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 uid 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 uctuations. 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 inam - 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- Quantication 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 ties 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 dened 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 uid’s resistance to ow. 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 uid from the seminal vesicles and the acidic uid from the pros - Calculating the Concentration tate. The pH of the two uids 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 uid. 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.