3 Semen Analysis

Meaghanne K. Caraballo, Alyssa M. Giroski, Rakesh Sharma, and Ashok Agarwal

Introduction Semen Collection Conceiving a child is often one of the most desired goals in a Semen samples should ideally be collected after abstaining from couple’s relationship, but unfortunately this cannot be achieved ejaculation for a minimum of 2 days but not more than 1 week [3]. naturally in all cases. Infertility is a couple problem that affects Longer abstinence times can result in hyperspermia or a semen 15%–20% of the population. Of these, 30%–40% can be attrib- volume greater than 5 mL. When the patient is instructed to uted to male-factor infertility, and 30%–40% can be attributed to provide a semen sample, they should be given the appropriate female-factor infertility. In the remaining 20%, there is a com- instructions to ensure that the semen sample is collected prop- bination of both male and female factors [1,2]. To assess male erly and free of contaminants that may affect semen quality [4]. factor infertility, semen analysis is the primary test that a physi- It is recommended that semen samples be collected via masturba- cian will order. A formal laboratory evaluation of semen analysis tion only into a sterile, wide-mouthed plastic container. Samples requires adherence to strict guidelines according to the World collected via coitus interruptus should not be accepted because Health Organization (WHO) guidelines [3]. they will be contaminated with cellular debris and bacteria. One Evaluation of semen is performed using standardized refer- of the major concerns with this method of collection is that the ence ranges (Table 3.1) established by WHO in 2010 [3]. Routine ™rst portion of ejaculate, which usually contains the highest con- semen analysis includes macroscopic evaluation, including color, centration of spermatozoa, is lost. Additionally, the acidic pH of volume, pH, viscosity, and liquefaction. Microscopic evaluation, the vaginal šuid can adversely affect sperm motility. In special includes round cell count, concentration, total motility includ- cases, patients may use a specialized collection condom, such ing progressive motility retrograde semen analysis, sperm mor- as a MaleFactor-Pak. These types of condoms are made of inert phology, sperm viability; and sperm-function testing including polyurethane and do not contain spermicides and, therefore, fructose testing, hypo-osmotic swelling, and antibody testing. maintain semen motility. No lubricants or saliva should be used Advanced sperm testing is available in specialized andrology in the collection process because they may adversely affect the laboratories for evaluating oxidative stress (OS) and includes sample quality. Immediately after collection, semen samples measurement of reactive oxygen species (ROS), total antioxidant should undergo a standard liquefaction period. If the patient capacity (TAC), oxidation reduction potential (ORP) and sperm collects off site, the sample must be delivered to the laboratory DNA fragmentation (SDF). within 1 hour of collection. It must be kept at body temperature to protect against temperature šuctuations. After 60 minutes, sperm motility may be compromised [2] and, thus, it is recommended that the analysis be performed as soon as possible after liquefac- tion has occurred. TABLE 3.1 Normal Values (™fth percentile) for Semen Analysis According to WHO 2010 Guidelines Macroscopic Evaluation of Semen Parameter References The macroscopic evaluation of semen includes semen volume, Volume (mL) 1.5 pH, viscosity, liquefaction, and age of the sample. pH Sperm concentration (×106/mL) 15 Motility (% total) 40 Appearance Morphology ( normal) 4 % After liquefaction, semen samples undergo a macroscopic Vitality ( ) 58 % analysis. Normal semen sample is opaque in appearance White blood cells ( 106/ml) 1.0 106 × < × and homogenous. If the specimen has a clear appearance,

19 20 Male Infertility in Reproductive Medicine the patient’s sperm concentration may be too low or possi- bly even azoospermic. On the other hand, if it has a reddish- Microscopic Evaluation of Semen brown appearance, then there may be red blood cells present. Hematospermia (i.e., appearance of blood in the semen) can be Round Cells attributed to a variety of factors such as infection or inšam - Round cells in the semen sample consist of immature germ cells mation in any area of the male reproductive system, excessive and leukocytes. The presence of excessive immature germ cells masturbation, obstruction, tumors, or polyps. Certain phar- in the ejaculate may indicate an environment of oxidative stress maceutical drugs and medical conditions (e.g., jaundice or (OS) during spermatogenesis [6]. Therefore, it is important to dif- prostate infection) can cause an abnormal appearance of the ferentiate between these two round cells. To distinguish between semen. Urine contamination can alter the appearance of semen the two, round cells are counted in a high-power ™eld. Presence to a yellowish color. of more than 5 round cells or >1 × 106 round cells on the wet prep indicates testing for presence of white blood cells accord- 6 Semen Volume ing to WHO guidelines. Presence of <1.0 × 10 round cells may contain activated leukocytes, which can produce the ROS that are The volume of the ejaculate is largely comprised of the secre - harmful for the spermatozoa [7,8]. tions of the accessory glands such as the seminal vesicles and the prostate gland. A small portion of the volume also comes Leukocytospermia from the bulbourethral glands and the epididymis. The stan- Quanti™cation of leukocytes in semen is performed on a wet prep dard volume for the ejaculate is ≥1.5 mL by WHO reference range [3]. and is tested by the myeloperoxidase or Endtz Test [9]. This test iden- When a semen sample measures less than 0.5 mL it is ti™es the peroxidase positive granulocytes that stain dark brown: referred to as hypospermia. This abnormality can be the result i.e. macrophages and neutrophils. Presence of >1 × 106 wbc/mL of many different underlying factors such as hypogonadism, ret- of the sample is indicative of leukocytospermia [3]. rograde ejaculation, and obstruction of the lower urinary tract or a congenital bilateral absence of the vas deferens. It is also Concentration and Motility important to note that the ™rst couple of drops of ejaculate typi- The concentration of a semen analysis is calculated by the cally have a high sperm concentration. Any incomplete sample number of spermatozoa in 1 mL of semen. The reference or split ejaculates must be reported by the patient and should 6 value is ≥15 × 10 /mL. The total sperm number or count is be documented in the analysis report. An incomplete sample the total number of spermatozoa in the entire semen ejaculate. can account for reduced semen volumes. Some patients do Oligozoospermia and azoospermia are diagnosed by the total not produce an ejaculate after orgasm and instead produce dry number of sperm count. Oligozoospermia is de™ned as sperm ejaculate. This is more commonly referred to as aspermia. An 6 concentration <15 × 10 /mL. Azoospermia or cryptozoospermia accurate record of semen volume is essential. The laboratory is referred to as the absence of spermatozoa in the ejaculate. may use a graduated serological pipette, a graduated 15 mL Calculating the concentration of semen is done by properly conical centrifuge tube or weight for accuracy. The WHO rec- mixing the semen through pipetting or vortexing the ejaculate. ommends using a preweighed sterile collection container for the The sample is placed on a ™xed counting chamber. A minimum most accurate volume [3]. of 200 spermatozoa are observed under 20×. A minimum of 200 spermatozoa should be counted as suggested by WHO; a 10 × 10 Semen Viscosity grid is placed in the eye piece for counting the sperm using a phase contrast microscope and 20× objective (Figure 3.1). It is comprised Semen viscosity measures the seminal šuid’s resistance to šow. of a total of 100 squares in 10 rows and 10 columns. A row factor is High viscosity is determined by the elastic property of the semen applied to obtain the concentration as shown in Table 3.2. sample when it is free dropped from a pipette. A thread length greater than 2 cm is considered abnormally viscous. High viscos- TC = Total number of sperm counted ity after complete liquefaction can affect motility, concentration, F = Total number of ™elds and antibody-coated spermatozoa. In cases of moderate or severe RF = Row factor viscosity, a preweighed vial of trypsin powder (5 mg) can be used MF = Microscope factor to break the viscosity. S = Number of squares on a grid Concentration = (TC/F) × RF × MF/S Semen pH TABLE 3.2 The semen sample is primarily made up of the alkaline šuid from the seminal vesicles and the acidic šuid from the pros - Calculating the Concentration tate. The pH of the two šuids combined should be in the range Number of Rows Counted Row Factor of 7.2–8.2. A pH of <7.0 in a semen sample may indicate an obstruction in the ejaculatory duct [5]. This results in an 1 10 absence of alkaline secretions from the seminal vesicle šuid. 2 5 5 2 A higher pH could indicate the presence of an underlying 10 1 infection [5]. Semen Analysis 21 40X

FIGURE 3.1 Measurement of sperm concentration and motility by a ™xed counting chamber and a phase contrast microscope.

Example: TABLE 3.3 Calculating Forward Progression Concentration = (167/5) × 5 × (20/100) = 33.40 × 106/mL Grade Letter Total sperm count of the sample is calculated by multiplying the 1 D total volume by the concentration of the sample. 2 C 3 B Example: 4 A

Concentration = 33.40 × 106/mL AB ++ C Percentage forward progression = ×100 Volume of the sample = 2.0 mL AB ++ CD +

Total sperm count = 33.40 × 106/mL × 2.0 mL

= 66.80 106 × Computer-Assisted Semen Analysis Computer-assisted sperm analysis (CASA) is an alternative Motility and Forward Progression method to the manual semen analysis. The CASA technology Assessment of sperm motility is important in the routine semen was developed in the late 1980s, which followed the rise of analysis and is related to the probability of pregnancy [10,11]. If the personal computer. The development of the digitization of the majority of sperm are non-motile, the chance of fertilization video helped to analyze motility and the various types of sperm of the oocyte will be minimal. WHO recommends that total nor- kinematics (i.e., amplitude of lateral head displacement [ALH], mal motility for a semen analysis is ≥40% [3]. linearity [LIN], and curvilinear velocity [VCL]) of the sperm. Sperm are classi™ed in four different grades of motility or forward CellSoft was ™rst introduced in 1985 and was soon followed by progression. Grade 1 is the least motile grade and is classi™ed as hav- Hamilton-Thorne in 2000 and was used primarily as a research ing weak šagellar and twitching movement. Grade 2 is de™ned as tool [12]. having poor to moderate forward progression. Grade 3 is described CASA in the laboratory setting must meet the WHO criteria as having “good” to “rapid” unidirectional forward progression. and must meet the Uncertainty of Measurement (UoM) require- Lastly, grade 4 has rapid multidirectional forward progression and is ment of ±20% compared to the manual count. The laboratory thought to be the most ef™cient passage through the cervical mucus. staff must be fully trained and competent to meet the WHO crite- Calculating forward progression is based on the total motile ria. A daily quality control of CASA must be performed by using sperm in the semen sample. Calculating the motility of semen is a suspended latex bead solution that is run in accordance with done by properly mixing the semen by either pipetting or vortex- Clinical Laboratory Improvement Amendment (CLIA) regula- ing the ejaculate, placing the sample on a hemocytometer cham- tions. This test solution kit includes two level solutions that are ber or on a ™xed sperm counting chamber, and observing it under prepared with small latex particles or beads that are suspended in 20×, as described in Table 3.3. an isopycnic medium that allow the latex particles to freely šoat 22 Male Infertility in Reproductive Medicine evenly throughout the solution. The two levels that are tested have sample to collect and pellet at the bottom of the tube(s). When the an upper range and a lower range to verify that the CASA can centrifugation is complete, the supernatant should be removed. pick up high and low sperm concentrations. These beads were Another 1–2 mL of HTF will be added to the urine sample to designed primarily for capillary-loading ™xed-counting cham- reconstitute the pellet at the bottom of the tube [15]. A portion bers (e.g., Leja chambers or MicroCell ™xed-cell counting cham- of this reconstituted pellet will then be analyzed microscopically bers) but may also be used for validation of manual sperm counts for any sperm present in the postejaculate urine sample. In cases using the hemocytometer. Weekly quality control should also be when the patient is unable to provide a semen sample because of performed by the lab with an unknown semen sample; this sample no ejaculate, the patient should masturbate until orgasm is reached should be tested on the CASA and read by a technologist to make and then immediately urinate into the designated container. sure that the UoM is met and acceptable for quality control. For those individuals that are diagnosed with retrograde ejacu- The CASA system is temperature controlled at 37°C and allows lation, their samples can be processed for intrauterine insemina- the laboratory to have accurate readings for sperm concentra- tion, sperm cryopreservation, and used in in vitro fertilization tion, motility, velocity, linearity, and amplitude of lateral head (IVF) or intracytoplasmic sperm injection (ICSI) if there are a displacement. After liquefaction of the collected semen sample, suf™cient number of sperm. the technologist loads the sample on the capillary-loading cham- ber and places it on the CASA stage. Once the slide is loaded on the stage, it is analyzed by the technologist [13]. Technologists Sperm Viability manually choose the ™elds that they would like selected for an analysis to be performed on. The CASA will analyze the selected Eosin-Nigrosin (E/N) is a staining technique that assesses the ™elds and create a report. The CASA results will then be com- vitality of a semen sample. It is recommended when the initial pared to the manual concentration of the technologist, and if the motility is less than 25%. Nigrosin increases the contrast between results are within the UoM of ±20%, then the CASA results will the background and sperm heads, making sperm easier to visu- be used. If the results differ by >20% then the manual reading for alize [16]. This staining technique must be performed immedi- concentration or motility will be reported. ately after motility is assessed using sperm from the same semen sample. Stained slides can be stored for reevaluation and quality control purposes. Retrograde Semen Analysis The staining solution consists of Eosin–Y (1%) and Nigrosin (10%). The slides are labeled and one drop of lique™ed semen For some men, infertility is a result of an anatomic abnormality. sample is placed into a Boerner slide well. Two drops of Eosin Normally, the bladder neck muscles tighten right before ejacula- are added and mixed well followed by two drops of Nigrosin. tion causing the semen to exit the male reproductive system via After mixing well, two smears are made and air dried and a the urethra. In the case of retrograde ejaculation, which can also coverslip is placed and sealed with a cytoseal mounting media. be referred to as a dry orgasm because of a lack of semen, semen The slide is observed under 100× objective. Eosin stains only retropulses into the bladder upon ejaculation instead of through the dead sperm, turning them a dark pink, whereas live sperm the urethra. There are several medical conditions that may result in appear white or faint pink (Figure 3.2). The normal range is 58% retrograde ejaculation, including but not limited to Parkinson dis- according to WHO guidelines [3]. For specimens with motility ease, diabetes, multiple sclerosis, spinal cord injuries, and enlarged <25%, viability should be ≥25%. prostate. These medical conditions damage and impair the nerves and muscles that control ejaculation, and some surgeries disrupt these nerves and muscles. Even medications that treat high blood pressure, benign prostatic hypertrophy (BPH), and depression may be the culprit behind a man’s retrograde ejaculation [14]. If retrograde ejaculation is suspected, the diagnosis can be con™rmed by a urinalysis of a postejaculate urine sample. A microscopic analysis of a spun down and reconstituted urine sample can be evaluated for any sperm that may be present. To complete a retrograde urinalysis, the patient will need to ™rst col- lect their ejaculate in one container and then immediately urinate into a secondary container. This secondary container should be given to the patient before collection and should contain warm human tubal šuid (HTF) with 5% human serum albumin. HTF is formulated on the chemical composition of HTF, which aids in the manipulation of gametes or, in this case, spermatozoa. Once the patient collects their semen and postejaculatory urine samples, the semen should be allowed to undergo the standard liquefaction period of at least 20 minutes, while the urine mixed with HTF is transferred into 15-mL conical tubes and is further processed by centrifugation. During centrifugation, the HTF acts FIGURE 3.2 Measurement of sperm vitality by the Nigrosin/Eosin test. as a washing medium and causes any sperm present in the urine Viable sperm appear white and nonviable sperm appear pink. Semen Analysis 23

Hypo-osmotic Swelling Sperm Morphology The hypo-osmotic swelling (HOS) test evaluates the functional For some men and couples who are dealing with infertility, the integrity of the sperm membrane by evaluating the maintenance issue does not always lie with the concentration, volume, and motil- of equilibrium between the sperm cell and its environment. ity of the sperm alone. Sperm morphology may also play a role in This test is an alternative to the Eosin/Nigrosin test and is used male infertility due to a high number of morphologically abnormal when samples are needed for therapeutic purposes as in assisted forms in their ejaculates. Staining of a patient’s seminal smear using reproductive techniques such as intracytoplasmic sperm injec- a rapid staining process, such as Diff-Quik, allows for the quantita- tion. A functional membrane is required for a spermatozoa to tive evaluation of normal and abnormal morphological forms in an fertilize an egg. The functional integrity can be demonstrated by ejaculate [17]. Most laboratories quantify morphology using WHO allowing sperm to react in a hypo-osmotic medium. The HOS guidelines or the Kruger’s strict criteria classi™cation [3]. test presumes that only cells with intact membranes will swell in Kruger’s classi™cation strictly de™nes spermatozoa as normal hypotonic solutions. only if they fall within the set parameters of shape. Any sper- The HOS solution is prepared by combining 100 mL of dis- matozoa that does not meet this criteria is considered abnormal. tilled water with 0.735 g sodium citrate dihydrate with 1.351 g A spermatozoa is classi™ed as normal only if it meets all of the fructose [20]. Testing the spermatozoa in the semen collection, following sperm parameter requirements: Sperm head must the technologist combines 0.1 mL well-mixed semen with 1 mL appear as a smooth oval with a well-de™ned acrosome that cov- HOS solution. Gently mix the solution by drawing the sample ers 40%–70% of the sperm head with a normal length of 5–6 μm in and out of the pipette. Incubate the HOS solution test at 37°C and a width of 2.5–3.5 μm. There should be no abnormalities in for 30–60 minutes. After incubation, 1 drop of the semen mix- the neck, midpiece, and tail. The tail should be thinner than the ture is placed on a glass slide and a coverslip is placed on top. midpiece (which should be less than 1 μm in width and length), The slide is observed for tail swelling under 40× phase contrast uncoiled and approximately 45 μm. Finally, there cannot be objective (Figure 3.3) [20]. The WHO lower reference limit for any extraresidual cytoplasm greater than 20% of the area of the membrane-intact spermatozoa is 58% [3]. sperm head. The reference range according to the WHO strict criteria is 4 normal forms [3]. ≥ % Sperm Antibodies WHO criteria is considered to be less stringent when it comes to sperm morphology classi™cation. Here abnormal spermato- Anti-sperm antibodies (ASAs) are thought to coat the sperm sur- zoa are classi™ed into speci™c categories based on the speci™c face and impair motility and the fertilization process. The sper- head, tail, and midpiece abnormalities that may occur. Head and matozoa agglutinate and stick to one another by head to head, tail shape abnormalities are often classi™ed as large, small, tapered, to tail, or head to tail. The antibodies may be present in seminal or double-headed [2]. Although pregnancy is achievable with plasma secretions and reproductive tract secretions. poor morphology, the absence of acrosomes is a high predictor ASAs belong to two immunoglobulin classes: IgG and IgA. for failure during fertilization [18]. IgG antibodies on the spermatozoa form agglutinates between the particles and the motile spermatozoa. When performing the Direct SpermMar Test, a 10 μL of fresh semen sample is Sperm-Function Testing placed on a glass slide and is mixed with 10 μL of IgG suspen- sion, and 10 μL of antiserum to IgG is added and mixed [ 21]. Fructose The antiserum binds to the IgG on the surface of the beads Fructose is produced in the male reproductive tract by the semi - and on the surface of the sperm if it is present. A coverslip is nal vesicles and is released into the semen during ejaculation. placed on top, and the slide is placed in a humid chamber for Fructose is the energy source for sperm motility and is present 3 minutes. The technologist observes the slide under a 40 × in all semen specimens except for males with congenital bilat- eral absence of vas deferens or bilateral ejaculatory duct obstruc- tion. A qualitative fructose is usually performed on azoospermic specimens with a semen volume of less than 1.5 mL. Resorcinol is the reagent that is used to conduct a fructose test within the laboratory. A positive and a negative control is used when running the patient’s sample. A positive test will show an orange-brown or orange-red color, a negative test will have no change in color. To perform the test 1 mL of resorcinol solution is added to a glass tube and 0.1 mL of semen sample is added. The sample is mixed gently. The negative control is prepared by adding 1 mL of resorcinol solution in a glass test tube, and semen sample is not added. In the positive control, 0.1 mL of semen of a previously tested patient who tested positive is added to 1 mL of resorcinol solution in a glass test tube [19]. All three tubes are placed in a beaker of water on a hot plate. Once the water starts to FIGURE 3.3 Hypo-osmotic swelling test showing spermatozoa with dif- boil, the change in color is observed and documented. ferent types of swelling. 24 Male Infertility in Reproductive Medicine wobjective and score 100 motile spermatozoa that are bound ROS levels can be measured in a given sample by chemilumi- or free of the beads. The results are observed in duplicate and nescence assay using the luminometer and using luminol as the the percentage of bound spermatozoa is reported. In addition, probe. The reagents used for global measurement of ROS are the the binding site of the bead on the motile spermatozoa (sperm stock luminol probe (100 mM), the working luminol (5 mM), and head, tail, midpiece, or total sperm) is also recorded. the dimethylsulfoxide (DMSO) solution. The test is light sensitive IgA antibodies have a more critical relevance in male infer- and performed under subdued light. A luminometer is attached to tility than IgG. Patients who are presenting with IgA antibodies a computer (Figure 3.4). A total of 11 tubes are used, including have a reduced chance of natural pregnancies [22,23]. IgA anti- 3 blank tubes that contain only phosphate buffer saline (PBS), 3 bodies mainly have agglutinating properties and rarely occur negative controls that contain PBS + luminol (working solution), without the presence of IgG antibodies. These antibodies are 2 tubes that have the patient sample and + luminol, and 3 positive secreted by the accessory glands; seminal vesicles, prostate controls that contain PBS + hydrogen peroxide (50 μL) + luminol gland, and the bulbourethral glands. They are present on the (Figure 3.5). The tubes are loaded into the luminometer (Autolumat spermatozoa and occasionally in the seminal plasma but are Plus LB 953, Berthold, Oak Ridge, TN) and a real-time plot of the absent in the serum. When performing the Direct SpermMar ROS levels produced in each sample is visualized on the computer Test for IgA a fresh semen sample or spermatozoa is isolated monitor and analyzed. The number of free radicals produced is from the seminal plasma by one cycle of suspension, centrifu- measured as relative light units/sec/106 sperm [25,26]. gation, and resuspension in media. A 10-μL spermatozoa sus- pension is mixed with 10 μL of antihuman anti-IgA suspension Oxidation Reduction Potential and mixed [21]. A coverslip is placed on top, and the slide is placed in a humidi™ed chamber for 3 minutes. The technolo- Oxidation reduction potential or ORP is measured by the gist will observe the wet prep slide with a coverslip under 40× MiOXSYS System for detecting OS; this is a direct evaluation objective and read 100 motile sperm [21]. The bead-binding site of the redox balance between ROS and antioxidants. This novel on the spermatozoa (sperm head, tail, midpiece, or total sperm) system is designed to monitor the amount of oxidative or reduc- is also recorded. tive stress present in semen samples. ORP is calculated using the Nernst equation: ORP = E°−RT/nF ln([Red]/[Ox]), where E = standard reduction potential, R = universal gas constant, Reactive Oxygen Species ° T = absolute temperature, n = number of moles of exchanged

Free oxygen radicals such as superoxide anion (O2¯), hydrogen electrons, F = Faraday’s constant, [Red] = concentration of peroxide (H2O2), hypochlorite (OHCl), and hydroxyl radical reduced species, and [Ox] = concentration of oxidized species. (OH) are called ROS. Free radicals have a very short half-life The MiOXSYS System is simple and user friendly test that and are continuously produced, mainly by white blood cells uses one sensor for each patient sample [27,28]. Each test sen- (WBC) or by granulocytes and abnormal sperm [24]. Excessive sor consists of three electrodes is inserted into a galvanostatic amounts of ROS impair sperm quality. ROS levels can be mea- MiOXSYS analyzer (Figure 3.6a and b). The semen sample sured with the chemiluminescence method using luminol as a (30 μL) is loaded onto the sample port of the sensor and the sen- probe. Luminol is extremely sensitive and reacts with a variety of sor is gently inserted into the analyzer. Once the sample drop ROS at neutral pH [25]. It can measure extracellular and intracel- is loaded onto the port, the sample moves to the reference elec- lular ROS. The free radical combines with luminol to produce a trode and signals the analyzer to emit a low-voltage oxidizing light signal that is converted to an electrical signal (photon) by a current between the electrodes. The ORP value appears on the luminometer. display unit and is expressed as millivolts. It is normalized by

FIGURE 3.4 Measurement of reactive oxygen species by chemiluminescence method using a luminometer. Semen Analysis 25

Collect semen specimen Exam for volume, color and pH Perform Endtz test Perform analysis by CASA

Liqueÿed semen

123456 78 91011

Blank Negative control Test sample Positive control Add 400 μL PBS Add 400 μL PBS Add 400 μL Add 400 μL PBS + Sperm suspension + 10 μL Luminol (5 mM) + 50 μL H2O2 10 μL Luminol (5 mM) + working solution 10 μL Luminol (5 mM) working solution

FIGURE 3.5 Setup showing the preparation of blank, negative control, test sample, and positive control tubes. CASA, computer-assisted semen analyzer

H2O2, hydrogen peroxide water; phosphate buffered saline (PBS).

Soclet insertion end

Display screen

Reference cell Sensor socket Sensor module Sample port (a)(b)

FIGURE 3.6 Measurement of oxidation reduction potential (ORP) using (a) MiOXSYS analyzer and (b) sensor.

the sperm concentration and expressed as millivolts/106 sperm/ Seminal plasma TAC measurement can be done using the anti- mL of semen. Values greater than 1.37 millivolts/106 sperm/mL oxidant assay kit and reagents (Cat #709001; Cayman Chemical, of semen are considered as abnormal and indicative of OS [27]. Ann Arbor, MI). The principle of the assay is based upon the ability of all anti - oxidants in the seminal plasma to inhibit the oxidation of the Total Antioxidant Capacity ABTS (2,20-Azino-di-[3-ethylbenzthiazoline sulphonate]) to Antioxidants present in the seminal plasma provide a good indi- ABTS+ resulting in the change of the absorbance at 750 nm to cation of the ability of the antioxidants to scavenge the excessive a degree that is proportional to the concentration of the anti - production of ROS and reduce OS in a given semen sample [29]. oxidant in the sample [30]. The capacity of the antioxidants 26 Male Infertility in Reproductive Medicine present in the sample to prevent ABTS oxidation is compared with that of standard 6-hydroxy-2,5,7,8- tetramethylchroman- Sperm DNA 2-carboxylic acid (Trolox), a water-soluble tocopherol ana- logue. Ten microliters of metmyoglobin and 150 μL of chromogen are added to all standard and sample wells. The reaction is initiated by adding 40 μL of hydrogen perox- ide as quickly as possible. The plate is incubated for 5 minutes DNA breaks at room temperature on a horizontal shaker and absorbance monitored at 750 nm using a microplate reader. Results are 3’–OH 3’–OH reported as micromoles of Trolox equivalent. Values less than 3’–OH 1,900 micromoles of TAC are considered as abnormal and fre- quently reported in men who are infertile and are indicative dUTP TdT enzyme of OS [29].

TUNEL Terminal Deoxynucleotidyl Transferase assay dUTP Nick End Labeling The terminal deoxynucleotidyl transferase dUTP nick end label- ing (TUNEL) assay is advantageous because it is a direct test that can measure “true” single- and double-stranded DNA fragmen- tation by directly incorporating modi™ed nucleotides into the site of damage. The TUNEL test uses a template-independent DNA poly- merase called terminal deoxynucleotidyl transferase (TdT) that nonpreferentially adds deoxyribonucleotides to 3′ hydroxyl (OH) single- and double-stranded DNA. Deoxyuridine triphosphate (dUTP) is the substrate that is added by the TdT enzyme to the FIGURE 3.7 Measurement of sperm DNA fragmentation using a bench free 3′-OH break-ends of DNA. The šuorescence is directly pro- top šow cytometer. TUNEL, terminal deoxynucleotidyl transferase dUTP portional to the number of strand breaks [31]. Two laser detectors nick end labeling. are used: FL1 (488) with a standard 533/30 Band Pass (BP) that detects green šuorescence šuorescein isothiocynate (FITC) and FL2 with standard 585/40BP that detects red or propidium iodide (PI) šuorochrome. Semen samples are tested for sperm DNA fragmentation using Conclusion an Apo-Direct kit (BD Pharmingen, CA, USA) with Accuri C6 Conducting a routine semen analysis is the ™rst step of labo - šow cytometer (BD Biosciences, MI, USA) [32]. Each sample ratory evaluation of semen analysis. It is comprised of both is run along with negative and positive controls. Results are macroscopic and microscopic tests conducted using the rec- expressed as the percentage of sperm with DNA fragmentation ommendations set forth by WHO. In addition, advanced sperm (%DNA fragmentation) using the šow cytometer software. tests comprised of ROS, TAC, SDF, and ORP can be included. A minimum of 10,000 events are recorded. To perform this test, These tests can provide additional information on the qual- 6 2.5 × 10 sperm are ™xed in 3.7 % paraformaldehyde solution. ity of the semen to the clinician in the management of male This is replaced by ice cold 70% ethanol. After removal of the infertility. ethanol and washing with the “Wash buffer,” the samples are incubated with 50 μL of the staining solution for 1 hour at room temperature. A positive sample is prepared by incubating the REFERENCES sperm with 2% of hydrogen peroxide for 1 hour at 50°C. TdT 1. Winters BR, Walsh TJ. The epidemiology of male infertility. is omitted from the negative control. At the end of incubation Urol Clin North Am. 2014;41:195–204. period, the excess stain is removed by washing with the “Rinse 2. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique buffer” twice. The rinse solution is replaced by propidium view on male infertility around the globe. Reprod Biol iodide and the tubes are ready for measurement of SDF. Endocrinol 2015;13:37. The samples are run on a bench top šow cytometer (Figure 3.7) 3. World Health Organization. WHO laboratory manual for and the data is acquired in the “Collect mode.” The alignment the examination and processing of human semen. Geneva strategy is adopted using the “virtual alignment strategy” as 2010. per the manufacturer’s instructions. The ™nal SDF is expressed 4. Agarwal A, Gupta S, Sharma R. Basic Semen Analysis. In: as percentage of DNA fragmentation. A cut-off value of 16.7% Andrological Evaluation of Male Infertility: A Laboratory has been recently shown to have 91.6% speci™city and positive Guide. Editors: Agarwal A, Gupta S, Sharma R. Springer predictive value of 91.4% [31]. Publishing, New York, 2016. Chapter 4, pp. 39–46. Semen Analysis 27

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