Studies on Men's Health and Fertility || Methods for the Detection of ROS in Human Sperm Samples

257A. 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_13, © Springer Science+Business Media, LLC 2012

Abstract Male-factor signifi cantly contributes to infertility in couples of reproductive age. Several studies have shown that oxidative stress (OS) is involved in the pathophysiology of male infertility. It is caused by an imbalance between the forma-tion of reactive oxygen species (ROS) and the ability of the antioxidants to scavenge them. There are several methods to measure seminal ROS in the clinical setting, most notably among them is the chemiluminescence assay. This technique mea-sures the global ROS, i.e., both the intra- and extracellular ROS. The measurement of seminal ROS employing the chemiluminescence technique in clinical andrology labs is explained in this chapter. Through a deeper understanding of ROS and its measurement, clinical andrology labs can better assist patients to achieve increased rates of fertility and pregnancy.

Keywords Use of chemiluminescence assay • Measurement of seminal reactive oxygen levels • Clinical andrology labs • Free radicals • Sources of ROS

13.1 Introduction

Infertility currently affects approximately 15% of couples of reproductive age and the incidence is growing, with male-factor contributing to as much as 50% of infer-tility issues [ 1, 2 ] . As infertility continues to develop into a growing concern, there is a need to better understand and identify the causes of male-factor infertility.

D. Benjamin, BA • R. K. Sharma, PhD • A. Moazzam, MD Center for Reproductive Medicine , Cleveland Clinic , Cleveland , OH 44195 , USA e-mail: [email protected]

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

Chapter 13 Methods for the Detection of ROS in Human Sperm Samples

David Benjamin , Rakesh K. Sharma , Arozia Moazzam , and Ashok Agarwal

258 D. Benjamin et al.

Suboptimal sperm quality is a major concern for male-factor infertility, and one of the underlying causes of suboptimal sperm quality is attributed to the presence of reactive oxygen species (ROS) [ 3 ] . Although the human body naturally produces free oxygen radicals and ROS in physiological amount, essential for capacitation, hyperactivation, acrosome reaction, and oocyte fusion, an excess of ROS can be harmful to male gametes. Several studies have demonstrated that an excess of ROS, which leads to the state of oxidative stress, affects spermatozoa motility and viabil-ity, oocytes, as well as rates of fertilization and pregnancy [ 3– 6 ] . There are several techniques currently used in the clinical andrology laboratory to quantify the amount of ROS in a patient’s semen sample, including the chemiluminescence assay. Several recent studies have identifi ed reference values for ROS in order to diagnose male infertility, and these reference values continue to be refi ned with the publication of new studies. Measurement of ROS can be used as a diagnostic tool in identifying the underlying etiology of idiopathic or unexplained infertility. In order for clinical andrology labs to better assist patients experiencing issues with infertility, it is important to understand ROS and the techniques, such as the chemiluminescence assay, which are employed to detect ROS.

13.2 What Are Free Radicals?

Free radicals are a group of atoms or molecules that are highly reactive due to hav-ing one or more unpaired electrons [ 7 ] . As a result of having an incomplete outer valance shell, these molecules attempt to react with other molecules in their vicinity in order to gain one or more electrons. However, once a molecule loses an electron to a free radical, a chain reaction is created, as now the former molecule becomes a free radical itself (Fig. 13.1 ). The chain reaction created from free radicals can have catastrophic effects for living cells. ROS are a collection of radicals and nonradical derivatives of oxygen. Free radicals derived from nitrogen are called reactive nitro-gen species (Table 13.1 ). The most common ROS that is produced by spermatozoa is the superoxide anion radical; this in turn forms hydrogen peroxide (strong oxi-dizer) on its own or by the action of superoxide dismutase [ 8 ] .

Plasma membrane of the spermatozoa is rich in polyunsaturated fatty acids (PUFA). Because their cytoplasm contains low concentrations of scavenging enzymes, they are particularly susceptible to the damage induced by excessive ROS [ 9, 10 ] . The seminal plasma, however, contains two different types of antioxidants to minimize free radical-induced damage: enzymatic and nonenzymatic antioxi-dants. Enzymatic antioxidants are: superoxide dismutase, catalase, glutathione reductase, and peroxidase. Nonenzymatic antioxidants are comprised of vitamins (vitamin C, E), proteins (albumin, transferrin, haptoglobin, and ceruloplasmin), and other molecules (glutathione, pyruvate, and ubiquinol). Both enzymatic and nonen-zymatic antioxidants can help minimize free radical-induced damage. Excessive generation of ROS damages cells, tissues, and organs and is involved in the patho-genesis of a wide range of diseases.

25913 Methods for the Detection of ROS in Human Sperm Samples

13.3 Sources of ROS

Morphologically defective spermatozoa and leukocytes found in semen are the pri-mary causes of ROS production [ 11 ] . Several studies have shown that human sper-matozoa can produce ROS, especially those with an excess of cytoplasm, also known as cytoplasmic droplets [ 12– 15 ] . ROS production due to excess cytoplasm occurs due to the cytosolic enzyme glucose-6-phosphate-dehydrogenase (G6PD). Spermatozoa may generate ROS in two ways: fi rst, through the NADPH-oxidase system found in the sperm plasma membrane, and secondly, through the NADH-dependent oxidoreductase in mitochondria [ 16 ] . Peroxidase-positive leukocytes, which are derived from the prostate and seminal vesicles, are also major contribu-tors to ROS production [ 16– 19 ] . Activation of leukocytes, stemming from infl am-mation and infection, can lead to ROS production as a result of increased NADPH production [ 3, 20 ] . Leukocytospermia (>1 × 10 6 WBC/mL of semen) causes sperm damage by ROS production, and removal of seminal plasma during sperm prepara-tion for assisted production can further deprive the sperm of important nutritional support (antioxidants) and further cause sperm damage through ROS production [ 17, 18 ] .

Fig. 13.1 Illustration is showing the oxygen molecule and the generation of a free radical

Table 13.1 Examples of free radicals

Reactive oxygen species Reactive nitrogen species

Superoxide anion (O 2 •− ) Nitric oxide (NO • )

Hydrogen peroxide (H 2 O

2 ) Nitric dioxide (NO

2 • )

Hydroxyl radical (OH • ) Peroxynitrite (ONOO − )


13.4 Importance of Oxidative Stress and ROS in Male Infertility

Sperm dysfunction is one of the leading causes of infertility. Despite much research, there is limited knowledge about the causes of sperm dysfunction. However, oxida-tive stress (OS) has been identifi ed as one of the causes of abnormal sperm function. OS refers to the state in which free radicals overwhelm the body’s antioxidant capacity [ 21 ] ; an excess of ROS can have harmful effects on the tissues and organs [ 9, 10 ] . ROS has been shown to negatively affect sperm quality and function [ 22, 23 ] . For normal male fertility, a fi ne balance between the generation of ROS and its removal is very important. Excess of ROS also has a negative impact on the preg-nancy outcome as well as the assisted reproductive techniques [ 24, 25 ] . Moreover, excess ROS can damage DNA in spermatozoa, induce cell apoptosis, and cause lipid peroxidation, which leads to morphological abnormalities, decrease in fertility, and increased sperm membrane permeability [ 26– 28 ] . It is important to effectively detect the amount of ROS in a semen sample in order to better treat subfertile male patients.

13.5 Methods for Detecting ROS

There are several techniques to detect ROS. These include the direct and indirect methods (Table 13.2 ). Direct methods include cytochrome c reduction, electron spin resonance, and nitroblue tetrazolium technique (NBT, and xyenol orange-based assay and fl ow cytometry [ 7, 22 ] ). Indirect methods of measurement include the Endtz test, redox potential (GSH/GSSG), measurement of lipid peroxidation prod-ucts levels, chemokines, measurement of oxidative DNA damage, and measurement of reactive nitrogen species by Greiss reaction and fl uorescence spectroscopy [ 7 ] .

13.6 Chemiluminescence Measurement of Extracellular and Intracellular ROS

Chemiluminescence depends on the measurement of light emitted after reagents are added to a sample of human spermatozoa, causing a reaction. The chemiluminescence assay is one of the most commonly used techniques to measure seminal ROS levels in clinical andrology laboratories [ 7, 12, 29, 30 ] . The two major probes used to measure ROS generation in the chemiluminescence assay are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione and 3-aminophthalic hydrazide) and lucigenin ( N , N ¢ -dimethyl-9,9 ¢ -biacridinium dinitrate). Both H

2 O

2 and O

2 •− are involved in luminol-dependent

chemiluminescence because both catalase and SOD can disrupt the luminol signal very effi ciently. The uncharged luminol molecule is membrane-permeant and can


react in the form of luminol or as a univalently oxidized luminol radical with a variety of ROS, including O

2 •− , H

2 O

2 , and OH • [ 12, 30 ] . Luminol is sensitive to H

2 O

2 and this

sensitivity can be greatly increased by addition of horseradish peroxidase. The lumi-nol signal generated by human spermatozoa is initiated by a one-electron oxidative event that is mediated by H

2 O

2 . A luminescent signal is produced with luminol through

a one-electron oxidative event mediated by H 2 O

2 and either endogenous peroxidase or

by addition of HRP [ 31 ] . Oxidation of luminol (one-electron) leads to the creation of a radical species which interacts with ground state oxygen to produce O

2 •− , which

participates in the oxygenation of luminol radical species to create an unstable endoperoxide, which breaks down and leads to light emission. In this, the O

2 •− is an

essential intermediate for the luminol-dependent chemiluminescence. Also, the redox cycling activity associated with this probe allows the signifi cant amplifi cation of the signal and allows easy measurement of H

2 O

2 .

Lucigenin works through a one-electron reduction unlike luminol which requires one-electron oxidation that creates a radical from lucigenin; this radical gives up its electron to the grounds state oxygen to create O

2 •− , thus returning the lucigenin to its

parent state [ 12, 30, 31 ] . The luminol probe is more advantageous than the lucigenin probe for several reasons. Luminol measures both intracellular and extracellular ROS such as hydrogen peroxide, superoxide anion, and hydroxyl radical; on the other hand, lucigenin measures only the extracellular ROS, and in particular, super-oxide anion [ 22, 30 ] . Lucigenin is an excellent probe for evaluating O

2 •− production

as a nonspecifi c redox marker for the enhanced electron transfer activity associated with defective sperm function. Both the sensitivity and specifi city of this probe are enhanced by its redox cycling activity [ 7, 29, 30 ] .

Table 13.2 Tests for detection of reactive oxygen species (direct) or their oxidized products (indirect)

Assay Probe Extracellular/intracellular

Direct measurement Tetrazolium nitroblue Ferricytochrome C Extracellular Chemiluminescence Luminol Both

Lucigenin Extracellular Indirect measurement Lipid peroxidation levels Thiobarbituric Measures oxidized component

in the body fl uids Antioxidants, micronutrients,

vitamins High-performance liquid

chromatography Serum and seminal plasma

Ascorbate High-performance liquid chromatography

Seminal plasma

Antioxidants enzymes Superoxide dismutase Seminal plasma Catalase Seminal plasma Glutathione peroxidase Spermatozoa Glutathione reductase Spermatozoa

Chemokines ELISA Seminal plasma Antioxidant-pro-oxidant status Total antioxidant Low chain-breaking levels


13.7 Measurement of ROS by Chemiluminescence Assay

Herein we describe the details of measurement of ROS in unprocessed, i.e., liquefi ed seminal ejaculate without any further processing by chemiluminescence assay.

1. Equipment and material

(a) Disposable polystyrene tubes with caps (15-mL) (b) Eppendorf pipettes (5-, 10-, 50-, and 1,000- m L) (c) Serological pipettes (1-, 2-mL) (d) Desk-top centrifuge (e) Disposable MicroCell slides (f) Dimethyl sulfoxide (DMSO; Catalog # D8779, Sigma Chemical Co., St.

Louis, MO) (g) Luminol (5-amino-2,3 dihydro-1,4 phthalazinedione; Catalog # A8511,

Sigma Chemical Co., St. Louis, MO) (h) Polystyrene Round bottom tubes (6-mL) (i) Luminometer (Model: LKB Autoplus 953) (j) Dulbecco’s Phosphate-buffered saline solution 1× (PBS-1×; Catalog #9235,

Irvine Scientifi c, Santa Ana, CA)

2. Preparation of reagents

(a) Stock luminol ( 100 mM ): Weigh 177.09 mg of luminol and add it to 10 mL of DMSO solution in a polystyrene tube. The tube must be covered in an aluminum foil due to the light sensitivity of the luminol. It can be stored at room temperature in the dark until the expiration date.

(b) Working luminol ( 5 mM ): Mix 20 m L luminol stock solution with 380 m L DMSO in a foil-covered polystyrene tube. This must be done prior to every use. Store the solution at room temperature in the dark until needed.

(c) DMSO solution : Provided ready to use; store at room temperature until the expiration date.

3. Specimen preparation

Upon arrival of the semen specimen, allow it to liquefy in the incubator at 37°C for approximately 20 min. A complete record of patient’s name, allocated identity number, period of sexual abstinence, and date and time of collection is noted. Manual semen analysis is performed for assessment of sperm con-centration and motility.

First, allow the semen sample to undergo liquefaction for 20 min in a 37°C incu-bator. Then record the initial physical characteristics such as volume, pH, and color and manually verify sperm count and motility. After complete liquefac-tion is ensured, set up the luminometer for ROS measurement.

4. ROS measurement by luminometer

This procedure is performed in a dark room. Set up the luminometer and the computer attached to it (Fig. 13.2a–c ).


Fig. 13.2 Autolumat 953 plus luminometer used in the measurement of ROS by chemilumines-cence assay. ( a ) External view and ( b ) internal view. Multiple tubes can be loaded simultaneously for measuring ROS. ( c ) The luminometer can be connected with the computer and a monitor and all the steps can be observed on the screen

Table 13.3 Setup for the measurement of ROS

Labeled tubes (no.) PBS-1× ( m L) Specimen volume ( m L)

Probe luminol (5 mM) ( m L)

Hydrogen peroxide ( m L)

Blank (tubes S1–3) 400 – – Negative control (tubes S4–6) 400 – 10 – Patient (tubes S78) – 400 10 – Positive control (tubes S9–11) 400 10 50

(a) Label 11 Falcon tubes (12 × 75 mm) in duplicates and add the following reagents as indicated in Table 13.3 (Fig. 13.3 ).

Note: To avoid contamination, change pipette tips after each addition. (b) Gently vortex the tubes to mix the aliquots uniformly.


(c) Place all the labeled tubes in the luminometer in the following order: Blank (tubes labeled 1–3).

(d) Negative control (tubes labeled 4–6), patient sample (tubes labeled 7–8), and positive control (tubes labeled 9–11) (Fig. 13.3 ).

5. Instrument setup

(a) Turn on the instrument and the computer. From the desktop, click on “Berthold tube” master icon to start the program.

(b) From the “Setup menu,” select “Measurement Defi nition” and then “New Measurement.” You will be prompted to the following:

“Measurement Name” (Initials, Date, Analyte, and Measurement). – It will show “Measurement Defi nition-on the ‘Tool bar.’” – Click “Luminometer Measurement” protocol and from the drop menu –click on “Rep. assay.”

Fig. 13.3 Preparing the tubes for the ROS measurement. A total of 11 tubes are labeled from S1 to 12: Blank, negative control, patient sample, and positive control. Luminol is added only to all tubes except blank. Hydrogen peroxide is added only to the positive control


Defi ne each “Parameter” as follows: –

Read time 1 s Background read time 0 s Total time 900 s Cycle time 30 s Delay “Inj M read (s)” 0 s Injector M ( m L) 0 s Temperature (°C) 37°C Temperature control (0 = OFF) 1 = ON

Press “Save” – (c) From the “Setup” menu, select “Assay Defi nition” and then “New Assay”: It will ask for the following:

“Assay Name” (Initials, Date, Analyte, Assay). Click “OK.” – Select “Measurement Method” and from the drop-down menu select the –measurement from Step 2a above. Go to “Column Menu.” Hide everything except the following: – Sample ID Status RLU mean Read date Read time Go to “Sample Type” menu and select “Normal.” – Press “OK” – Go to fi le, “New” click “Workload” Press “OK.” –

(d) Save your “Work Load” (Date, Initial, Sample or experiment ID) in “Work Load” fi le.

(e) Click “File name” (f) After saving the “Work Load,” the name of the fi le will show in the “Title

Bar.” (g) The specimens are ready to be analyzed.

6. Analyzing the samples

(a) Load the tubes into the instrument and click “Start.” It will start scanning for tubes.

(b) After scanning, it will show how many tubes are detected by the instrument in each batch, press “Next.”

(c) Select the “Assay Type” and type fi le name and then click “Finish.” (d) The “Excel spreadsheet” will open, measurement of the tubes will start. (e) Do not touch or change the screen, wait (3–5 min) to make sure everything

is working fi ne. (f) After fi nishing measurements, it will ask for “Save Excel Spread Sheet,”

save it in the “My Document” under “ExcelSheet” folder.


(g) Select “Excel Sheet,” name the fi le, and save type as “Measurement Files” (*.txr). Save the “Excel Sheet” (Fig. 13.4 ).

7. Printing ROS results

(a) Print Excel as well as the “chart 1” (Figs. 13.4 and 13.5 ). (b) Close the “Excel sheet” (c) Print the “Work Load” sheet (Fig. 13.6 ), save, and close it.

8. Calculating results

(a) Calculate the “average RLU” for Negative control, Samples, and Positive control.

(b) Calculate sample ROS by subtracting its average from negative control average.

(c) Sample ROS = Average “RLU mean” for sample − Average “RLU mean” for negative control.

(d) Correct the sample ROS by dividing it with “Sperm concentration/mL”

Corrected sample ROS

= × 6Calculated sample ROS/sperm concentration XX (RLU/s/ 10 sperm)

A typical example of calculating ROS values is illustrated in Fig. 13.6 .

Fig. 13.4 A representative display of the readings showing the number of signals generated in each of the above 12 tubes (S1–11). The measurement is for a total of 900 s. As seen here, blanks have the least number of ROS produced and the positive control to which hydrogen peroxide is added has the highest number of ROS generated


Fig. 13.5 A typical graph showing the ROS levels in the 11 tubes (S1–11). As seen here, only positive controls have signifi cantly higher levels of ROS. Those producing low levels (tubes S1–8) of ROS are seen very close to the X axis

Fig. 13.6 A representative print out generated after the ROS measurement is complete. The chart shows the 11 tubes (S1–11), each representative of the type of sampler and the mean RLU value for each tube. At the bottom is also an example illustrating how to calculate the fi nal amount of ROS generated in a given test (patient) sample


Reference values Normal range: <20 RLU/s/× 10 6 sperm Critical Values: >20 RLU/s/× 10 6 sperm

9. Quality control

(a) Criteria for acceptance : control reads 20 RLU/s/× 10 6 sperm. (b) Criteria for rejection : control reads >20 RLU/s/× 10 6 sperm. The assay must

be repeated and results shown to the Director. (c) The reagent lot numbers and expiration dates are recorded in the assay

Quality Control book located in the laboratory.

13.8 Types of Samples for ROS Measurement

ROS measurement can be performed in various types of samples such as:

1. Neat or unprocessed , whole seminal ejaculate . In this method, the seminal plasma is not removed. All components of the seminal ejaculate are intact and ROS levels indicate the actual de novo levels in the ejaculate. This is the ideal setup for assessing the clinical relevance of ROS in the seminal ejaculate and is indicative of oxidative stress and sperm dysfunction.

2. Processed sample : Liquefi ed semen specimens are centrifuged at 300 × g for 7 min and seminal plasma is removed. The sperm pellet is washed and resus-pended to 1 mL volume in PBS. This is also called the simple “wash and resuspend” method. With this method, the seminal plasma that confers protection to the sperm and other dissolved components is removed. However, all the cellular components such as debris, round cells, white blood cells, and leukocytes are still present in the sample.

3. Sperm preparation by the swim-up procedure : This method is used to measure ROS in highly motile sperm prepared by swim up. After liquefaction, an aliquot of specimen is mixed with sperm wash media using a sterile Pasteur pipette. It is centrifuged at 330 × g for 10 min. The supernatant is carefully aspirated and the pellet resuspended in 3 mL of fresh sperm wash media. The resuspended sample is carefully transferred in equal parts to two 15-mL sterile round-bottom test tubes and centrifuged at 330 × g for 5 min. Motile sperm are allowed to swim up during the incubation of test tubes at a 45° angle in 5% CO

2 at 37°C for 1 h.

Supernatant is aspirated into a clean test tube and centrifuged at 330 × g for 7 min. The fi nal supernatant is aspirated and the sperm pellet is resuspended in sperm wash media and is used for measurement of ROS.

4. Sperm preparation by density gradients : This method is used to measure ROS in immature (morphologically abnormal, poor motility) and mature (highly motile and morphologically normal) sperm. A double density gradient (40% “Upper phase” and 80% “Lower phase”) is used. Both the density gradient and the sperm wash media is brought to 37°C or room temperature. Using a sterile pipette, 2.0 mL of the “Lower phase” is transferred into a 15-mL conical centrifuge tube.


Similarly, 2.0 mL of the “Upper phase” is carefully placed on the top of the lower layer. The liquefi ed semen sample (1–2 mL) is placed on top of the upper layer, and the tube is centrifuged for 20 min at 330 × g or 1,600 revolutions per minute (rpm). The uppermost layer containing clear seminal plasma and dead white cells and other debris are discarded. For separating the immature sperm, the interphase between the upper and lower layer is carefully transferred to a conical tube. For separating the mature sperm, the pellet is carefully aspirated and trans-ferred into another centrifuge tube. Using a transfer pipette, 2–3 mL of sperm wash media is added, and the immature and mature fractions are centrifuged for 7 min at 330 × g or 1,600 rpm. The supernatant is discarded, and the pellet is suspended in 1.0 mL of sperm wash media. Sperm count, motility, and ROS levels are measured in the recovered fractions.

13.9 Luminometers

Several types of luminometers, varying ranging in features, design, and pricing, can be used in measuring the emitted light from the chemiluminescence assay reaction (Table 13.4 ) [ 32 ] . The measurements from the chemiluminescence assay are expressed as either counted photons per minute (cpm) or as relative light units (RLU). There are two different kinds of processing designs for luminometers. While direct current luminometers measure electric current, photon counting luminome-ters count individual photons [ 33 ] . Moreover, there are currently three types of luminometers available for commercial uses. First is single and double tube lumi-nometers, which measure one or two samples at a time. These are inexpensive and are typically used by small research laboratories. Secondly, multiple tube luminom-eters that measure several tubes at a time. They are more expensive than single and double tube luminometers and are used by research centers that depend on chemilu-minescence for research projects. Lastly, plate luminometers measure multiple tubes at a given time and are typically used by core research laboratories and commercial enterprises.

Table 13.4 Commercially available luminometers shown by type, sensitivity, and manufacturer

Model Type Sensitivity and dynamic range Manufacturer

GloMax 20/20 Single tube 0.1 fg luciferase Promega Corporation (Sunnyvale, CA, USA)

FB-12 Single tube 1,000 molecules of luciferase Zylux Corporation (Oak Ridge, TN, USA)

Triathler Single tube <10 amol ATP/vial Hidex (Turku, Finland) Optocomp-2 Multiple tube 0.1 pg ATP MGM Instruments

(Hamden, CT, USA) Autolumat Auto

Plus LB 953 Multiple tube 5 amol of ATP Berthold Technologies

(Oak Ridge, TN, USA)


13.10 Factors Affecting Chemiluminescent Reactions

It is important to have a reliable and valid method of detection of ROS [ 29 ] . The luminol assay is robust; however, there are various factors that can affect ROS detec-tion by the chemiluminescent reactions [ 7, 30, 34– 36 ] . Both the probes luminol and lucigenin have great sensitivities, although the specifi city is limited because of the interference of other components such as infi ltrating leukocytes. Some of these con-founding factors are:

1. The luminometer instrumentation, its calibration, determination of sensitivity, dynamic range, and units used.

2. The concentration and the type of probe used. 3. The concentration and volume of the semen sample, use of reagent, and tempera-

ture of the luminometer. 4. Semen age, i.e., time to analysis after sample, is collected to the time of ROS

measurement. 5. Viscous samples and poor liquefaction interferes with chemiluminescent

signals. 6. Repeated centrifugation: Artifi cial increase in chemiluminescent signal because

of the shearing forces generated by centrifugation [ 35 ] . 7. Serum albumin. Use of media that contain bovine serum albumin can generate

spurious signals in the presence of human seminal plasma. 8. Medium pH. Luminol is sensitive to pH changes. 9. Nonspecifi c interference: Many compounds can artifi cially increase (cysteine,

thiol-containing compounds) or decrease (ascorbate, uric acid) the chemilumi-nescent signal generated by the spermatozoa. Hence, it is necessary to run sperm-free controls as integral part of the chemiluminescent assay.

13.11 Diagnostic Application of Chemiluminescence Assays

Chemiluminescent assays provide clinically signifi cant results that correlate with the quality of the ejaculate and the fertilizing potential of the spermatozoa both in vitro and in vivo. This is especially true in patients presenting with poor sperm quality such as in cases of oligozoospermia, or in samples where low-density imma-ture spermatozoa recovered following sperm preparation using a density gradient technique. Defective sperm populations such as those exhibiting presence of excess residual cytoplasmic are associated with high redox activity [ 6, 13, 36– 38 ] . This assay can also be used as an important diagnostic screening tool in the evaluating patients with unexplained or idiopathic infertility as these patients demonstrate high ROS levels in their seminal ejaculates.


13.12 Conclusion

Oxidative stress is implicated in the etiology of male infertility and sperm dysfunc-tion resulting in induction of lipid peroxidation, abnormal sperm function, and increase in mitochondrial and nuclear DNA damage. Therefore, there is an intense interest in the development and evaluation of simple, convenient techniques for monitoring the generation of ROS by human spermatozoa. The chemiluminescence assay provides andrology laboratories with a reliable, heavily-tested and used method in detecting ROS levels in patient semen samples. As infertility rates con-tinue to rise, and male-factor plays a key role in contributing to infertility in couples of reproductive age, it is critical for andrology laboratories to fully understand the chemiluminescence assay in order to provide better care for infertile patients. While there are only two probes that can be used in the assay for different measurements of ROS, there is a multitude of luminometers that are available for commercial purchase and suitable for differing laboratory settings. Andrology laboratories must review the latest literature in order to learn about the latest studies that estab-lish more precise and accurate reference levels for ROS in semen samples. Chemiluminescent probes can be used to monitor redox activity associated with sperm suspensions and differentiate this from the redox activity associated with con-taminating leukocytes as these can disrupt fertilization and DNA integrity in the spermatozoa exposed after sperm preparation and increasing their susceptibility to oxidative stress. These chemiluminescent signals are also linked to the presence of excessive residual cytoplasm in defective spermatozoa and are indicative of aber-rant spermatogenic process. The redox activity measured in defective spermatozoa by chemiluminescent increases our understanding of the etiology of male infertility. The chemiluminescence assay is a key technique in the andrology laboratory that will continue to develop and be improved upon and used in testing for suboptimal sperm quality.

References

1. Stephen E, Chandra A (1998) Updated projection of infertility in the United States: 1995–2025. Fertil Steril 70:30–34

2. Bhasin S, de Kretser DM, Baker HW (1994) Clinical review 64: pathophysiology and natural history of male infertility. J Clin Endocrinol Metab 79:1525–1529

3. Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ, Agarwal A (2000) Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing infertility investigation. Fertil Steril 73:459–464

4. de Lamirande E, Gagnon C (1992) Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl 13:379–386

5. Mammoto A, Masumoto N, Tahara M et al (1996) Reactive oxygen species block sperm-egg fusion via oxidation of sperm sulfhydryl proteins in mice. Biol Reprod 55:1063–1068


6. Aitken RJ, Irvine DS, Wu FC (1991) Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 164:542–551

7. Agarwal A, Cocuzza M, Abdelrazik H, Sharma RK (2008) Oxidative stress measurement in patients with male or female factor infertility. In: Popov I, Lewin G (eds) Handbook of chemi-luminescent methods in oxidative stress assessment. Tranworld Research Network, Trivandrum, Kerala, India, pp 195–218

8. Alvarez JG, Touchstone JC, Blasco L et al (1987) Spontaneous lipid peroxidation and produc-tion of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 8:338–348

9. Aitken RJ, Baker HW (1995) Seminal leukocytes: passengers, terrorists or good samaritans? Hum Reprod 10:1736–1739

10. Pasqualotto FF, Sharma RK, Kobayashi H, Nelson DR, Thomas AJ, Agarwal A (2001) Oxidative stress in normospermic men undergoing infertility evaluation. J Androl 22:316–322

11. Kessopoulou E, Tomlinson MJ, Barrat CLR et al (1992) Origin of reactive oxygen species in human semen spermatozoa or leukocytes. J Reprod Fertil 94:463–470

12. Aitken RJ, Buckingham DW, West KM (1992) Reactive oxygen species and human spermato-zoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemi-luminescence. J Cell Physiol 151:466–477

13. Gil-Guzman E, Ollero M, Lopez MC et al (2001) Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Reprod 16:1922–1930

14. Hendin B, Kolettis P, Sharma RK et al (1999) Varicocele is associated with elevated spermato-zoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol 161:1831–1834

15. Said TM, Agarwal A, Sharma RK, Thomas AJ, Sikka SC (2005) Impact of sperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucle-otide phosphate. Fertil Steril 83:95–103

16. Aitken RJ, West KM (1990) Analysis of the relationship between reactive oxygen species production and leukocyte infi ltration in fractions of human semen separated on Percoll gradi-ents. Int J Androl 3:433–451

17. Ochsendorf FR (1999) Infections in the male genital tract and reactive oxygen species. Hum Reprod 5:399–420

18. Shekarriz M, Sharma RK, Thomas AJ et al (1995) Positive myeloperoxidase staining (Endtz Test) as an indicator of excessive reactive oxygen species formation in semen. J Assist Reprod Genet 12:70–74

19. Wolff H (1995) The biologic signifi cance of white blood cells in semen. Fertil Steril 63:1143–1157

20. Blake DR, Allen RE, Lunec J (1987) Free radical in biological systems—a review oriented to infl ammatory processes. Br Med Bull 43:371–385

21. Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ, Agarwal A (1999) The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod 14:2801–2807

22. Sharma RK, Agarwal A (1996) Role of reactive oxygen species in male infertility. Urology 48:835–850

23. Sikka SC, Rajasekaran M, Hellstrom WJG (1995) Role of oxidative stress and antioxidants in male infertility. J Androl 16:464–468

24. Kefer JC, Agarwal A, Sabanegh E (2009) Role of antioxidants in the treatment of male infertil-ity. Int J Urol 16:449–457

25. Lanzafame FM, La Vignera S, Vicari E, Calogero AE (2009) Oxidative stress and medical antioxidant treatment in male infertility. Reprod Biomed Online 19:638–659


26. Tremellen K (2008) Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 14:243–258

27. Saleh RA, Agarwal A (2002) Oxidative stress and male infertility: from research bench to clinical practice. J Androl 23:737–752

28. Agarwal A, Said TM (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9:331–345

29. Kobayashi H, Gil-Guzman E, Mahran AM et al (2001) Quality control of reactive oxygen spe-cies measurement by luminol-dependent chemiluminescence assay. J Androl 22:568–574

30. Aitken RJ, Baker MA, O’Bryan M (2004) Shedding light on chemiluminescence: the applica-tion of chemiluminescence in diagnostic andrology. J Androl 25:455–465

31. Faulkner K, Fridovich I (1993) Luminol and lucigenin as detectors for O 2 −• . Free Radic Biol

Med 15:447–451 32. Stanley PE (1999) Commercially available fl uorometers, luminometers and imaging devices

for low-light level measurements and allied kits and reagents: survey update 6. Luminescence 14:201–213

33. Agarwal A, Allamaneni S, Said T (2004) Chemiluminescence technique for measuring reac-tive oxygen species. Reprod Biomed Online 9:466–468

34. Berthold R, Herick K, Siewe RM (2000) Luminometer design and low light detection. Methods Enzymol 305:62–87

35. Shekarriz M, DeWire DM, Thomas AJ Jr, Agarwal A (1995) A method of human semen cen-trifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol 28:31–35

36. Aitken RJ, Clarkson JS (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 81:459–469

37. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC (1989) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 10:214–220

38. Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, Thomas AJ Jr, Alvarez JG (2001) Characterization of subsets of human spermatozoa at differ-ent stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod 16:1912–1921

Documents

Studies on Men's Health and Fertility || Methods for the Detection of ROS in Human Sperm Samples