Received: 6 February 2020 | Accepted: 1 April 2020 DOI: 10.1111/and.13614 INVITED REVIEW Diagnostic value of routine semen 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 semen analysis 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, male infertility, 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 ejaculation, 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 epididymis, seminal vesicles, advanced optional tests and methods applied for clinics and re- bulbourethral and prostate 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)