperm morphological defects and leukocytospermia. Fertil Steril. 2004;82(3):621–7.

43. Lackner J, Agarwal A, Mahfouz R, du Plessis SS, Schatzl G. The association between leuko-cytes and sperm quality is concentration dependent. Reprod Biol Endocrinol. 2010;8:12.

44. Pasqualotto F, Sharma RK, Nelson DR, Thomas AJ, Agarwal A. Relationship between oxida-tive stress, semen characteristics, and clinical diagnosis in men undergoing infertility investi-gation. Fertil Steril. 2000;73(3):459–64.

45. Maruyama DJ, Hale RW, Rogers BJ. Effects of white blood cells on the in vitro penetration of zona-free hamster eggs by human spermatozoa. J Androl. 1985;6(2):127–35.

46. Tomlinson M, Barratt CL, Cooke ID. Prospective study of leukocytes and leukocyte subpopu-lations in semen suggests they are not a cause of male infertility. Fertil Steril. 1993;60(6):1069–75.

47. Aitken J, Krausz C, Buckingham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and pre-cursor germ cells in human sperm suspensions. Mol Reprod Dev. 1994;39(3):268–79.

48. Agarwal A, Prabakaran S, Allamaneni S. What an andrologist/urologist should know about free radicals and why. Urology. 2006;67(1):2–8.

49. Ochsendorf F. Infections in the male genital tract and reactive oxygen species. Hum Reprod Update. 1999;5(5):399–420.

50. Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod Biomed Online. 2004;8(6):616–27.

51. Rowe P, Comhaire F, Hargreave T, Mahmoud A, Rowe P, Comhaire F, Hargreave T, Mahmoud A. WHO manual for the standardized investigation, diagnosis and management of the infertile male. 1st ed. Cambridge: Cambridge University Press; 2000.

52. Comhaire F, Mahmoud AMA, Depuydt CE, Zalata AA, Christophe AB. Mechanisms and effects of male genital tract infection on sperm quality and fertilizing potential: the androlo-gist’s viewpoint. Hum Reprod. 1999;5(5):393–8.

53. Shibata K, Kajihara J, Kato K, Hirano K. Purifi cation and characterization of prostate specifi c antigen from human urine. Biochim Biophys Acta. 1997;1336(3):425–33.

54. Kvist U, Björndahl L. Zinc preserves an inherent capacity for human sperm chromatin decon-densation. Acta Physiol Scand. 1985;124(2):195–200.

55. Kundu T, Rao M. DNA condensation by the rat spermatidal protein TP2 shows GC-rich sequence preference and is zinc dependent. Biochemistry. 1995;34(15):5143–50.

56. Comhaire F, Vermeulen L, Pieters O. Study of the accuracy of physical and biochemical mark-ers in semen to detect infectious dysfunction of the accessory sex glands. J Androl. 1989;10(1):50–3.


57. Kjellberg S, Björndahl L, Kvist U. Sperm chromatin stability and zinc binding properties in semen from men in barren unions. Int J Androl. 1992;15(2):103–13.

58. Pasqualotto F, Sharma RK, Kobayashi H, Nelson DR, Thomas Jr AJ, Agarwal A. Oxidative stress in normospermic men undergoing infertility evaluation. J Androl. 2001;22(2):316–22.

59. Alkan I, Simşek F, Haklar G, Kervancioğlu E, Ozveri H, Yalçin S, Akdaş A. Reactive oxygen species production by the spermatozoa of patients with idiopathic infertility: relationship to seminal plasma antioxidants. J Urol. 1997;157(1):140–3.

60. Saleh R, Agarwal A, Sharma RK, Nelson DR, Thomas Jr AJ. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study. Fertil Steril. 2002;78(3):491–9.

61. Fraga C, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res. 1996;351(2):199–203.

62. Traber M, van der Vliet A, Reznick AZ, Cross CE. Tobacco-related diseases. Is there a role for antioxidant micronutrient supplementation? Clin Chest Med. 2000;21(1):173–87.

63. Pasqualotto F, Sobreiro BP, Hallak J, Pasqualotto EB, Lucon AM. Cigarette smoking is related to a decrease in semen volume in a population of fertile men. BJU Int. 2006;97(2):324–6.

64. Koizumi T, Li ZG. Role of oxidative stress in single-dose, cadmium-induced testicular cancer. J Toxicol Environ Health. 1992;37(1):25–36.

65. De Rosa M, Zarrilli S, Paesano L, Carbone U, Boggia B, Petretta M, et al. Traffi c pollutants affect fertility in men. Hum Reprod. 2003;18(5):1055–61.

66. Fowler B, Whittaker MH, Lipsky M, Wang G, Chen XQ. Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: an overview. Biometals. 2004;17(5):567–8.

67. Kumar S. Occupational exposure associated with reproductive dysfunction. J Occup Health. 2004;46(1):1–19.

68. Skakkebaek N, Jørgensen N, Main KM, Rajpert-De Meyts E, Leffers H, Andersson AM, et al. Is human fecundity declining? Int J Androl. 2006;29(1):2–11.

69. Hauser R. The environment and male fertility: recent research on emerging chemicals and semen quality. Semin Reprod Med. 2006;24(3):156–67.

70. Agarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Androl. 1994;33(3):157–62.

71. Saleh R, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl. 2002;22(6):737–52.

72. Zorn B, Vidmar G, Meden-Vrtovec H. Seminal reactive oxygen species as predictors of fertil-ization, embryo quality and pregnancy rates after conventional in vitro fertilization and intra-cytoplasmic sperm injection. Int J Androl. 2003;26(5):279–85.

73. Bedaiwy M, Falcone T, Mohamed MS, Aleem AA, Sharma RK, Worley SE, et al. Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil Steril. 2004;82(3):593–600.

74. Høst E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand. 2000;79(7):559–63.

75. Aitken R. The Amoroso lecture. The human spermatozoon—a cell in crisis? J Reprod Fertil. 1999;115(1):1–7.

76. Agarwal A, Allamaneni SS, Said TM. Chemiluminescence technique for measuring reactive oxygen species. Reprod Biomed Online. 2004;9(4):466–8.

77. Pasqualotto F, Sharma RK, Pasqualotto EB, Agarwal A. Poor semen quality and ROS-TAC scores in patients with idiopathic infertility. Urol Int. 2008;81(3):263–70.

78. Sharma R, Pasqualotto FF, Nelson DR, Thomas Jr AJ, Agarwal A. The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999;14(11):2801–7.

79. Aitken R, Harkiss D, Buckingham DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev. 1993;35(3):302–15.

80. Oral O, Kutlu T, Aksoy E, Fiçicioğlu C, Uslu H, Tuğrul S. The effects of oxidative stress on outcomes of assisted reproductive techniques. J Assist Reprod Genet. 2006;23(2):81–5.


81. Tavilani H, Doosti M, Saeidi H. Malondialdehyde levels in sperm and seminal plasma of asthenozoospermic and its relationship with semen parameters. Clin Chim Acta. 2005;356(1–2):199–203.

82. Hsieh Y, Chang CC, Lin CS. Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility. Int J Biol Sci. 2006;2(1):23–9.

83. Suleiman S, Ali ME, Zaki ZM, el-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: protective role of vitamin E. J Androl. 1996;17(5):530–7.

84. Keskes-Ammar L, Feki-Chakroun N, Rebai T, Sahnoun Z, Ghozzi H, Hammami S, et al. Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men. Arch Androl. 2003;49(2):83–94.

85. Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl. 2005;26(3):349–53.

86. Greco E, Romano S, Iacobelli M, Ferrero S, Baroni E, Minasi MG, et al. ICSI in cases of sperm DNA damage: benefi cial effect of oral antioxidant treatment. Hum Reprod. 2005;20(9):2590–4.

87. Thiele J, Friesleben HJ, Fuchs J, Ochsendorf FR. Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen. Hum Reprod. 1995;10(1):110–5.

88. Ricci G, Perticarari S, Boscolo R, Simeone R, Martinelli M, Fischer-Tamaro L, et al. Leukocytospermia and sperm preparation—a fl ow cytometric study. Reprod Biol Endocrinol. 2009;7:128.

89. Mortimer D. Sperm preparation methods. J Androl. 2000;21(3):357–66. 90. Agarwal A, Deepinder F, Cocuzza M, Agarwal R, Short RA, Sabanegh E, Marmar JL. Effi cacy

of varicocelectomy in improving semen parameters: new meta-analytical approach. Urology. 2007;70(3):532–8.

91. Chen SS, Huang WJ, Chang LS, Wei YH. Attenuation of oxidative stress after varicocelec-tomy in subfertile patients with varicocele. J Urol. 2008;179(2):639–42.

clinical consequences of oxidative stress in male infertility · 2014. 1. 31. · culture media...

Documents