Clinical assessment of the male fertility

Article information

Obstet Gynecol Sci. 2018;61(2):179-191
Publication date (electronic) : 2018 March 05
doi :
Department of Animal Science and Technology, Chung-Ang University, Anseong, Korea.
Corresponding author: Myung-Geol Pang. Department of Animal Science and Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong 17546, Korea.
Received 2017 June 01; Revised 2017 September 06; Accepted 2017 September 26.


The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.


Male factor infertility can be a health issue for males and is primarily responsible for inability to conceive a child after 1 year of regular, unprotected intercourse [1]. Particularly, male factor infertility affects nearly 50% of infertile couples worldwide who want a child and who require empirical therapy [2]. In a fertility clinic, physiologically abnormal spermatozoa are the prime target for male fertility evaluation and are tested for each step of successful conception, such as movement, fertilization, embryonic progress, and pregnancy. For these evaluations, distinct aspects of semen analysis such as sperm concentration, motility, morphology, acrosomal integrity, DNA damage, chromatin stability, oxidative stress, and genomic and proteomic composition have been thoroughly investigated. Nonetheless, the present understanding of abnormal sperm functions including their different physiological and pathological aspects remains limited and not well defined. Therefore, more extensive evaluation techniques are required to clarify the associations among certain diagnostic strategies and their evaluation of fertilizing capacity for males. Conventional semen analysis has been considered as the initial choice for fertility assessment and commonly tailored by more intensive and comprehensive sperm function tests. Recently, concerns have arisen to establish tests for selecting mature spermatozoon containing regular number of chromosomes. Thus, SpermSlow, sperm aneuploidy analysis, proteomic and genomic investigations are getting more attention, which could provide highly accurate assessment of male fertility by determining the true fertilizing potential of spermatozoa. However, both comprehensive and conventional sperm function tests still lack accuracy and reproducibility. Successive introduction of intracytoplasmic sperm injection (ICSI), intracytoplasmic morphological sperm injection (IMSI), and physiological intracytoplasmic sperm injection (PICSI) for successful reproductive outcome encouraged to go for newer tests which should expect the successful fertilization in vitro and in vivo.

Sperm biology

Spermatogenesis is a differentiation process that transforms a spermatogonial stem cell into spermatozoa within 74 days in the seminiferous tubules of the testes [3]. Over 1 mitotic and 2 meiosis divisions, the spermatogonial cell is transformed through subsequent proliferation and differentiation into a primary spermatocyte, secondary spermatocyte, spermatid, and finally a spermatozoon. This spermatogenesis process is controlled by follicle-stimulating hormone. However, at the end of the spermatogenesis process, spermatozoa move forward from the lumen of the seminiferous tubule to the proximal end of the epididymis [4]. During the next journey from the proximal to distal end of the epididymis, spermatozoa undergo a process where they acquire maturity, motility, and fertilizing capacity. Eventually, matured spermatozoa are stored in the epididymis tail until ejaculation. After ejaculation, spermatozoa undergo the capacitation process in the female reproductive tract. During capacitation, spermatozoa undergo a series of biochemical and physiological modifications through which they gain fertilizing ability [5]. Subsequently, capacitation triggers hyperactivated motility. Once spermatozoa reach an oocyte, they start the acrosome reaction (AR) before penetrating the zona pellucida. Fertilization occurs through subsequent zygote formation and development. Any abnormality that could befall spermatozoa during movement from testes to oocyte can lead to infertility.

Conventional semen analysis

Spermatogenesis and maturation processes can be affected by fluctuations in hormones, temperature, dietary balance, and exposure to toxins due to habits or environmental pollutants (i.e., smoking, alcohol, cadmium, lead, radiation, pesticide, endocrine disruptor chemicals) [6789]. Eventually, these factors can affect semen quality and result in abnormal spermatozoa. Conventional semen analysis is commonly used to define semen quality and to predict only quantitative values. Semen samples should be tested twice after an abstinence period of 2–7 days. After collection, the sample should be liquefied at room temperature and analyzed within 1 hour. Particularly, the number of spermatozoa present in each ejaculate, the percentage of motile spermatozoa or progressive motility, and the proportion of morphologically normal spermatozoa are evaluated based on standard reference values proposed by the World Health Organization (WHO) (Table 1) [10]. A large study was performed to evaluate male fertility based on sperm concentration, motility, and morphology among 765 males of infertile couples and 696 males of fertile couples. In that study, significant overlap was found between the fertile and infertile groups in all parameters (sperm concentration, motility, and morphology) [11]. Despite of the inaccuracy of these conventional semen analyses to evaluate male fertility was acknowledged in the mid-1980s [12], these are corner stone of infertility evaluation. Several recent studies have focused on establishing effective reference values for clinical use of semen analysis [131415]. In 1998, Bonde et al. [16] proposed that sperm count and morphology were correlated with conception. Although a range of semen analysis methods are commonly used throughout the fertility clinics and laboratories across the world, the current quality assessment tools of semen are unable to provide accuracy for predicting fertility status of a man [10171819]. Therefore, lower reference limits for semen parameters have been modified several times (1987, 1992, 1999, 2010) in the WHO manual to increase the clinical value of these parameters for evaluating male fertility.

Standard reference values for semen characteristics World Health Organization (WHO) (2010)

Sperm motion kinematics

Motile spermatozoa follow various specific movement patterns that are attained based on the sperm's functional requirement. Human spermatozoa ideally tend to generate propulsive forces, i.e., linear and progressive trajectories, during cervical barrier penetration. Therefore, several specific movement attributes (average path velocity, straightness, and amplitude of lateral sperm head displacement) including conventional analysis criteria (sperm count and morphology) are considered features of semen quality that can facilitate cervical barrier penetration [2021]. Consequently, computer-assisted semen analysis (CASA) has been established for mechanical analysis of sperm kinematics of ejaculated semen [22]. Based on serial digital imaging, CASA determines the sperm head trajectory and movement by measuring the motion patterns of the sperm head in 2 dimensions. Several researchers have suggested that quantitative evaluation of sperm kinematics using CASA can assess human sperm fertility under in vitro conditions, and that conventional semen analysis provides values of limited accuracy [2324]. In addition, many studies have demonstrated that quantitative assessment of sperm motion kinematics has diagnostic value for evaluating unexplained infertility [2526].

Although CASA was initially accepted as an important method for semen diagnosis, the individual motion kinematic value for fertility assessment remains questionable. Moreover, a wide range of errors due to object selection, setup procedures, and different kinetic values among populations render this application unacceptable [27]. Hyperactivation is another form of spermatozoa movement defined by high velocity and asymmetrical flagellar waves. Hyperactivated motility is an indication of the capacitation process believed to facilitate the mechanical thrust from the tubal epithelium to penetrate the zona pellucida [28]. Although hyperactivated motility can have significant effect on sperm fertility, there is no established value for the proportion of hyperactivated spermatozoa that should be present in each semen sample of a fertile male; as a result, the universal criteria for hyperactivation remain unknown. Therefore, although a previous study has established a relationship between hyperactivated sperm percentage and successful fertilization in vitro, this quantitative parameter is not recommended for clinical use [29].

Sperm morphology

Sperm morphology is an integral part of routine analysis of human semen. Kruger et al. [30] proposed strict criteria for evaluation of sperm morphology, where spermatozoa with any slight defect in the head, neck, body, and tail regions are considered to have abnormal morphology under the strict criteria. Eventually, these strict criteria were included in the latest WHO laboratory manual for the prediction of normal sperm morphology [31323334353738]. If more than 4% of sperm show a normal sperm morphology, the semen is considered within 95% of the fertile reference range [32]. One recent study has shown that the percentage of morphologically normal spermatozoa influenced the time to pregnancy (TTP) [39]. After evaluating 501 couples, investigators reported that sperm head width and coiled tails were important predictors of TTP [40]. In addition, percentage of morphologically normal spermatozoa independent of sperm concentration provided significant predictive value for couple fecundity as measured by TTP [4041]. However, after 1987, studies revealed that sperm morphology is a vital indicator of male fertility, controversies are also increasing. A study evaluated the consequences for men with 0% morphologically normal spermatozoa to determine the relationship between sperm morphology and reproductive success independent of assisted reproductive technologies (ART). Men with 100% abnormal spermatozoa achieved pregnancies in which only 25% required in vitro fertilization (IVF). Therefore, to refine the evaluation process, additional functional tests have been developed to explain the unanswered questions pertaining to male fertility [42].

Sperm viability

Sperm viability is an evaluation parameter for investigating the percentage of viable spermatozoa in an ejaculate that contains <5%–10% motile spermatozoa. Ultrastructural defects in human spermatozoa produce dead or non-motile spermatozoa. Moreover, sperm viability is used to identify viable spermatozoa that are appropriate for ICSI. Correa and Zavos [43] proposed that a positive correlation between viable and motile spermatozoa percentage in human semen could predict sperm fertility. Two methods are commonly used for sperm viability testing, the hypo-osmotic sperm swelling test (HOST) to differentiate dead or live sperm and flow cytometry to investigate sperm membrane integrity. The HOST procedure consists of placing spermatozoa into media with a lower osmotic potential than the spermatozoa. Thus, water enters the cytoplasm of live spermatozoa to achieve osmotic pressure equilibrium, which swells the spermatozoa tails. Supravital dyes (e.g., eosin or trypan blue) are also used to investigate sperm viability. Since the mixture of spermatozoa and supravital dye kills spermatozoa, the cells cannot later be used for ICSI. Therefore, the HOST is considered more suitable and allows the spermatozoa to subsequently be used for ICSI. Flow cytometry is another technique used to investigate the viability of spermatozoa by examining the integrity of both the plasma and mitochondrial membranes [44]. According to the WHO (5th edition), HOST is normal for a semen sample if >58% of spermatozoa undergo tail swelling, indicating an intact membrane. Semen consisting of <50% viable spermatozoa is considered abnormal. A recent study focused on both sperm viability and DNA fragmentation by testing 3,049 semen samples from 2008 to 2013 and showed a strong negative correlation between sperm viability and DNA fragmentation rate. The study reported that men with sperm viability ≥75% or ≤30% do not require DNA fragmentation index (DFI) [45]. Therefore, sperm viability tests are a valuable cost-effective measure for investigating male fertility, while DNA fragmentation tests are expensive as a routine test. Although motile spermatozoa could be defined as viable, viable spermatozoa are also related to an undamaged plasma membrane since the plasmalemma is essential for interactions between spermatozoa and oocyte. Therefore, sperm viability assay methods not only focus on cell viability, but also assess whether the plasmalemma is intact.

Acrosomal integrity

Evaluation of acrosomal status in human spermatozoa is another method to predict male fertility. Ionophore A23187-induced AR (acrosome reaction to ionophore challenge [ARIC]) is a good indicator of male fertility [46]. An infertile male who undergoes the ARIC test shows a significantly reduced number of acrosome-reacted spermatozoa. A study of 86 males with good fertilization rate (≥30%) and 39 males with poor fertilization rate (<30%) undergoing IVF and embryo transfer was conducted to verify the efficacy of the ARIC test. Significant reduction in induced AR rate and ARIC value was found in the poor fertilization group. Using the cutoff value of 8.5 for the ARIC test, sensitivity, specificity, positive predictive value, and negative predictive value were 83.7%, 92.3%, 96.0%, and 72.0%, respectively [47]. When using the ARIC test with ionophore A23187, clinicians can determine the difference between complete acrosome-reacted spermatozoa with and without treatment [48]. This percentage difference provides the evaluative value; a difference ≤5% indicates male infertility. If the acrosome-reacted spermatozoa represent 5%–10% of a given sample, high fertility and reproductive outcomes might be indicated [49]. Thus, this evaluative parameter could be useful to explain male infertility if a couple fails IVF, but is not yet suitable for assessing male infertility as the primary stage.

Hemizona assay

Sperm-zona binding triggers the AR in mammalian spermatozoa. Therefore, during IVF, imperfect binding and penetration of spermatozoa with the zona pellucida result in unsuccessful fertilization. Overstreet and Hembree [50] introduced an assay for assessing human sperm-oocyte interactions. Because sperm-zona binding is a species-specific event, the bioavailability of this assay is limited [51]. However, 2 available assays are used to assess sperm-zona binding ability, the hemizona assay and the sperm–zona binding ratio. Human oocytes are used to isolate zona pellucida, which is divided in half during the hemizona assay. One half of the zona pellucida is incubated with a fertile male's spermatozoa (control group), and the other half is incubated with the patient's spermatozoa (evaluated group).

The binding ratio of patient spermatozoa to that of a fertile donor is evaluated; a ratio <30% is considered abnormal/infertile [51]. In addition, the sperm-zona binding ratio is another method to assess male fertility. The patient and fertile male spermatozoa are labeled with 2 distinct fluorochromes. After incubation with intact oocytes, the total number of bound spermatozoa is counted [49]. The lower binding ratio of spermatozoa with the zona pellucida has been correlated with lower fertilization rates during IVF. Therefore, these tests are beneficial mainly for male patients that have failed standard IVF and have limited utility in cases of primary infertility. However, the use of human oocytes raises ethical issues for these tests, while intra- and inter-assay variability during testing are 2 major factors that affect the assay efficiency.

Sperm penetration assay

In an earlier study, basic semen analysis showed a large percentage of false data with low accuracy to predict fertility potential in terms of both spontaneous and assisted conception [21]. Therefore, to obtain more efficient assessment tool, researchers focused on the ability of spermatozoa to penetrate an oocyte. Hence, sperm penetration assay (SPA) introduced by Yanagimachi et al. [52] in 1976 is considered the most reliable bioassay for explaining non-defined male infertility [5354]. Subsequently, this assay was utilized due to the reduced false data results and high accuracy [5354]. However, hamster egg retrieval, semen sampling and liquefaction, sperm washing and preincubation, insemination, sperm-egg coincubation, as well as the protein source in the media are the main factors that affect the SPA test [55]. Various protein sources and their different concentrations have significant effects on SPA test results (Fig. 1) [56]. Several factors such as period of abstinence, sperm concentration during coincubation, media constituents, trypsinization, and clinician experience have been reported as inter and intra- assay variables those can influence SPA accuracy [57]. Therefore, several studies have been done to optimize and standardize SPA test using other mammalian animal trials [565859]. In the early stage of SPA, several studies have focused on the correlation (positive/negative) among quantitative parameters and SPA. Additionally, SPA combined with other tests, including strict sperm morphology criteria and the ARIC test, can provide more accurate values regarding fertilizing capacity [60]. Nonetheless, SPA is still difficult to standardize due to variation of culture condition. Moreover, this is a cumbersome, costly, time consuming method, which had lost its clinical usefulness after introducing ICSI.

Fig. 1

Effects of sperm treatment based on TEST-yolk buffer (TYB), Biggers-Whitten-Whittingham (BWW), and human serum albumin (HSA) on the outcome of the sperm penetration assay (SPA). (A) Effect of TYB and BWW on the outcome of SPA tests. (B) Human serum albumin concentrations in fertilization media. (C) Human serum albumin concentration in swim up method. The figure has been modified, and citing the original source published by Oh et al. [59].

PI, penetration index; PR, plasticity range.

Reactive oxygen species

In recent years, oxidative stress has been recognized as one of the main factors affecting sperm functions [5253]. In the male reproductive system, production of antioxidant scavengers and reactive oxygen species (ROS) is required to maintain equilibrium; low ROS levels regulate capacitation, and elevated ROS levels affect sperm physiology by increasing oxidative stress [61]. Elevated ROS levels such as superoxide anions, hydrogen peroxide, and hydroxyl radicals or decreased antioxidant levels are the main factors for sperm malfunctions by affecting the sperm cell membrane lipid peroxidation, sperm motility, and DNA integrity [62]. Since human spermatozoa are very sensitive, these dysfunctions impair their fertilizing ability. To assess ROS levels in semen, the chemiluminescence assay is used [63], and total antioxidant capacity is detected using the colorimetric assay [64]. A recent study proposed an optimal cutoff value to differentiate between controls and infertile males of 102.2 relative light unit/s/106 sperm, showing 76.4% sensitivity and 53.3% specificity [65]. However, the accuracy of this method to evaluate male fertility is not well-established. Further investigations are required to establish highly accurate ROS cutoff values in both fertile and infertile patients.

Sperm DNA damage

DNA damage is responsible for causing apoptosis in spermatozoa and loss of embryo development and pregnancy. A considerable number of studies have shown the number of DNA-damaged spermatozoa is higher in infertile than fertile males [66]. Therefore, DNA damage is an important factor for sperm quality evaluation that correlates with both in vivo and in vitro development of an embryo [67]. DNA damage can be assessed by several assays including single-cell electrophoresis assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay, alkaline gel electrophoresis, sperm chromatin structure assay (SCSA), and quantitative polymerase chain reaction of nuclear and mitochondrial DNA. Previous studies have indicated that sperm DNA damage affects male fertility potential and has a higher predictive value for reproductive outcome in natural fertility [666869] In addition, poor results after intrauterine insemination (IUI) are associated with a high percentage of damaged DNA in a group of spermatozoa; however, the factors affecting fertilization rate or pregnancy can be avoided by ICSI [7071]. Study results have indicated that SPA optimized with a sperm head chromatin pattern was highly accurate [72], showing 96% sensitivity and 56% specificity, which could be useful for evaluating infertile males. Moreover, results from ICSI or routine IVF demonstrated that a DFI value <30% can decrease fertility success in infertile couples by 1.6-fold [73]. Conversely, a recent meta-analysis confirmed a minor but statistically significant relationship between sperm DNA damage and pregnancy rate in IVF and ICSI cycles, although the clinical utility of sperm DNA damage has not been determined [74].


The SCSA was proposed by Evenson et al. [75] in 1980 to investigate sperm DNA integrity. After its introduction, SCSA was considered a method that could potentially help with male factor infertility and obtain success in ART. SCSA is a flow cytometric method that tests the vulnerability of DNA to acid-induced denaturation by exposure to acridine orange media [76]. Double-stranded DNA binds with acridine orange and fluoresces green, while single-stranded DNA binds with acridine orange and fluoresces red. Using flow cytometry, the ratio of red to green fluorescence can be analyzed. The percentage of spermatozoa with red fluorescence as the ratio of red/red and green fluorescence is termed the DFI and can be visualized on a histogram. Conversely, spermatozoa with green fluorescence show the percentage of sperm with high DNA integrity (mature sperm). DNA damage in spermatozoa is considered to be associated with poor semen quality, which can lead to a lower preimplantation rate, higher abortion rate, and higher childhood cancer [77]. Combining the SCSA test with conventional sperm analysis can result in higher accuracy when assessing male fertility. If one of the traditional sperm parameters is abnormal, fertility is reduced, with a DFI >10%. Infertile males with a high DFI percentage have less potential for natural fertility and IUI outcome. However, ICSI does not consider DNA damage as a factor affecting the reproductive outcome [7578].

ICSI, IMSI, and PICSI for male fertility assessment

Following all sperm function tests, the percentage of sperm that bind and penetrate the zona pellucida in oocytes cannot explain the approximately 25% of male infertility worldwide [69]. For couples who suffer from a low fertilization rate over several IVF cycles, ICSI helps to alleviate male factor infertility [6974]. However, the outcome of ICSI depends primarily on the quality of the oocyte, female age, and sperm morphology. Although ICSI has been successful for overcoming unexplained infertility, studies showed that sperm morphology, motility, sperm-zona pellucida binding, sperm-zona pellucida penetration, zona pellucida-induced AR, and sperm DNA damage are still useful predictors before patients commence ICSI treatment [798081]. However, substantial incidence of failed (0%) and suboptimal (<50%) fertilization after ICSI remains a challenge due to morphological defects in spermatozoa [8283].

Motile sperm organelle morphology examination has provided an opportunity for intensive selection of spermatozoa for ICSI [84]. This examination offers improved morphological assessment of mitochondria, nucleus, acrosome, post-acrosomal region, neck, and tail of spermatozoa using high magnification >6,000× [85]. Thus, the inclusion of this method into ICSI led to a new technique termed IMSI [86]. After introduction of IMSI, numerous comparative and randomized studies were conducted, although the comparison between IMSI and conventional ICSI provided controversial data. A wide range of factors can contribute to the efficiency of IMSI over conventional ICSI such as dissimilar study design, lack of homogenous inclusion criteria, and non-classified high magnification sperm morphology [87]. However, a recent study showed that IMSI in infertile couples enhanced the reproductive outcomes compared with conventional ICSI by increasing implantation rate (3 times), pregnancy rate (2 times), and miscarriage reduction rate (70%) [87]. However, a meta-analysis of current trials determined that IMSI has no significant effects on clinical pregnancy rate and live birth [88].

Using hyaluronic acid-containing media for selection of spermatozoa provided a new opportunity to sort mature spermatozoa with lower biological risk [89]. Binding ability of spermatozoa with hyaluronic acid depends on plasma membrane maturity and fertilizing ability of spermatozoa. This novel method of sperm selection based on the ability to bind with hyaluronic acid led to a new method termed PICSI [9091]. Many recent studies have indicated that PICSI is effective for selecting spermatozoa by excluding fragmented DNA and abnormal nucleus [9293]. However, conflicting results showed that PICSI does not improve the fertilization and cleavage rate after ICSI [94959697].

SpermSlow for assessment of spermatozoa

SpermSlow is used to decelerate the movement of spermatozoa to allow the selection of viable, mature, and non-fragmented DNA-containing spermatozoon for ICSI [98]. This technique allows the selection of the most mature spermatozoa during ICSI. A plastic culture dish with microdots of hyaluronic acid is used as a device for ICSI, and SpermSlow is used as a viscous media containing hyaluronic acid. A significant enhancement of embryo quality was observed in a study when SpermSlow was used to select spermatozoon [98]. Controversy has increased because several studies did not find any significant difference in fertilization rate or embryo quality after injecting SpermSlow-selected spermatozoa compared to other physiologic ICSI treatments [99100].

Sperm aneuploidy analysis for ICSI, IMSI, and PICSI

The presence of inadequate numbers of chromosomes in spermatozoa causes chromosomal abnormalities, defined as aneuploidy, reportedly occurring 3 times more frequently in spermatozoa of infertile males with azoospermia [99101]. Notable improvements have been observed using ICSI, IMSI, and PICSI for infertility treatment in recent years since the treatments required spermatozoa without any cytogenetic abnormalities to ensure pregnancy and healthy live birth [102103]. Therefore, to decrease aneuploid fertilization, combined HOST and fluorescence in situ hybridization (FISH) are used simultaneously as a cytogenetic assay to evaluate the rates of chromosomal abnormalities. Only a few studies reported a significantly reduced aneuploidy frequency in spermatozoa with tail-tip swelling pattern (using HOST) and FISH [104105]. Due to the increased rate of aneuploid fertilization during ICSI [106], FISH in functionally live spermatozoa is considered the most accurate evaluation tool for excluding unhealthy fertilization in infertile men. Future studies are needed to evaluate both the HOST and FISH for consideration as routine tests of cytoplasmic abnormalities in human spermatozoa.

Future diagnostic tests for male fertility: omics

Recently, more advanced research methods have provided an opportunity to investigate new prediction techniques based on genomics, proteomics, transcriptomics, and metabolomics. Combination of the current omics and conventional semen analysis could provide new methods for exploring potential predictors of male fertility [101]. Research focuses on the differentially expressed proteins and genes found under different conditions including fertility/infertility [107108]. During the past decade, protein biomarkers have been the subject of extensive research for diversified diseases as well as male fertility. Several seminal plasma proteins have already been evaluated as potential biomarkers for genital duct patency. For example, a current study showed that the cysteine-rich secretory protein level in seminal plasma is a predictable proteomic biomarker that can identify fertile and infertile men with 85% specificity and 92% sensitivity [109]. Another study proposed that the level of lipocalin-type prostaglandin D synthase is significantly lower in infertile men with obstructive azoospermia compared to men with normal semen parameters [110]. However, intensive proteomic analysis of both semen and sperm is discovering functionally important proteins, protein-protein interactions during the path to the oocyte, and other various altered proteins associated with the fertilization process. Similarly, transcriptional profiling of spermatozoa is also a valuable method for males with unexplained infertility problems, and further studies are required to establish a true fertility/infertility predictor by investigating omics of human semen.


Conventional semen analysis is considered as the initial step to investigate semen quality and male factor infertility; however, this method cannot always provide valid information regarding specific defects of sperm physiology. Therefore, novel predictors are needed for assessing semen quality to determine the reason for non-pregnancy in infertile couples. The current assays can determine specific imperfections based on sperm physiology, but newer predictive tests might reveal the precise reason for male infertility. Thus, the addition of new predictive methods can aid researchers to better understand sperm potency. Implementation of such evaluation procedures might help clarify the unknown characteristics of spermatozoa to maximize successful reproduction.


This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (iPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (116172-3).


Conflict of interest: No potential conflict of interest relevant to this article was reported.


1. Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril 2009;92:1520–1524. 19828144.
2. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol 2015;13:37. 25928197.
3. Samplaski MK, Agarwal A, Sharma R, Sabanegh E. New generation of diagnostic tests for infertility: review of specialized semen tests. Int J Urol 2010;17:839–847. 20887631.
4. Rahman MS, Pang MG. Sperm biology: towards understanding global issue of male infertility. Austin Androl 2016;1:1003.
5. Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci USA 2009;106:667–668. 19144927.
6. Watanabe T, Ohkawa K, Kasai S, Ebara S, Nakano Y, Watanabe Y. The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats. Congenit Anom (Kyoto) 2003;43:57–64. 12692404.
7. Gorpinchenko I, Nikitin O, Banyra O, Shulyak A. The influence of direct mobile phone radiation on sperm quality. Cent European J Urol 2014;67:65–71.
8. Bretveld R, Brouwers M, Ebisch I, Roeleveld N. Influence of pesticides on male fertility. Scand J Work Environ Health 2007;33:13–28. 17353961.
9. Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. Gestational exposure to bisphenol A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 2017;125:238–245. 27384531.
10. Esteves SC. Clinical relevance of routine semen analysis and controversies surrounding the 2010 World Health Organization criteria for semen examination. Int Braz J Urol 2014;40:443–453. 25254609.
11. Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 2001;345:1388–1393. 11794171.
12. Glazener CM, Coulson C, Lambert PA, Watt EM, Hinton RA, Kelly NJ, et al. The value of artificial insemination with husband’s semen in infertility due to failure of postcoital sperm-mucus penetration--controlled trial of treatment. Br J Obstet Gynaecol 1987;94:774–778. 3311134.
13. Swan SH. Semen quality in fertile US men in relation to geographical area and pesticide exposure. Int J Androl 2006;29:62–68. 16466525.
14. Nallella KP, Sharma RK, Aziz N, Agarwal A. Significance of sperm characteristics in the evaluation of male infertility. Fertil Steril 2006;85:629–634. 16500330.
15. Haugen TB, Egeland T, Magnus O. Semen parameters in Norwegian fertile men. J Androl 2006;27:66–71. 16400080.
16. Bonde JP, Ernst E, Jensen TK, Hjollund NH, Kolstad H, Henriksen TB, et al. Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet 1998;352:1172–1177. 9777833.
17. Aitken RJ, Best FS, Warner P, Templeton A. A prospective study of the relationship between semen quality and fertility in cases of unexplained infertility. J Androl 1984;5:297–303. 6540770.
18. Lewis SE. Is sperm evaluation useful in predicting human fertility? Reproduction 2007;134:31–40. 17641086.
19. Wang C, Swerdloff RS. Limitations of semen analysis as a test of male fertility and anticipated needs from newer tests. Fertil Steril 2014;102:1502–1507. 25458617.
20. Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73:441–449. 3989795.
21. Aitken RJ. Sperm function tests and fertility. Int J Androl 2006;29:69–75. 16466526.
22. Mortimer ST. CASA--practical aspects. J Androl 2000;21:515–524. 10901437.
23. Paston MJ, Sarkar S, Oates RP, Badawy SZ. Computer-aided semen analysis variables as predictors of male fertility potential. Arch Androl 1994;33:93–99. 7818377.
24. Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, et al. Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro . J Assist Reprod Genet 2001;18:213–218. 11432113.
25. Peedicayil J, Deendayal M, Sadasivan G, Shivaji S. Assessment of hyperactivation, acrosome reaction and motility characteristics of spermatozoa from semen of men of proven fertility and unexplained infertility. Andrologia 1997;29:209–218. 9263571.
26. Shibahara H, Obara H. Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertility. Int J Androl 2004;27:63–68. 15149462.
27. Davis RO, Katz DF. Operational standards for CASA instruments. J Androl 1993;14:385–394. 8288492.
28. Ho HC, Suarez SS. Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 2001;122:519–526. 11570958.
29. Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25 Hz using in-vitro fertilization as a functional end-point. Hum Reprod 1995;10:2928–2937. 8747047.
30. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck LL, et al. New method of evaluating sperm morphology with predictive value for human in vitro fertilization. Urology 1987;30:248–251. 3629768.
31. Moon SY, Choi YM, Kim SH, Oh SK, Suh CS, Lee JY, et al. Analysis of strict morphology of human spermatozoa. Korean J Obstet Gynecol 1998;41:2923–2931.
32. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update 2010;16:231–245. 19934213.
33. Coetzee K, Kruge TF, Lombard CJ. Predictive value of normal sperm morphology: a structured literature review. Hum Reprod Update 1998;4:73–82. 9622414.
34. Menkveld R, Holleboom CA, Rhemrev JP. Measurement and significance of sperm morphology. Asian J Androl 2011;13:59–68. 21076438.
35. Van Waart J, Kruger TF, Lombard CJ, Ombelet W. Predictive value of normal sperm morphology in intrauterine insemination (IUI): a structured literature review. Hum Reprod Update 2001;7:495–500. 11556497.
36. Check ML, Bollendorf A, Check JH, Katsoff D. Reevaluation of the clinical importance of evaluating sperm morphology using strict criteria. Arch Androl 2002;48:1–3. 11789677.
37. Kiefer D, Check JH, Katsoff D. The value of motile density, strict morphology, and the hypoosmotic swelling test in in vitro fertilization-embryo transfer. Arch Androl 1996;37:57–60. 8827348.
38. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586–592. 2394790.
39. Slama R, Eustache F, Ducot B, Jensen TK, Jørgensen N, Horte A, et al. Time to pregnancy and semen parameters: a cross-sectional study among fertile couples from four European cities. Hum Reprod 2002;17:503–515. 11821304.
40. Buck Louis GM, Sundaram R, Schisterman EF, Sweeney A, Lynch CD, Kim S, et al. Semen quality and time to pregnancy: the Longitudinal Investigation of Fertility and the Environment Study. Fertil Steril 2014;101:453–462. 24239161.
41. Zinaman MJ, Brown CC, Selevan SG, Clegg ED. Semen quality and human fertility: a prospective study with healthy couples. J Androl 2000;21:145–153. 10670528.
42. Kovac JR, Smith RP, Cajipe M, Lamb DJ, Lipshultz LI. Men with a complete absence of normal sperm morphology exhibit high rates of success without assisted reproduction. Asian J Androl 2017;19:39–42. 27751992.
43. Correa JR, Zavos PM. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 1994;42:351–360. 16727543.
44. Evenson DP, Darzynkiewicz Z, Melamed MR. Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J Histochem Cytochem 1982;30:279–280. 6174566.
45. Samplaski MK, Dimitromanolakis A, Lo KC, Grober ED, Mullen B, Garbens A, et al. The relationship between sperm viability and DNA fragmentation rates. Reprod Biol Endocrinol 2015;13:42. 25971317.
46. Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters. J Androl 1991;12:98–103. 2050585.
47. Ryu BY, Kim SH, Park SY, Jee BC, Jung BJ, Kim HS, et al. Efficacy of acrosome reaction after ionophore challenge (ARIC) test in evaluation of fertilization capacity of human spermatozoa. Korean J Obstet Gynecol 1998;41:2562–2570.
48. Krausz C, Bonaccorsi L, Maggio P, Luconi M, Criscuoli L, Fuzzi B, et al. Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization. Hum Reprod 1996;11:1661–1667. 8921113.
49. Sigman M, Baazeem A, Zini A. Semen analysis and sperm function assays: what do they mean? Semin Reprod Med 2009;27:115–123. 19247913.
50. Overstreet JW, Hembree WC. Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro . Fertil Steril 1976;27:815–831. 820576.
51. Arslan M, Morshedi M, Arslan EO, Taylor S, Kanik A, Duran HE, et al. Predictive value of the hemizona assay for pregnancy outcome in patients undergoing controlled ovarian hyperstimulation with intrauterine insemination. Fertil Steril 2006;85:1697–1707. 16682031.
52. Yanagimachi R. Specificity of sperm-egg interaction. In : Edidin M, Johnson MH, eds. Immunobiology of gametes Cambridge (GB): Cambridge University Press; 1977. p. 255–295.
53. Pang MG, Oh SK, Shin CJ, Kim JG, Moon SY, Chang YS, et al. Study on the clinical validity of sperm penetration assay. Korean J Fertil Steril 1993;20:1–7.
54. Pang MG, Jung BJ, Moon SY. Influence of sperm fertilizing capacity on embryonic development and pregnancy in in vitro fertilization. Korean J Fertil Steril 2003;30:105–109.
55. Chang YS, Lee JY, Moon SY, Kim JG, Pang MG, Shin CJ. Factors affecting penetration of zona-free hamster ova. Arch Androl 1990;25:213–224. 2285345.
56. Kim SH, Kim MH, Jee BC, Jung BJ, Kim HS, Ryu BY, et al. Efficacy of zona-free hamster ova sperm penetration assay (SPA) in evaluation of fertilization capacity of human spermatozoa. Korean J Obstet Gynecol 1998;41:2401–2410.
57. Aitken RJ, Ross A, Lees MM. Analysis of sperm function in Kartagener's syndrome. Fertil Steril 1983;40:696–698. 6605262.
58. Kim SH, Pang MG, Shin CJ, Kim JG, Moon SY, Lee JY, et al. Establishment of normal fertile range of sperm zona-free hamster ova penetration assay in Korean male. Korean J Fertil Steril 1991;18:63–71.
59. Oh SA, You YA, Park YJ, Pang MG. The sperm penetration assay predicts the litter size in pigs. Int J Androl 2010;33:604–612. 19538520.
60. Moon SY, Ryu BY, Pang MG, Oh SK, Lee JH, Suh CS, et al. Comparison of sperm morphology evaluation using strict criteria, acrosome reaction following ionophore challenge and zona-free hamster ova sperm penetration assay as prognostic factors in diagnosis of male infertility and in vitro fertilization. Korean J Fertil Steril 2002;29:57–66.
61. Aitken RJ, Baker MA, O'Bryan M. Shedding light on chemiluminescence: the application of chemiluminescence in diagnostic andrology. J Androl 2004;25:455–465. 15223833.
62. Aitken RJ, Ryan AL, Curry BJ, Baker MA. Multiple forms of redox activity in populations of human spermatozoa. Mol Hum Reprod 2003;9:645–661. 14561808.
63. Kobayashi H, Gil-Guzman E, Mahran AM. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Androl 2001;22:568–574. 11451353.
64. Said TM, Agarwal A, Sharma RK, Mascha E, Sikka SC, Thomas AJ Jr. Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility. Fertil Steril 2004;82:871–877. 15482762.
65. Agarwal A, Roychoudhury S, Bjugstad KB, Cho CL. Oxidation-reduction potential of semen: what is its role in the treatment of male infertility? Ther Adv Urol 2016;8:302–318. 27695529.
66. Cho C, Jung-Ha H, Willis WD, Goulding EH, Stein P, Xu Z, et al. Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Biol Reprod 2003;69:211–217. 12620939.
67. Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322:33–41. 15912407.
68. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14:1039–1049. 10221239.
69. Zini A, Meriano J, Kader K, Jarvi K, Laskin CA, Cadesky K. Potential adverse effect of sperm DNA damage on embryo quality after ICSI. Hum Reprod 2005;20:3476–3480. 16123087.
70. Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C, et al. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 2000;15:830–839. 10739828.
71. Høst E, Lindenberg S, Kahn JA, Christensen F. DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynecol Scand 1999;78:336–339. 10203303.
72. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003;79(Suppl 3):1597–1605. 12801566.
73. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007;22:174–179. 16921163.
74. Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, François Guerin J. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril 2007;87:93–100. 17074327.
75. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;210:1131–1133. 7444440.
76. Evenson D, Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod Biomed Online 2006;12:466–472. 16740220.
77. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 2008;89:823–831. 17644094.
78. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002;23:25–43. 11780920.
79. Liu DY, Baker HW. Evaluation and assessment of semen for IVF/ICSI. Asian J Androl 2002;4:281–285. 12508129.
80. Palermo GD, Neri QV, Schlegel PN, Rosenwaks Z. Intracytoplasmic sperm injection (ICSI) in extreme cases of male infertility. PLoS One 2014;9e113671. 25437298.
81. Practice Committees of the American Society for Reproductive Medicine and Society for Assisted Reproductive Technology. Intracytoplasmic sperm injection (ICSI) for non-male factor infertility: a committee opinion. Fertil Steril 2012;98:1395–1399. 22981171.
82. Liu DY, Baker HW. Tests of human sperm function and fertilization in vitro . Fertil Steril 1992;58:465–483. 1521638.
83. Liu DY, Lopata A, Johnston WI, Baker HW. A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro . Fertil Steril 1988;50:782–788. 3141221.
84. Palermo GD, Hu JC, Rienzi L, Maggiulli R, Takeuchi T, Yoshida A, et al. Thoughts on IMSI. In : Racowsky C, Schlegel PN, Fauser BC, Carrell DT, eds. Biennial review of infertility New York (NY): Springer; 2011. p. 277–289.
85. Flaherty SP, Payne D, Matthews CD. Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1998;13(Suppl 1):155–164. 9663780.
86. Mansour R. Intracytoplasmic sperm injection: a state of the art technique. Hum Reprod Update 1998;4:43–56. 9622412.
87. Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 2002;23:1–8. 11780915.
88. Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, et al. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril 2003;80:1413–1419. 14667877.
89. Perrin A, Nguyen MH, Douet-Guilbert N, Morel F, De Braekeleer M. Intracytoplasmic morphologically selected sperm injection or intracytoplasmic sperm injection: where are we 12 years later? Expert Rev Obstet Gynecol 2013;8:261–270.
90. Setti AS, Braga DP, Figueira RC, Iaconelli A Jr, Borges E. Intracytoplasmic morphologically selected sperm injection results in improved clinical outcomes in couples with previous ICSI failures or male factor infertility: a meta-analysis. Eur J Obstet Gynecol Reprod Biol 2014;183:96–103. 25461360.
91. Teixeira DM, Barbosa MA, Ferriani RA, Navarro PA, Raine-Fenning N, Nastri CO, et al. Regular (ICSI) versus ultra-high magnification (IMSI) sperm selection for assisted reproduction. Cochrane Database Syst Rev 2013;:CD010167. 23884963.
92. Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod Update 2011;17:719–733. 21873262.
93. Ebner T, Filicori M, Tews G, Parmegiani L. A plea for a more physiological ICSI. Andrologia 2012;44(Suppl 1):2–19. 22211911.
94. Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M. “Physiologic ICSI”: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertil Steril 2010;93:598–604. 19393999.
95. Prinosilova P, Kruger T, Sati L, Ozkavukcu S, Vigue L, Kovanci E, et al. Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology. Reprod Biomed Online 2009;18:177–183. 19192336.
96. Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, Ward D, et al. Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies. Fertil Steril 2005;84:1665–1673. 16359962.
97. Castillo-Baso J, Garcia-Villafaña G, Santos-Haliscak R, Diaz P, Sepluveda-Gonzalez J, Hernandez-Ayup S. Embryo quality and reproductive outcomes of spermatozoa selected by physiologic-ICSI or conventional ICSI in patients with kruger <4% and >4% normo-morphology. Fertil Steril 2011;96:S159.
98. Worrilow KC, Eid S, Woodhouse D, Witmyer J, Khoury C, Liebermann J. Increased clinical pregnancy rates (CPR) and statistically significant decrease in loss rates using hyaluronan in sperm selection: prospective, multi-center, double-blind, randomized clinical trial. Fertil Steril 2011;96:S179.
99. World Health Organization. WHO laboratory manual for the examination and processing of human semen 5th edth ed. Geneva (CH): World Health Organization; 2010.
100. Lee K, Hyslop JM, Nanassy L, Machaty Z. Incidence of apoptosis in parthenogenetic porcine embryos generated by using protein kinase or protein synthesis inhibitors. Anim Reprod Sci 2009;112:261–272. 18547754.
101. Van Den Bergh MJ, Fahy-Deshe M, Hohl MK. Pronuclear zygote score following intracytoplasmic injection of hyaluronan-bound spermatozoa: a prospective randomized study. Reprod Biomed Online 2009;19:796–801. 20031019.
102. Menezo Y, Junca AM, Dumont M, De Mouzon J, Cohen-Bacrie P, Ben Khalifa M. “Physiologic” (hyaluronic acid-carried) ICSI results in the same embryo quality and pregnancy rates as with the use of potentially toxic polyvinylpyrrolidone (PVP). Fertil Steril 2010;94:S232.
103. O'Flynn O'Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review. Fertil Steril 2010;93:1–12. 20103481.
104. Ferlin A, Raicu F, Gatta V, Zuccarello D, Palka G, Foresta C. Male infertility: role of genetic background. Reprod Biomed Online 2007;14:734–745. 17579990.
105. In't Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet 1995;346:773. 7658889.
106. Van Opstal D, Los FJ, Ramlakhan S, Van Hemel JO, Van Den Ouweland AM, Brandenburg H, et al. Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum Reprod 1997;12:682–686. 9159424.
107. Pang MG, You YA, Park YJ, Oh SA, Kim DS, Kim YJ. Numerical chromosome abnormalities are associated with sperm tail swelling patterns. Fertil Steril 2010;94:1012–1020. 19505688.
108. You YA, Park YJ, Kwon WS, Yoon SJ, Ryu BY, Kim YJ, et al. Increased frequency of aneuploidy in long-lived spermatozoa. PLoS One 2014;9e114600. 25490252.
109. Drabovich AP, Saraon P, Jarvi K, Diamandis EP. Seminal plasma as a diagnostic fluid for male reproductive system disorders. Nat Rev Urol 2014;11:278–288. 24709963.
110. Kwon WS, Rahman MS, Lee JS, Yoon SJ, Park YJ, Pang MG. Discovery of predictive biomarkers for litter size in boar spermatozoa. Mol Cell Proteomics 2015;14:1230–1240. 25693803.

Article information Continued

Funded by : Ministry of Agriculture, Food and Rural Affairs
Award ID : 116172-3

Table 1

Standard reference values for semen characteristics World Health Organization (WHO) (2010)

Volume 2 mL or more
pH 7.0–8.0
Sperm concentration 15 million or more/mL
Total No. of spermatozoa 39 million or more spermatozoa/ejaculate
Motility 40% or more progressive motility or 32% (a+b) (within 1 hour after ejaculation)
Morphology 4.0% or more (normal forms)
Viability 58% or more live spermatozoa
Leukocytes (106/mL) <1.0
Mixed antiglobulin reaction Less than 50% spermatozoa with adherent particle

Source: WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 5th edition. Geneva (Switzerland): World Health Organization; 2010.

Fig. 1

Effects of sperm treatment based on TEST-yolk buffer (TYB), Biggers-Whitten-Whittingham (BWW), and human serum albumin (HSA) on the outcome of the sperm penetration assay (SPA). (A) Effect of TYB and BWW on the outcome of SPA tests. (B) Human serum albumin concentrations in fertilization media. (C) Human serum albumin concentration in swim up method. The figure has been modified, and citing the original source published by Oh et al. [59].

PI, penetration index; PR, plasticity range.