Received: 6 February 2020 | Accepted: 1 April 2020 DOI: 10.1111/and.13614

INVITED REVIEW

Diagnostic value of routine analysis in clinical andrology

Saradha Baskaran1 | Renata Finelli1 | Ashok Agarwal1 | Ralf Henkel1,2

1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, Abstract USA Infertility is a major health issue affecting over 48.5 million couples around the world, 2 Department of Medical Bioscience, with the male factor accounting for about 50% of the cases. The conventional semen University of the Western Cape, Bellville, South Africa analysis recommended by the World Health Organization (WHO) is the cornerstone in the evaluation of male fertility status. It includes macroscopic and microscopic Correspondence Ashok Agarwal, American Center for evaluation of the ejaculate, which reflects the production of spermatozoa in the tes- Reproductive Medicine, Cleveland Clinic, tes, the patency of the duct system and the glandular secretory activity. Evaluation Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH 44195, USA. of seminal fructose, sperm vitality and leucocytes (Endtz test) are useful adjuncts Email: [email protected] to that provide information on specific clinical conditions. Though several computer-assisted sperm analysis (CASA) systems have been developed, con- ventional methods for semen analysis are still widely accepted in clinical practice. This review summarises the conventional techniques used in routine semen analysis and their diagnostic value in clinical andrology.

KEYWORDS diagnostic value, , semen analysis, spermatozoa

1 | INTRODUCTION chromosome Y), cancer, systemic diseases, medical therapies or exposure to endocrine disruptors (Fainberg & Kashanian, 2019; Infertility is defined as the inability of a couple to conceive after Miyamoto et al., 2017). In addition, lifestyle-associated factors such 12 months of regular, unprotected sexual intercourse. It is one of the as smoking, drinking, drug abuse, high energy-based diet, obesity major health issues affecting about 48.5 million couples around the and psychological stress negatively impact the male reproductive world, with the male factor accounting for about 50% of the cases potential (Durairajanayagam, 2018). Evidence suggests that oxida- (Mascarenhas, Flaxman, Boerma, Vanderpoel, & Stevens, 2012). tive stress plays an important role in the establishment of male infer- Reportedly, the global prevalence of male infertility ranges be- tility, accounting as a causative factor in 30%–80% of infertile men tween 2.5% and 12%, with a higher prevalence in Central/Eastern (Agarwal et al., 2019). Europe and Australia (Agarwal, Mulgund, Hamada, & Chyatte, 2015). Semen analysis is the most fundamental step in the evalua- A cross-sectional population survey revealed a strong associa- tion of the male fertility potential. It reflects the testicular func- tion between increased incidence of male infertility with high so- tionality of spermatozoa production as well as the patency of the cio-economic status and delayed parenthood (Datta et al., 2016). duct system and the secretory activity of the accessory glands Furthermore, it is estimated that around 50% of men requiring treat- (WHO, 2010). Semen analysis helps in investigating male infer- ment for infertility seek for help (Datta et al., 2016). tility and provides essential information regarding the contribu- Several factors can cause male infertility, which include testicular tion of the male factor in an infertile couple (Pfeifer et al., 2015). and hypothalamic-pituitary diseases (such as cryptorchidism, orchi- Traditional semen characteristics provide a predictive value for in tis, genital tract infections, varicocele, obstructions of the male gen- vivo conception, success of in vitro fertilisation as well as manage- ital tract and hypogonadism), genetic conditions (such as Kallmann ment of couples requiring assisted reproductive techniques (ART) or Klinefelter syndromes, globozoospermia and microdeletions of (Bonde et al., 1998; Buck Louis et al., 2014). It also helps in the

Andrologia. 2020;00:e13614. wileyonlinelibrary.com/journal/and © 2020 Blackwell Verlag GmbH | 1 of 12 https://doi.org/10.1111/and.13614 2 of 12 | BASKARAN et al. identification of reversible medical conditions which can impact groups (Homonnai, Matzkin, Fainman, Paz, & Kraicer, 1978; Zhao fertility (Jungwirth et al., 2018). et al., 2016). In this review, the World Health Organization (WHO, 2010) Finally, the bulbourethral glands contribute <5% (<0.15 ml) of guidelines for the conventional methods used in routine semen the total ejaculate, producing a clear secretion rich in mucoproteins analysis and adjunct tests (e.g. fructose test, sperm vitality tests and (Puppo & Puppo, 2016). Endtz test) are discussed with reference to clinical interpretation of each variable. Furthermore, the diagnostic value and limitations of standard semen analysis as well as technological advances in the 3 | SEMEN ANALYSIS field are summarised. 3.1 | WHO 2010 (5th edition)

2 | SEMEN The WHO laboratory manual has been the standard reference handbook for semen analysis since 1980. The latest, updated ver- Semen is a heterogeneous fluid released at the time of , sion (5th edition) of the WHO guidelines, published in 2010, pro- which is composed of cellular and noncellular fractions. The cel- vides recommendations to improve the standardisation of semen lular fraction includes mature spermatozoa, leucocytes, immature quality evaluation and the comparability of results obtained by dif- germ cells and epithelial cells (in rare pathological cases even eryth- ferent laboratories. The 2010 edition is composed of three parts: rocytes), while the noncellular fraction is a water-based seminal (a) the analysis of semen quality, including routine methods, more plasma constituted by the secretions of , , advanced optional tests and methods applied for clinics and re- bulbourethral and glands (Milardi, Grande, Vincenzoni, search; (b) preparation of sperm, including ejaculated spermatozoa Castagnola, & Marana, 2013). as well as those retrieved surgically, with a focus on sperm cryo- Testis and epididymis account for about 5% of the total seminal preservation; and (c) a final section on quality control, to ensure plasma volume (~0.15 ml). The epididymal fraction is rich in neutral a strict control of such analysis. In comparison with previous edi- α-glucosidase, the main marker of epididymal functionality (Qiu, Chu, tions, the 5th edition focuses mainly on preparation of solutions, Zhang, Luo, & Quan, 2018), and L-carnitine, a molecule involved in procedures, mathematical calculations and interpretations of re- the mitochondrial β-oxidation pathway (Li, Li, & Huang, 2007). Other sults in order to explain how to carry out each procedure and re- molecules include microRNAs, glyceryl phosphorylcholine and mal- duce the intra- and inter-laboratories variability. The section on the tase (Hu, Wu, Guo, Li, & Xiong, 2014; Rosecrans, Jeyendran, Perez- evaluation of sperm count has been revised to consider a minimum Pelaez, & Kennedy, 1987). of at least 200 spermatozoa for the assessment of correct sperm Seminal vesicles contribute to 50%–65% of the total ejaculate concentration. The evaluation of sperm motility was changed in the (1.5–2.0 ml), while the prostatic secretions account for 20%–30% 5th edition to progressive, nonprogressive and immotile spermato- (0.6–0.9 ml) of the ejaculate. The secretion of seminal vesicles is zoa in order to reduce the bias linked to the subjectivity of the mi- rich in fructose (1.5–6.5 mg/ml), which is involved in sperm energy croscopic evaluation, while previous editions differentiated sperm metabolism and motility (Buckett & Lewis-Jones, 2002). Seminal motility in grades from a to d. vesicles also produce prostaglandins, which enhance sperm motil- ity and inhibit the female immune response against spermatozoa (Adefuye, Adeola, Sales, & Katz, 2016), lactoferrin having anti- 3.1.1 | Reference values microbial activity (Buckett, Luckas, Gazvani, Aird, & Iwan Lewis- Jones, 1997), and the glycoproteins semenogelins 1 and 2. The The 5th edition provides updated reference values (Table 1) ob- semenogelins are involved in the prevention of premature sperm tained from semen parameters of 1953 proven fertile men in 8 dif- capacitation and in the formation of the seminal coagulum fol- ferent countries located on three continents (WHO, 2010). Due lowing ejaculation (De Lamirande, Yoshida, Yoshiike, Iwamoto, & to the highly right-skewed distribution of semen parameters, the Gagnon, 2001). 5th percentile has been considered as the lower reference value The prostatic secretion is rich in citric acid, which binds Ca2+ for each variable. However, it must be considered that having a and regulates semen liquefaction and coagulation in synergy with normal semen evaluation does not guarantee the fertility status. proteolytic enzymes (lysozyme, α-amylase, β-glucuronidase) and Decreased reference values have been reported in the 5th edition prostatic specific antigen (PSA), a trypsin-like protease, which compared to the 4th for volume (0.5 ml lower), sperm concentra- cleaves the semenogelin proteins. Furthermore, cholesterol and tion (5 million/ml lower), motility and morphology (10% lower). phospholipids help in the stabilisation of the sperm membrane, These changes in the reference values have had a great impact while a number of cations (zinc, calcium, magnesium) are import- on the classification of male fertility, especially for those patients ant cofactors of seminal enzymes, pH regulation, bactericidal who were previously classified as having ‘abnormal’ semen qual- activity and protection of sperm chromatin by stabilising the S-S ity but are being regarded as ‘normozoospermic’ according to BASKARAN et al. | 3 of 12

TABLE 1 Lower reference values for semen parameters according to the WHO 5th edition guidelines (WHO, 2010)

Lower reference value (5th Semen parameter percentile)

Macroscopic parameters Appearance Grey-opalescent Viscosity Forms small discrete drops (thread < 2 cm long) Volume 1.5 ml pH 7.2 Microscopic parameters Sperm concentration 15 × 106 sperm/ml Total sperm count 39 × 106 sperm/ml Total motility 40% Progressive motility 32% Morphology 4% Vitality 58% FIGURE 1 Semen analysis: macroscopic and microscopic semen Peroxidase-positive leucocytes 1 × 106 cells characteristics Seminal fructose 13 µmol/ejaculate

can be short, bent, coiled or double. Any borderline morphology the current 5th edition reference values (Alshahrani et al., 2018; should be considered as ‘abnormal’, in order to reduce the degree Murray et al., 2012). of operator subjectivity in the microscopic evaluation (Menkveld et al., 2011).

3.1.2 | Strict criteria classification— sperm morphology 3.2 | Sample collection

In agreement with the previous editions, the 5th edition of 3.2.1 | Abstinence time and liquefaction the WHO laboratory manual reinforces the sperm morphology ‘strict criteria’ defined in 1988 (Kruger et al., 1988; Menkveld, The WHO guidelines recommend sexual abstinence for 2–7 days Stander, Kotze, Kruger, & van Zyl, 1990). It describes the sper- before sample collection (WHO, 2010). Such range of time may be matozoa that successfully penetrate the cervical mucus as mor- considered arbitrary as the influence of abstinence time on semen phologically ‘normal’ (Kruger et al., 1988). According to the ‘strict parameters is still debated. Evidence shows that a shorter period criteria’, a morphologically normal is defined as hav- of abstinence is associated with an improvement of sperm motil- ing a smooth oval-shaped head (length: 3–5 µm; width: 2–3 µm) ity, morphology and DNA integrity, while a longer abstinence time with an acrosome, accounting for 40%–70% of the volume, an axi- improves semen volume and sperm count (Hanson, Aston, Jenkins, ally attached midpiece of 1.5 times the length of the head and a Carrell, & Hotaling, 2018). uniform 45 µm long tail, thinner than the intermediate midpiece Semen evaluation must be carried out after liquefaction, which (Menkveld, Holleboom, & Rhemrev, 2011). Several studies have can occur within 15 min at room temperature after ejaculation, al- shown that the normal spermatozoa defined as per the ‘strict cri- though it may need up to 60 min. Continuous gentle mixing or ro- teria’ were able to bind selectively to the zona pellucida, there- tation of the sample (room temperature/incubator at 37°C) aids to fore associating good morphology to better reproductive function obtain a homogeneous watery sample. If the liquefaction of semen (Liu & Baker, 1992; Menkveld, Franken, Kruger, Oehninger, & does not occur within 60 min, then it is referred to as delayed lique- Hodgen, 1991). faction, for which enzymatic or mechanical digestion are considered The 5th edition has several tables showing abnormalities of (WHO, 2010). sperm head, neck and tail (WHO, 2010). Head defects include abnormal shapes classified as tapered, pyriform, round, amor- phous and vacuolated as well as double heads. Abnormal neck 3.2.2 | Sample handling—the do's and the don'ts and midpiece can be categorised as asymmetrical insertion of the midpiece into the head, abnormally thin, thick, sharply bent or The sample handling is crucial for proper evaluation of macro- and mi- having excessive cytoplasmic residue. Finally, abnormal tail forms croscopic features (Figure 1) of semen (WHO, 2010). Once the sample 4 of 12 | BASKARAN et al. is collected in a sterile, nontoxic container, it should be kept at 37°C prostate and seminal vesicles (Harchegani, Rahmani, Tahmasbpour, to favour liquefaction. If the sample is collected at home, it must be & Shahriary, 2019). Furthermore, infection and inflammation of ac- transported to the laboratory within one hour. However, the collection cessory glands are also associated with semen hyperviscosity (Elia in a private room close to the laboratory is preferable in order to avoid et al., 2009). Conversely, absence of coagulum may be due to ob- fluctuations in temperature and record the exact time of collection. struction in the ejaculatory duct or congenital absence of seminal The collection during sexual intercourse is allowed only if the subject vesicles (Andrade-Rocha, 2005). is unable to collect the sample by masturbation. However, commer- cial latex condoms should be avoided as they contain spermicidal sub- stances (Jones, Kovacs, Harrison, Jennings, & Baker, 1986). The entire 3.3.4 | Volume ejaculated fraction should be collected, as the loss of a part may result in a false evaluation of semen parameters. Semen evaluation should Semen volume reflects the overall functionality of the accessory sex be conducted within an hour of liquefaction to avoid dehydration or glands. The measure of semen volume is a critical step as it deter- thermal variations from affecting semen quality. Like any other biologi- mines the calculation of the total number of spermatozoa and other cal fluid, semen should be considered potentially infectious due to the nongerminal cell counts. The WHO (2010) recommends weighing potential presence of agents such as HIV, hepatitis or herpes simplex the sample in the same container in which it is collected in order to virus. Therefore, sterile materials, safety guidelines and good labora- avoid the loss of sample during the transfer. Then, the volume would tory practices must be strictly adhered to reduce biological risk. be calculated assuming a semen density equal to 1g/ml. Otherwise, the sample can be collected into a graduated cylinder and directly measured. The sample is considered to be normal if the volume of 3.3 | Macroscopic evaluation of semen— the ejaculate is about 1.5–6.0 ml (WHO, 2010). Incomplete collec- Assessment and clinical interpretation tion, obstruction of ejaculatory ducts, congenital bilateral absence of the vas deferens (CBAVD), partial or hypo- 3.3.1 | Semen age gonadism can result in low semen volume. On the other side, higher semen volume () may indicate inflammation of the ac- Semen age is defined as the period between sample collection and cessory glands. initiation of semen analysis following liquefaction. It should not ex- ceed 60 min in order to prevent variation of semen quality due to external factors (WHO, 2010). 3.3.5 | pH

The pH of semen is an indicator of balance between acidic prostatic 3.3.2 | Appearance and colour secretion and alkaline seminal vesicular secretion. For evaluation of the pH, a drop of sample is placed on a pH paper and the result- Normal liquefied semen should appear homogeneous with a grey- ant colour is compared against the calibration strip. According to the opalescent colour. In case of high sperm concentration, semen WHO manual, a pH of 7.2 is set as the lower threshold value. In case appears opaque, while it appears more transparent in case of low of CBAVD and obstruction of ejaculatory ducts, the loss of seminal sperm concentration. The ejaculate may also appear in red-brown vesicles’ fraction results into a more acidic pH (pH < 7), while a more nuances due to the presence of erythrocytes (haematospermia), alkaline pH is usually related to poor sample handling and delay in while a yellowish colour could be due to liver diseases or consump- analysis. tion of certain vitamins/drugs.

3.4 | Microscopic evaluation of semen— 3.3.3 | Viscosity Assessment and clinical interpretation

Normally, semen coagulates upon ejaculation and liquefies within 3.4.1 | Agglutination 15 min. Viscosity reflects the seminal fluid's resistance to flow. It is assessed by aspirating the sample into a pipette and allowing it Adherence of spermatozoa to each other forming clumps is known to drop by gravity. A normal semen sample forms small discrete as agglutination. This phenomenon affects sperm motility and is drops, while formation of thread >2 cm is indicative of hyperviscos- graded according to the number of spermatozoa involved and the ity. High viscosity interferes with the evaluation of sperm motility site of agglutination (head-to-head, tail-to-tail or mixed). Presence of and concentration. It can be reduced by diluting the sample with agglutination interferes with the assessment of sperm motility and a physiological medium or by the addition of proteolytic enzymes concentration. It is indicative of the presence of antisperm antibod- (Du Plessis, Gokul, & Agarwal, 2013). Hyperviscosity is indicative ies and further testing is required to deduce an immunological cause of accessory gland dysfunction resulting in inadequate secretion of of infertility (Krapez, Hayden, Rutherford, & Balen, 1998). BASKARAN et al. | 5 of 12

3.4.2 | Presence of round cells progressive, nonprogressive and immotile sperm, with the lower ref- erence value of 40% and 32% for total and progressive motility, re- Besides spermatozoa, round cells can be observed in the ejacu- spectively (WHO, 2010). For the evaluation of motility, an aliquot of lated sample, which include leucocytes and immature germ cells. If liquefied sample is loaded into the counting chamber and examined the round cell concentration exceeds 1 x 106/ml, then a differen- under phase contrast microscope at ×200 or ×400 magnification. A tial staining technique should be performed on the semen smear minimum of 200 spermatozoa in a total of at least 5 fields in each to distinguish leucocytes and immature germ cells (WHO, 2010). replicate is evaluated. The total number of progressive and nonpro- Leucocytospermia is reported when the seminal leucocyte con- gressive motile spermatozoa as well as the total number of immotile centration is higher than 1 × 106 cells/ml. Furthermore, round sperm is counted to calculate the percentage of total motility. cells can be precisely identified and quantified by detecting Although it is not indicative of any specific condition, a reduc- peroxidase activity and the antigen CD45 (immunohistochemis- tion in sperm motility under the lower reference value (astheno- try) (WHO, 2010). The presence of round cells in the ejaculated zoospermia) could be due to infection/inflammation of accessory sample is a possible indication of spermatogenic insult and uro- sex glands, testicular (e.g. varicocele, cryptorchidism and testicu- genital infection (Brunner, Demeter, & Sindhwani, 2019; Palermo lar cancer) or related to lifestyle and environmental risk factors, et al., 2016). including pollution exposure, alcohol and drug consumption, smoking, psychological stress (Shahrokhi, Salehi, Alyasin, Taghiyar, & Deemeh, 2019). These factors increase seminal oxidative stress 3.4.3 | Sperm concentration resulting in the impairment of sperm motility due to peroxida- tive damage of the sperm plasma membrane or flagellar proteins. Sperm concentration is defined as the number of spermatozoa per Complete absence of motility is observed in men with primary cil- unit volume of semen and is directly proportional to the number of iary dyskinesia or antisperm antibodies (Cui et al., 2015; Munro sperm discharged and the amount of fluid diluting them. Therefore, et al., 1994). sperm concentration is influenced by the functionality of repro- ductive organs. Sperm concentration is usually assessed by load- ing an aliquot of liquefied sample into a haemocytometer chamber. 3.4.5 | Sperm morphology Depending on the chamber used, a specific pattern of gridlines is used to count at least 200 spermatozoa per replicate under the Sperm morphology is one of the most challenging sperm param- phase contrast microscope at × 200 or × 400 magnification. eters to analyse and interpret as there is a significant variation in Often the terms ‘sperm concentration’ and ‘total sperm number’ the shape of spermatozoa within an ejaculate as well as between are confused or misunderstood as synonyms. The total sperm num- different ejaculates of the same individual. Nevertheless, the ber is a measure of the testicular sperm productivity and is described evaluation of sperm morphology is a critical component in deter- as the total number of spermatozoa in the entire ejaculate. It is cal- mining the sperm quality. The assessment of sperm morphology culated by multiplying sperm concentration and semen volume. The involves smearing a drop of a well-mixed sample on a slide fol- total number of spermatozoa and progressively motile spermatozoa lowed by air-drying, fixing, staining and microscopic examination. are useful parameters in recommending ART treatment as they indi- The recommended staining solutions include Papanicolau, Shorr cate the total number of potentially functional spermatozoa available. or Diff-Quick, which result in differential staining of sperm head, A semen sample is defined as oligozoospermic when the acrosome region, tail and cytoplasmic residue (Henkel et al., 2008; sperm concentration is lower than 15 × 106 spermatozoa per mL WHO, 2010). Atypical morphology is due to abnormal spermato- and as azoospermic when there are no spermatozoa in the ejacu- genesis or maturation, while specific morphological defects such as late (WHO, 2010). In patients suspected for , at least globozoospermia, multiple tail abnormalities, headless spermatozoa two samples should be collected on different days. The samples and fibrous sheath dysplasia are indicative of genetic abnormalities should be centrifuged, and the pellet examined for spermatozoa. (Gatimel, Moreau, Parinaud, & Léandri, 2017). When samples are Azoospermia can be classified as obstructive and nonobstructive evaluated according to strict criteria (lower reference value equal azoospermia based on the evaluation of the other semen parame- to 4%), an association with reproductive outcomes has been re- ters, such as volume, pH and fructose, which are generally lower in ported, although results are still contradictory due to the high rate case of obstruction (Wosnitzer, Goldstein, & Hardy, 2014). of inter-laboratory variations which makes it difficult to use sperm morphology in clinical investigation (Gatimel et al., 2017).

3.4.4 | Sperm motility 3.5 | Frequency of testing Sperm motility should be assessed immediately following liquefac- tion in order to avoid dehydration or variation induced by changes Due to high biological variability, at least two semen samples in pH or temperature. The WHO 5th edition classifies motility into should be evaluated to characterise semen quality, particularly 6 of 12 | BASKARAN et al. in case of azoospermia. However, one semen analysis is recom- 4.2 | Sperm vitality mended in case of normal parameters (Jungwirth et al., 2018; WHO, 2010). Sperm vitality is determined based on the membrane integrity of the cells and can differentiate live from dead immotile sperm. Sperm vi- tality assessment is recommended for all samples with less than 40% 4 | ADJUNCT TESTS progressive motile spermatozoa.

4.1 | Seminal fructose 4.2.1 | Eosin-Nigrosin staining 4.1.1 | Fructose test Sperm vitality is most commonly assessed by the Eosin-Nigrosin Under the influence of heat and low pH, fructose reacts with indole staining (Björndahl, Söderlund, & Kvist, 2003). This method is and forms a coloured complex, which absorbs light at a wavelength based on the principle that damaged plasma membrane, as seen of 470 nm (Toragall, Satapathy, Kadadevaru, & Hiremath, 2019). For in dead spermatozoa, allows the entry of membrane-imper- this test, 5 µl of seminal plasma is incubated with ZnSO4 (63 μmol/L) meant stain, while the vital sperms exclude the dye and remain and NaOH (0.1 mol/L) for 15 min at room temperature, to allow unstained. deproteinisation. Subsequently, the sample is centrifuged, and an A drop of semen sample is mixed with an equal volume of Eosin- aliquot of the supernatant is incubated with indole reagent and hy- Nigrosin solution and smeared on a glass slide. The slides are exam- drochloric acid at 50°C for 20 min and then cooled on ice for 15 min. ined with bright optics at ×1,000 magnification and oil immersion. The absorbance of the coloured complex is measured spectropho- Nonviable sperm stains light-to-dark pink in colour while the live tometrically at 470 nm. The fructose concentration is calculated by cells appear white (Figure 2a). The use of nigrosin facilitates the mi- comparing the absorbance against a standard curve (WHO, 2010). croscopic evaluation by producing a dark-colour background, thus enabling the counting of lightly stained spermatozoa. The number of stained (dead) and unstained (vital) cells are counted in a total of 4.1.2 | Indications for testing and reference value 200 spermatozoa evaluated in each replicate and the percentage of viable cells calculated. Fructose is mainly secreted by seminal vesicles and therefore con- sidered a seminal marker of gland functionality. According to WHO (2010), the lower reference value is 13 μmol per ejaculate. Evaluation 4.2.2 | Hypo-osmotic swelling (HOS) test of seminal fructose is suggested when an obstruction is suspected. Low levels of seminal fructose along with low sample volume and The HOS test is an alternative test used to assess sperm vitality. pH is indicative of obstructive azoospermia. Furthermore, low con- The test differentiates live and dead spermatozoa based on the oc- centration of seminal fructose can be noted in men with CBAVD or currence of flagellar swelling when spermatozoa are incubated in partial retrograde ejaculation (Daudin et al., 2000; WHO, 2010). a hypo-osmotic solution. The HOS test is based on the fact that

FIGURE 2 Sperm vitality tests (a) Eosin-Nigrosin staining (b) Hypo-osmotic swelling test BASKARAN et al. | 7 of 12

TABLE 2 Characteristics of sperm Kinematic kinematics assessed by CASAa Characteristics parameter Description

Sperm motion velocity Curvilinear The velocity along a curvilinear path, velocity (VCL) considering the movement of the head Straight-line The velocity along a straight-line path between velocity (VSL) the first detected position and the last one Average path The average velocity of the sperm head along motility (VAP) the entire path. Sperm velocity ratio Linearity (LIN) The linearity of a curvilinear path, calculated as VSL/VCL Wobble (WOB) A measure of oscillation of the actual path about the average path, calculated as VAP/ VCL Straightness (STR) Linearity of the average path, calculated as VSL/VAP Sperm wobble Amplitude of The magnitude of lateral displacement of the lateral head sperm head along its average path displacement (ALH) Beat-cross The average rate at which the curvilinear path frequency (BCF) crosses the average path Mean angular The time-averaged absolute values of the displacement instantaneous turning angle of the sperm (MAD) head along its curvilinear trajectory.

aAdapted from WHO guidelines (WHO, 2010). only live cells having an intact, semipermeable membrane can spermatozoa for intracytoplasmic sperm injection (ICSI) (Cincik swell under hypo-osmotic conditions. The swelling solution is pre- et al., 2007; WHO, 2010). The WHO manual reports 58% as the pared by mixing sodium citrate dihydrate (0.735 g) and d-fructose lower reference limit for sperm vitality and suggests to analyse the (1.351 g) in 100 ml of purified water. An aliquot of the sample is semen sample soon after liquefaction in order to avoid the adverse mixed with the hypo-osmotic solution and incubated for 30 min (in effects of dehydration or fluctuations in temperature or pH on vi- case of routine diagnostics) or 5 min (if the spermatozoa are pro- tality (WHO, 2010). cessed for ART) at 37°C. Following incubation, a wet preparation is subjected to examination under phase contrast optics at ×200 or ×400 magnification. A total of 200 spermatozoa are evaluated 4.3 | Endtz test in each replicate and the results are expressed as a percentage of viable cells. The swollen spermatozoa (viable) are identified by 4.3.1 | Principle and methodology change in the shape of the sperm cells as reflected by coiling of the tail (Figure 2b). Polymorphonuclear leucocytes (PMN, neutrophils) are the pre- dominant form of leucocytes in the human ejaculate. The Endtz test is a simple and inexpensive method used for screening granu- 4.2.3 | Indications for testing and reference value locytes based on the histochemical identification of peroxidase enzyme, which is characteristic of granulocytes. This test involves Eosin-Nigrosin staining is indicated in case having high percentage ortho-toluidine staining of peroxidase-positive cells and is use- of immotile sperm. It is clinically important to assess the vitality re- ful in distinguishing leucocytes from the other round cells, which sults in conjunction with motility results of the same sample. The are peroxidase free. Furthermore, this technique fails to detect presence of a high percentage of vital, but nonmotile spermato- activated polymorphs that have released their granules as well as zoa, may be suggestive of structural defects in flagella, whereas a other peroxidase-negative leucocytes, such as lymphocytes, mac- larger proportion of nonviable and immotile spermatozoa may indi- rophages and monocytes (WHO, 2010). In this method, an aliquot cate epididymal pathology (Chemes & Rawe, 2003; Correa-Pérez, of semen sample (20 µl) is mixed with twice the volume of Endtz Fernández-Pelegrina, Aslanis, & Zavos, 2004). Since the HOS test solution and incubated for 5 min at room temperature in the dark. does not kill or damage spermatozoa, it is indicated when stain- Following incubation, an aliquot of the mixture is loaded onto a ing of spermatozoa must be avoided, for example when selecting hemocytometer chamber and examined for dark brown-stained 8 of 12 | BASKARAN et al. peroxidase-positive cells under a phase contrast optics at ×200 or 5.2 | Parameters evaluated and their significance ×400 magnification. The majority of the CASA system allows analysis of classic sperm characteristics (sperm concentration, motility, morphology and vi- 4.3.2 | Indications and reference value tality) as well as sperm kinematics, which cannot be determined by microscopic evaluation. The characteristics of sperm kinematics The Endtz test is performed when the seminal concentration of round are defined by sperm motion velocity, velocity ratios and wobble cells is higher than 1 × 106 cells/ml (WHO, 2010). The condition of leu- (Table 2). The assessment of these multiple parameters of sperm cocytospermia is defined as a seminal leucocyte concentration that kinematics allows a detailed four grade categorisation [Grade 1— is higher than 1 × 106 cells/ml and has been associated with genital immotile; Grade 2—nonprogressive; Grade 3—slowly progressive; infections/inflammatory conditions, high antisperm antibodies and Grade 4—rapidly progressive] of sperm motility when compared to elevated levels of ROS (Lackner et al., 2006; Mahfouz et al., 2010). a three-grade classification proposed by the WHO for conventional assessment. Classifying progressive motility into rapid and slow by manual assessment is not possible without significant bias. 5 | COMPUTER-ASSISTED SPERM Generally, CASA systems classify the sperm head and midpiece ANALYSIS (CASA) as normal or abnormal based on the dimension of head and mid- piece, head ellipticity and regularity, and the measurement of the 5.1 | Manual assessment versus CASA acrosome area using different staining methods. The automated sys- tems have greater precision and reproducibility than manual assess- The CASA instruments were first introduced to the market in the ment. However, the accuracy of sperm morphometric assessment 1980s with the objective of reducing subjectivity, labour and time as- is compromised by methodological discrepancies and technical dif- sociated with semen evaluation as well as to decrease the inter- and ficulties in distinguishing sperm head from debris, especially when intra-observer variability (Mortimer, 2000). These systems are based sperm concentration is low. The pros and cons of CASA are depicted on capturing the image of the microscopic field as frames (series of in Table 3. pictures), which are digitalised and subsequently analysed using an algorithm. Several manufacturers have produced semi- or fully au- tomated CASA systems having different algorithms. Generally, these 6 | DIAGNOSTIC VALUE OF SEMEN systems can assess sperm motility and kinematics. Until recently, ANALYSIS measurement of sperm concentration using CASA was not possible due to difficulties in differentiating spermatozoa from particulate 6.1 | What can routine semen analysis offer in the debris. However, recent advancements in CASA equipped with a investigation of male infertility? fluorescent microscope can assess sperm concentration as well as concentration of progressively motile spermatozoa with the use of A standard semen analysis is the first test prescribed by a clinician to fluorescent DNA stain and a tail detection algorithm (Hu, Lu, Shao, assess male factor infertility in a couple. The lower reference values Huang, & Lü, 2013; Zinaman, Uhler, Vertuno, Fisher, & Clegg, 1996). provided by the WHO are used to guide the diagnosis and treat- High precision and detailed analysis of sperm kinematic parameters ment of the male partner. Diagnosis of normozoospermia may push are the two major advantages of CASA over manual methods. the investigation towards the female partner as the primary cause

TABLE 3 Pros and Cons of CASA Pros Cons

Rapid analysis of larger number of samples Expensive instrumentation and requires well- trained personnel Decreased subjectivity Multiple biological factors (semen hyperviscosity, presence of round cells, debris and sperm agglutination) interfere with CASA analysis Increased reproducibility Precision of sperm concentration measurement decreases outside an optimal range (2–50 × 106/ml) Allows detailed evaluation of sperm motion Low accuracy in sperm morphometric characteristics assessment Allows re-analysis of recorded videos and No comparability of results across instruments pictures at a later time point due to lack of standardisation of CASA systems BASKARAN et al. | 9 of 12 of infertility, while a semen sample with altered sperm parameters challenging due to inherent biological variability between semen suggests male factor as a putative cause for infertility and guide to- ejaculates (Leushuis et al., 2010). Hence, the WHO guidelines (2010) wards more advanced investigations, genetic testing and treatments recommend the examination of at least two semen samples to ob- (Wang & Swerdloff, 2014). Furthermore, morphological abnormali- tain a representative evaluation of semen quality (WHO, 2010). ties such as globozoospermia, macrocephaly, multiple tail defects and decapitated sperm syndrome are indicative of genetic disorders (Gatimel et al., 2017). 7 | TECHNOLOGICAL ADVANCES IN Semen analysis is also useful in monitoring spermatogenesis SEMEN ANALYSIS in men undergoing fertility regulation (WHO, 2010). Prospective studies conducted in couples who discontinued contraception and Technological advancements have been facilitated by the develop- were trying to conceive revealed a significant association between ment of several home-based semen analysis systems, which allow sperm parameters (sperm count and percentage of morphologically the in-house evaluation of semen (Yu et al., 2018). The home-testing normal sperm) and time to pregnancy, a marker of couple fecundity systems are based on antibody reaction, microfluidics or smartphone (Buck Louis et al., 2014; Slama et al., 2002). Sperm morphology can apps (e.g. Sandstone Diagnostics’ Trak device and the Yo Sperm Test) be highly predictive of fertilisation and pregnancy rates after in- (Vij & Agarwal, 2017; Yu et al., 2018). Although these systems are trauterine insemination (IUI) (Kruger & Coetzee, 1999; Van Waart, far from being perfect, they can minimise possible errors due to Kruger, Lombard, & Ombelet, 2001), although evidence is still con- sample transportation and handling. Other benefits include cost ef- tradictory (Lemmens et al., 2016; Thijssen et al., 2017). However, fectiveness, privacy and quick response to fertility-related queries. sperm abnormalities are not related to reproductive outcomes in Furthermore, home-based tests provide a preliminary idea on the case of ICSI, reducing the power of conventional semen analysis fertility status and indicate whether a user should or need not pur- in predicting fertility (Nagy et al., 1995; Oehninger et al., 1995). sue further testing, which is particularly useful for men who are hesi- tant to seek medical evaluation as well as post-vasectomy patients. However, most of these systems provide information about one 6.2 | Limitations of semen analysis semen parameter, such as sperm concentration (e.g. SpermCheck Fertility Test) or motile sperm concentration (e.g. Fertell Male Since the semen characteristics that distinguish fertile and infertile Fertility Home Test, SwimCount Sperm Quality Test) or a few (e.g. men are poorly defined, semen analysis cannot accurately predict Micra Sperm Test, paper-based devices or microfluidic devices), but male infertility. About 15% of infertile men have semen parame- not all sperm functional parameters pertinent to fertility testing (Yu ters within the normal reference range and are categorised as un- et al., 2018). Therefore, home-based semen analysis systems are not explained infertility (Esteves, Sharma, Gosálvez, & Agarwal, 2014; a replacement of conventional semen analysis as home-based tests Hamada, Esteves, Nizza, & Agarwal, 2012). Normally, spermatozoa provide only rudimentary results based on a few parameters and are undergo several changes during their transit through the female prone to false-negative results (Coppola et al., 2010; Yu et al., 2018). reproductive tract before fertilising the oocyte. However, stand- ard semen analysis cannot assess the ability of spermatozoa to un- dergo capacitation, acrosome reaction and hyperactivation as well 8 | CONCLUSION as its potential to fertilise the oocyte (Cho, 2012; Inoue, Ikawa, & Okabe, 2011). Therefore, conventional semen analysis has a limited The conventional semen analysis is the first and key step in the eval- predictive value for reproductive outcomes. Furthermore, the con- uation of the male fertility potential. However, it is a poor indicator ventional semen analysis suffers from subjectivity and poor stand- of reproductive outcomes. Alterations in multiple semen parameters ardisation of human manual analysis. A large number of laboratories are indicative of putative sperm dysfunction and associated impair- do not strictly adhere to guidelines, reducing the diagnostic power ment of male fertility. Although several CASA systems have been of semen analysis (Filimberti et al., 2013; Mallidis et al., 2012; developed with the objective of reducing time and subjectivity re- Punjabi et al., 2016). Improper sample collection as well as lack of lated to manual semen analysis, lack of standard guidelines and high adherence to the recommended length of abstinence and time of cost associated with CASA limits its application in clinical practice. delivery to the laboratory may cause bias in semen evaluation. Technological advancements have resulted in the development of The validity of the 2010 WHO reference values are extensively several home semen testing systems that serve as a quick and cost- debated. Several clinicians and researchers have criticised the lower effective self-screening tool for male fertility. cut-off values as being too low, with reference values being de- rived based on the semen characteristics of only fertile men, lack of consideration of a broader geographical diversity and ethnic dif- 9 | TAKE-HOME MESSAGE ferences, all of which could influence semen parameters (Cooper et al., 2010; Esteves, 2014; Esteves et al., 2012; Murray et al., 2012). • Semen analysis is the initial and fundamental step in the evalua- Finally, diagnosis of infertility based on semen analysis can be tion of the male fertility potential. 10 of 12 | BASKARAN et al.

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