Introduction

Over the years, there has been a worldwide decline in human semen parameters and overall decline in fertility outcomes due to which infertile men resort to assisted reproductive techniques (ART) [1]. Male factor infertility is the cause of almost 50% of couples wanting to have a child. Male fertility is affected by many conditions like genetic disorders, varicocele, infections, endocrine disturbances, lifestyle choices, and idiopathic factors [2,3,4]. The continuum of environmental exposures to which a person is exposed at a time includes air pollutants (NO2, Ozone, etc.), water pollutants like PCBs, and industrial chemicals like phthalates, which may also affect fertility [5]. Other known causes of male infertility include DNA fragmentation, reactive oxygen species (ROS) production, disruption in mitochondrial potential, abnormal epigenetic modifications, protamine depletion, and anti-sperm antibodies against sperm antigens. These factors, solely or in combination, may decrease sperm quality and also have an impact on blastocyst formation and embryo quality, and thereby affecting successful pregnancy [6, 7].

Semen contains secretion from the testis, epididymis, and various other accessory glands, which can convey the physiological/pathological information. Hence, conventional semen analysis is usually the first line of diagnostic tests used to evaluate the male fertility potential [8, 9]. The conventional semen analysis basically relies upon microscopic evaluation of parameters like sperm concentration, motility, and morphology in the ejaculate. Also, physical characteristics of semen like liquefaction time, volume, pH, odour, colour, and viscosity are evaluated. In order to maintain a uniform semen analysis procedure, World Health Organization (WHO) instituted a guideline and published first laboratory manual for semen analysis in 1980. The manual has been updated time to time with the latest version out in 2021 (Table 1) [10].

Table 1 Semen characteristics cut-off reference values as classified by world health organisation (WHO) and their method of detection
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The conventional manual semen analysis is subjective and susceptible to various errors, which include intra-individual variability, lack of standardization of protocols among laboratories, and absence of training. Additionally, the latest WHO guidelines are not uniformly adapted in different clinical/research settings, which make it even more difficult to analyse the result obtained from different settings [11]. More importantly, men with normal semen parameters can be both fertile and infertile, and thus advanced sperm function tests in adjunction with routine semen analysis is the need today.

In this systematic review, we have attempted to put together different sperm function tests available in the literature and assessed the clinical utility of these tests in predicting the male fertility potential.

Methods

Literature Search

This systematic literature search was conducted according to the PRISMA guidelines (Fig. 1) using PubMed, MEDLINE, Google Scholar, and Cochrane Library. Different keywords such as sperm DNA fragmentation tests, antisperm antibody test, mitochondrial dysfunction test, advanced sperm function test, hypo-osmotic swelling test, acrosome reaction test, capacitation, hemizona assay, seminal reactive oxygen species, sperm mitochondrial dysfunction, sperm agglutination, sperm nuclear chromatin decondensation, DNA damage and pregnancy, mitochondrial DNA copy number, sperm mitochondrial activity index, sperm intracellular ROS, antisperm antibody, mitochondrial membrane potential, sperm total antioxidant capacity, live birth rate, pregnancy outcome, spontaneous pregnancy, and assisted reproduction technology (IVF/IUI/ICSI) outcomes were used singly or in combination to retrieve the relevant articles.

Fig. 1
figure 1

Study selection criteria according to the PRISMA guidelines

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Inclusion and Exclusion Criteria

Full-text articles having adequate data on human subjects in English language were included in this systematic review. The non-related and non-human studies were excluded. Posters, abstracts, letters to the editor, and editorials were also not considered to compile this systematic review.

Data Extraction

The articles following inclusion criteria were further reviewed independently by two authors and information available on the following sperm function tests was extracted: “Sperm capacitation test, acrosome reaction test, hemizona binding assay, sperm DNA fragmentation test, seminal reactive oxygen species test, mitochondrial dysfunction tests, antisperm antibody test, hypo-osmotic swelling test, nuclear chromatin de-condensation (NCD) test.” Also, data on assisted reproduction outcomes was extracted and presented in the review.

Results

A total of 5169 articles were obtained, out of which 155 were excluded as non-English articles, 4403 articles were excluded as they were not directly related to the addressed topic and 517 articles were excluded as they dealt with non-human subjects.16 cross-references were included and finally 110 articles were included in writing the review (Fig. 1). The majorly investigated sperm function tests are hypo-osmotic swelling test, acrosome reaction test, sperm capacitation test, hemizona binding assay, sperm DNA fragmentation test, seminal ROS test, mitochondrial dysfunction tests, antisperm antibody test, and nuclear chromatin de-condensation (NCD) test. Table 2 represents the data extracted from these eligible articles, which enlist the details of various sperm function tests reported in the literature.

Table 2 Details of sperm function tests reported in literature
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Hypo-osmotic Swelling Test

The hypo-osmotic swelling test (HOS) is a simple, easy to perform, and accurate test that determines the sperm plasma membrane integrity. The test evaluates the ability of the spermatozoa to respond to changes in the osmolarity of the medium and its response to a hypo-osmotic environment. When the viable sperms are exposed to a hypo-osmotic medium, there is an influx of fluid, which causes the tail to swell and coil and this change in the tail morphology can be visualized under a phase contrast microscope. Higher the percentage of sperms with a swollen tail more likely is the fact that they have an intact plasma membrane. Sperm membrane integrity and its susceptibility to changes is very crucial for various events associated with fertilization like sperm capacitation, acrosome reaction, and spermatozoa binding to the zona pellucida layer of the ovum. Thus, an assay designed to evaluate the status of the spermatozoa membrane is of utmost importance in predicting fertility outcomes [40]. The spermatozoa are exposed to 50–100 mOsm lactose or sucrose solution and the fluid from the hypo-osmotic solution is exported across the plasma membrane and the sperm membrane integrity can be measured. The percentage of sperm with swollen or coiled tail (HOS+) are then evaluated [41]. If 60% of the spermatozoa are coiled, then it is considered as normal, and if 40% of the coiled spermatozoa are present in the semen sample, then it is considered as abnormal for fertility [42]. The HOS test induces several distinct categories of swelling, which are present in the sperm tail region. Tip swelling is when the rest of the tail is normal, but the tip of the tail is swollen; hairpin swelling is when the tail swells at the mid-piece and main piece junction and the tip may or may not show a swelling, shortened and thickened tail is when the tail swells that leads to the constriction of the surface causing shortening and partly or completely enveloped sperm tail where the tail balloons from swelling [40]. The HOS test is valuable in predicting the sperm variability, helps in infertility diagnosis, and widely used in sperm selection in assisted reproduction settings [40].

Sperm Capacitation Test

Capacitation is a biologically controlled phenomenon which is highly selective and is associated with a cascade of changes that occur once the sperm enters the female reproductive tract [43, 44]. The events involved in the capacitation of sperm renders it responsive to signals that originate from the cumulus-oocyte complex [43, 45]. The events involved in sperm capacitation are interconversion of various reactive nitrogen and oxygen species [43], loss of cholesterols from the plasma membrane involving a reversible lowering of the cholesterol/phospholipid ratio in the membranes leading to changes in membrane fluidity [43, 46], increase in tyrosine phosphorylation involving Src and cABL family kinases in the presence of ATP and appropriate pH [43, 47, 48] and calcium mediated hyper activated motility which is important for the penetration of the zona pellucida layer of the oocyte [49,50,51].

Moody et al. (2017) [12] established a laboratory-based protocol involving monosialotetrahexosylganglioside (GM1) localization measured by cap score sperm function test as a reliable, accurate and highly precise test that can be used to differentiate between sperms that respond to stimuli for capacitation and undergo various physiological changes required to fertilize the ovum from those that did not respond to cues of capacitation. Matamoros-Volante et al. (2018) [13] described another semi-automatized analysis using image-based flow cytometry as a powerful tool to study the intracellular events in a sperm to enable the study of various sperm factors associated with signalling with emphasis on tyrosine phosphorylation which is a hallmark in sperm capacitation. This study confirmed the role of PYK2 kinase and FYK2 kinase in tyrosine phosphorylation and provided evidence of the FYK2 expression and its subcellular localization in mammalian spermatozoa. Immunocytochemistry can also detect the subcellular localization of the tyrosine phosphorylation with the limitation that only a small number of cells can be analysed at a given time [52]. In conclusion, these techniques are useful in understanding the differential phosphorylation status of the sperm during various stages of capacitation and hence can help in predicting male fertility status.

Acrosome Reaction Test

Through the process of fertilization, the sperm must first undergo the acrosome reaction before it can penetrate the zona pellucida (ZP) layer and fuse with the oolemma. Progesterone secreted by the cumulus cells is an important factor influencing capacitation as well as the acrosome reaction [53]. Acrosome reacted sperm can only penetrate the ZP and premature occurrence of the acrosome reaction can lead to an inability to penetrate the ZP layer. Defects in ZP induced acrosome reaction have been a rising cause of male infertility [54, 55].

Acrosome reaction can be evaluated using flow cytometry, immunofluorescent assays using fluorescently labelled lectins and chlortetracycline assays. Several factors have been known to induce the acrosome reaction, which includes cumulus-corona complex, follicular fluid, progesterone and intact or acid solubilised ZP [14, 56,57,58]. A study by Liu and Baker (1996) [14] compared the chemically induced acrosome reaction by the calcium ionophore A23187 and by ZP. Sperm samples were collected from men with normal semen parameters according to the WHO criteria and subjected to acrosome reaction by A23187, intact ZP, and solubilised ZP. The acrosome reacted spermatozoa were distinguished by labelling with fluorescein labelled PSA and if more than half of the head showed a fluorescence, then the acrosome was considered to be intact and spermatozoa without any fluorescence was considered to be acrosome reacted. Sperm samples bound to intact ZP had a higher acrosome reaction than those subjected to solubilised ZP. So, the assessment of acrosome reaction should be done with sperms bound to intact ZP. A modification of the acrosome test, which does not require special reagents and equipment, has been described by Glazier et al. (2000) [16] which uses a combined approach of using HOS test and a double stain (Bismarck brown and rose bengal) and requires the usage of basic reagents and a light microscope. HOS test was used to detect viable sperms and the Bismarck brown and rose bengal was used to stain the post acrosome and acrosome region, respectively. A clear colour on the sperm head depicted a complete acrosome reaction. The cut-off set by this protocol was similar to that set by Carver-Ward et al. (1996) [17], in which CD46 present in the inner acrosome membrane was used as a marker to evaluate a viable acrosome reaction and analysed by flow cytometry. Zona induced acrosome reaction (ZIAR) is another method for induction of acrosome reaction by calcium ionophore. Here, the acrosomal status can be evaluated using microscopy, flow cytometry, and fluorescently labelled agglutinin.

Hemizona Binding Assay

Sperm zona pellucida binding tests most commonly includes the hemizona assay (HZA) and competitive intact zona sperm binding assay. HZA is an internally controlled assay and has been recommended as a diagnostic test to assess the binding of the sperm to zona pellucida to predict the fertilization outcome [15, 18]. In the HZA assay, each zona pellucida is cut into equal two halves or hemispheres by micromanipulation and one half is exposed to abnormal spermatozoa and the other half to spermatozoa from a normal sample. The results of the HZA are evaluated using the hemizona index [HZI = (bound sperm from sub-fertile men/bound sperm from fertile men) × 100]. The use of the HZA confers several advantages, which include the two halves of the zona pellucida, created by microdissection are equivalent a controlled comparison can be made, an internally controlled test can be carried out in the same oocyte, which circumvents the problem of using multiple oocytes and also ethical objections that might arise due to inadvertent fertilization of live viable oocyte is also eliminated [18]. Defective zona pellucida interactions have been seen in men with oligozoospermia and hence HZA assay should be performed in their case to predict their success rate in ART procedures.

Nuclear Chromatin De-condensation (NCD) Test

During the process of spermatogenesis there are several changes that occur in the haploid spermatid which includes elongation phase, cytoplasmic droplet ejection, and finally differentiation into mature spermatozoa [59]. During the process of mature sperm formation, diploid spermatogonia divide and differentiates into haploid round spermatids, which undergo cyto-differentiation including changes in chromatin. Testes specific histone variants, transition proteins and protamines replace the classical DNA bound histones and lead to a highly condensed paternal genome, which helps in the protection of the genome from various exogenic insults [60, 61]. The highly condensed nature of the nuclear chromatin in spermatozoa is due to the presence of S-S cross-links between the histone units [26]. In the NCD assay, the cleavage of the S-S links is induced by incubating the sperm, sodium dodecyl sulphate and ethylene diamine tetra-acetic acid together and the sample is visualized under a phase contrast microscope. Under the phase contrast microscope, the spermatozoa that do not decondense appear bright. Sperm nuclear chromatin condensation or chromatin integrity is also measured by analysing the levels of histone and protamine protein or transcript in sperm. A study done by Hamad 2019 [62] demonstrated the ratio of sperm’s nuclear histones/protamine transcripts (H2A+H2B)/(PRM1+PRM2) were higher in infertile patient samples as compared to the normal samples and there was a negative correlation between the histones/protamine transcript ratio and other sperm parameters like sperm count, motility, progressive motility, membrane integrity, normal morphology, and was positively correlated with chromatin decondensation [62]. The data suggests that histones/protamine ratios are important measures for sperm quality and could be used to predict male infertility.

Sperm DNA Fragmentation Test

Damaged DNA in human spermatozoa is the rising cause of male infertility according to recent clinical evidences. It has been observed that the amount of damaged DNA is much greater in infertile males than among fertile males [63]. The main cause of sperm DNA damage could be: Intrinsic factors such as defects during the spermatozoa maturation process, impaired protamination and defects in chromatin remodeling; Extrinsic factors include both dietary and various environmental effects and finally a defect in the apoptotic pathway, which increases the cellular stress and hence leads to increased sperm DNA damage [64]. Different assays are described for comprehensive analysis of sperm DNA fragmentation [65].

  1. a)

    Terminal deoxytransferase mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL): The TUNEL assay first described by Gorczyca et al. (1992) [19] utilizes the property of the terminal deoxyribonucleotide triphosphates (TdT) to non-preferentially add deoxyribonucleotide residues (dUTP) to the 3’OH groups of single and double stranded DNA. These cells are then subjected to flow cytometry and the spermatozoa positive for the TUNEL assay are shown to have more DNA damage. Sharma et al. (2016) [20] established a standard protocol, reference values, sensitivity, and specificity of the TUNEL assay using a new bench top flow cytometer which could make a prediction that there is an increased DNA damage associated with infertile men.

  2. b)

    Alkaline comet assay: First introduced by Ostling and Johanson (1984) [21], the alkaline comet assay is also known as single cell gel electrophoresis. In this assay, spermatozoa are lysed with detergent after they are embedded in the agarose gel and the migration of the fragments is noted with gel electrophoresis. After the electrophoresis is complete, the slides are stained with SYBR Green I and visualized with the help of a fluorescence microscope. Intact DNA without any fragmentation remains in the head and the fragmented DNA moves farther than the head in the gel forming a tail [25]. Comet assay is a sensitive, cheap, and reliable method that can be used as an advanced sperm function test in routine semen analysis. To make the comet assay more accurate and informative, Albert et al. (2016) [66] developed a standardized automated high throughput comet (HT-COMET) assay that could be used in fertility clinics as a diagnostic test to assay the degree of sperm DNA damage. The comet assay is a low throughput technique and cannot be used, when there is a large cohort of sample are to be analysed because of its manual selection procedure, focusing, imaging, and semi-automatized screening. The automated HT-COMET assay is at advantage because of its high accuracy, evenness, and ability to analyse a large cohort of samples in a much reduced time. HT-COMET assay uses the Komet KM software, which allows the automatic scoring and screening of comets. HT-COMET assay as established in this study could also be used in the assessment of DNA damage profiles which is a reflection of the extent of chromatin damage.

  3. c)

    Sperm chromatin structure assay (SCSA): SCSA is the most widely used technique that is routinely used in laboratories as a suitable test to detect the extent of sperm DNA damage. SCSA evaluates the ratio of single and double stranded DNA after the sperm nuclei is stained with Acridine Orange (AO) following denaturation by acid exposure. This is an indirect assay where; upon acid denaturation the DNA breaks are exposed which is then detected by AO. AO when bound to dsDNA (intact DNA) emits green fluorescence and when bound to ssDNA (denatured DNA) it emits red fluorescence. The SCSA evaluates the DNA fragmentation index (DFI), which is a ratio of the red to the total fluorescence intensity indicating the amount of denatured DNA over the total DNA present. DFI is a suitable indicator of the rate of fragmentation and is the most important parameter of SCSA. DFI value of 30% is considered to be clinically useful [22]. The SCSA is also used in the evaluation of high DNA stainable cells (HDS). HDS represents a distinct set of immature spermatozoa with incomplete chromatin condensation [22]. Another study by Enciso et al. (2013) [67] investigated the possibility of correlation between numerous chromosomal abnormalities and DNA damage in infertile men. Samples were evaluated for DNA damage using the SCSA assay and fluorescence in situ hybridization (FISH) with probes specific to chromosome 13 was used for detection of chromosomal abnormalities. The data obtained showed a significant correlation between the extent of DNA damage and the proportion of sperm with a numerical chromosome abnormality (p < 0.05) and concluded that the spermatozoa with chromosome abnormalities are more prone to DNA damage.

  4. d)

    Sperm Chromatin Dispersion Test (SCD) Test/Halo: SCD test is an indirect technique developed by Fernandez et al. (2003) [23]. In this technique, when relaxed DNA after acid denaturation is loaded into agarose, it produces halos/chromatin dispersion, which can be visualized by fluorescence microscopy. Since sperms with more damage have more ssDNA and fragmented DNA have a characteristic small halo. A study by Greze et al. (2019) [68] compared the efficacy of TUNEL and SCD assay, which are two sperm DNA damage tests to determine whether they can be used interchangeably and concluded that despite of a good correlation value the SCD assay gives slightly underestimated measurements as compared to the TUNEL assay and requires further validation from each laboratory because these results cannot be considered absolutely conclusive because of the small sample size used for the study.

  5. e)

    Toluidine blue staining test: This assay is a rapid, simple, and effective method that helps in the assessment of the integrity of the sperm chromosomal DNA. A thin smear of the semen sample after fixation and treatment with acid is stained with toluidine blue stain. The heads of the spermatozoa which have high chromatin DNA integrity are stained blue and the damaged DNA stains purple [25]. The toluidine blue (TB) test when compared to SCSA and TUNEL assay, showed a strong correlation between the proportion of damaged cells detected by TUNEL and SCSA assay and that detected by the TB test. The proportion of cells with defective DNA integrity as detected by TB test was almost equal to the sum of two SCSA parameters (DNA fragmentation index and the proportion of high DNA stainable cells) [24]. TB positive cells indicate that the DNA is both fragmented with an abnormal chromatin structure and hence being an inexpensive method can be used in andrology laboratories worldwide to test for DNA integrity along with other routine semen analysis parameters.

Antisperm Antibody Test

Antisperm antibodies, which were first described by Rumke et al. (1954) [28], are present in both the semen and the cervical mucus and have an inhibitory effect on the sperm function. Antisperm antibodies negatively impact the motility of the sperm through the cervical mucus, uterine cavities, and fallopian tubes and also block the events of fertilization like sperm capacitation, acrosome reaction, and sperm–egg interaction [69].

To detect the presence of antibodies on the surface of the sperm membrane mixed antiglobulin reaction (MAR) and direct immunobead test (D-IBT) are routinely used in andrology laboratories worldwide according to the standards set by WHO. Gel agglutination test (GAT) is also one of the most significant diagnostic tests to determine the presence or absence of antisperm antibodies in the semen of infertile men [29]. Immunobead test (IBT) allows for the rapid detection of IgA and IgG which are bound to washed spermatozoa and also detects the location of binding and provides an estimate regarding the proportion of spermatozoa which are bound to the antibodies [70]. Almeida et al. (1986) [30], in their study, evaluated whether IBT could be used as a screening test for antisperm antibodies in the men with accuracy and reliability. The majority of men who tested positive for IBT had both IgA and IgG on their surface and the immunobeads were predominantly localized over the head and the end piece of the tail but were also localized over the whole spermatozoa with identical binding pattern of IgA and IgG antibodies. IBT positive samples had impaired sperm penetration of the cervical mucus evaluated by cervical mucus in vitro penetration test (CMPT) and there was also an increased incidence of circulating antibodies in the serum. Thus, IBT can be used as a routine screening assay which would help in better selection of sperms for ART.

While evaluating male infertility, one of the parameters that should be evaluated before screening for ASA is sperm agglutination, which is due to binding of antibodies on the surface of the spermatozoa. Study by Gatimel et al. (2018) [71] suggests that the tests for the presence of antisperm antibodies should only be done when there is a presence of sperm agglutination during routine analysis.

Mitochondrial Dysfunction Tests

Mitochondrial dysfunctions in human spermatozoa are increasingly recognized as a cause of male infertility. The mitochondria present in the somatic cells are different from that present in the spermatozoa as during germ cell differentiation, there is a rapid loss of the sperm’s antioxidant defences. ROS such as hydroxyl radicals, superoxide anions, and hydrogen peroxide are generated in terms of oxidative stress in the mitochondria during cellular respiration. The antioxidant defences in the cell prevent the cell from succumbing to damages caused by ROS but when this balance is dismantled in the spermatozoa, there is an increased progression toward decreased motility. Mature spermatozoa contain 50–75 mitochondria, which are helically arranged around the mid-piece axoneme and are responsible for the generation of ATP by oxidative phosphorylation (OXPHOS) system, a key event required for sperm motility in the female reproductive tract [6, 72].

  1. a)

    Mitochondrial reactive oxygen species (ROS) tests: Numerous assays designed to measure the ROS levels in spermatozoa have gained significance over the years. Cassina et al. (2015) [73] have shown that sperm mitochondrial dysfunction evaluated using respiratory control ratio (RCR) measured by high resolution respirometer is correlated to sperm motility. Sperm mitochondrial oxygen consumption is dependent on various sperm parameters and this study showed that there is a positive correlation of RCR with sperm morphology, sperm viability, and sperm concentration. The study showed a positive correlation of both progressive and total motility with oxygen consumption measured as RCR with mean RCR values were lower in groups with <32% motility than those with motility greater than this value. MitoSOX; a fluorochrome has been routinely used in flow cytometric analysis for the detection of mitochondrial ROS in cells including sperm and semen [31, 74]. The study by Marques et al. (2014) [31] showed that the semen sample is actually heterogeneous with different categories of sperm population and can be divided into MItoSOX- (one that included better quality sperm and hence was not stained by the probe), MitoSOX+ and MitoSOX++ which had worsened sperm characters. Thus, MitoSOX can be used to predict sperm mitochondrial ROS levels and help in better selection of spermatozoa for ART.

  2. b)

    Mitochondrial membrane potential test: The reduction in mitochondrial membrane potential (MMP) has been considered a suitable prognostic indicator of abnormal sperm associated with reduced male fertility. A study by Espinoza et al. (2009) [75] recommended that the measurement of MMP as a routine test to evaluate semen quality. The study analysed three interrelated events namely ROS levels, phosphatidylserine (PS) externalization and sperm mitochondrial potential in fertile individuals and those undergoing fertility treatments using the fluorescent compounds dihydroethidium, JC-1 and annexin V-FITC, respectively. The results indicated that men with normal semen parameters had a higher MMP than men with abnormal semen parameters. MMP positively correlates with sperm motility. In addition, MMP showed a significant correlation with all sperm parameters analysed, namely, motility, viability, morphology, sperm count, and volume. The correlation between prohibitin (a major mitochondrial inner membrane protein) and mitochondrial membrane potential (MMP) has been described by Chai et al. (2017) [34] using JC-1 assay.

  3. c)

    Mitochondrial DNA copy Number: Sperm mitochondrial DNA copy number (mtDNAcn) accounts for mitochondrial DNA copies per nuclear DNA copy [76]. Several human studies have shown the association of high mtDNAcn with poor semen parameters. Few studies have also reported the association of mtDNAcn with sperm motility and lower pregnancy rate [77,78,79,80]. These findings indicate that sperm mtDNAcn may be a novel biomarker of semen quality and pregnancy outcome. To estimate the mtDNAcn, quantitative real-time PCR (qPCR) has been used most frequently and current gold standard method due to its low cost and quick turnaround time [35]. Few studies have reported digital PCR (dPCR) as the new gold standard for mtDNAcn estimation technique due to its sensitivity and absolute copy number quantify ability [36, 37].

Seminal Reactive Oxygen Species Test/Oxidative Stress Test

Oxidative stress is one of the most well-established factors associated with male infertility due to its effect on both DNA integrity and impairment of sperm motility [81]. Various tests for detection of oxidative stress include reactive oxygen species (ROS) assays, total antioxidant capacity (TAC) assay, and malondialdehyde (MDA) assay [38].

Nitro blue tetrazolium (NBT) assay is a photometric method of detection of coloured formation that is an indicator of seminal ROS production [82]. NBT assay is a simple and inexpensive test which can be used routinely but suffers from the lack of standardization protocol. Tunc et al. (2010) [82] developed a standardized protocol for the NBT assay and correlated the results obtained with those obtained from assessing sperm DNA integrity, apoptosis, and sperm motility. In their study, semen samples from both fertile and etiology infertile men were assessed for seminal ROS (NBT assay), sperm DNA integrity (TUNEL assay), apoptosis (Annexin V), and sperm motility. The production of seminal ROS was higher in infertile men than in fertile men. A negative correlation existed between formazan production and sperm motility, which suggested that with an increase in ROS production there is a decrease in the sperm motility. 8-Hydroxydeoxyguanosine(8-OHdG) is a DNA adduct that can act as a biomarker and can be commonly used for detection of DNA damage due to oxidative stress. Another study by Kao et al. (2008) [83] demonstrated that sperm which has higher oxidative damage (measured by levels of 8-OHdG and lipid peroxides) have a lower antioxidant capacity and negatively correlated with sperm motility. The levels of various antioxidants like retinol, α-tocopherol, ascorbate, and protein thiols were measured, and it was seen that the antioxidant capacities were lower in men diagnosed with infertility.

A study by Shen et al. (2000) [84] established 8-OHdG as a suitable and quantitative biomarker for the oxidative DNA damage induced by ROS in human sperm along with TUNEL, single cell gel electrophoresis (SCGE) and Comet assay. Majzoub et al. (2018) [85] assessed the relationship between seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) for the first time and also evaluated its effect on different sperm parameters. ORP was measured by the male infertility oxidative system (MiOXSYS) that measures the net balance between the oxidants and reductants in any given media. In the control patients, there was no correlation between SDF and ORP, but there was a significant correlation between ORP and SDF levels in infertile group.

Homa et al. (2019) [86] evaluated the efficacy of two different assays for measuring oxidative stress in seminal plasma, namely; chemiluminescence assay and oxidation-reduction potential (sORP). Semen samples collected were measured for seminal ROS using either a chemiluminescence assay or an electrochemical assay to measure the oxidation-reduction potential. In samples with abnormal semen parameters, ROS, sORP, DFI, and HDS were elevated as compared to the control samples but in samples which had polymorphonuclear leucocytes (PMN); there was an increased ROS level but there was no change in the sORP and DFI. This assay validated the need of using both tests in conjunction for the evaluation of oxidative stress in different samples with abnormal semen parameters with special emphasis on those samples where leucocytes are detected.

The Suitability of Sperm Function Tests in Predicting Male Fertility and Pregnancy Outcomes

Routine semen analysis is widely used in andrology laboratories for the male infertility diagnosis but its limited predictive value emphasizes on the need to include more advanced sperm function tests. Number of sperm function tests has been reported in the literature; however, very few studies report their predictive value for fertilization and pregnancy rates (Table 3).

Table 3 Studies reporting suitability of different sperm function tests in predicting male fertility and pregnancy outcomes
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A randomized control study (n = 79) by Sallam et al. (2005) [98] has concluded that sperm selected via modified HOS test is better suited for ICSI as compared to selection based on morphology alone. Low HOS test scores can act as a predictor of the formation of defective embryos which translates to a lower fertilization rate [101]. A study by Tartagni et al. (2002) [102] reported prediction of a successful pregnancy with the HOS test > 50%, had a sensitivity of 64%, a specificity of 75%, respectively.

The HZA can evaluate the relationship between the sperm-zona binding and sperm fertilizing ability in IVF settings [103]. Oehninger et al. (1989) [18] indicated that patients with poor fertilization rates in IVF showed a lower binding than successful fertilization cases which makes HZA is a useful tool for the evaluation of dysfunctional sperm-zona pellucida binding. Further, HZA along with other factors like sperm motility, sperm morphology and sperm viability can predict IVF outcome. Franken et al. (1989) [97] demonstrated that binding to the hemizona was significantly higher for the group with reported IVF success as compared to the failure group. Another study by Gamzu et al. (1994) [92] aimed to assess the prognostic value of HZA in determining the success of IVF with a large sample of patients to establish its validity. The threshold value for HZI that gave the best predictive value in this analysis was 23%, and thus this could be used as a pre selection test before IVF. HZA can also be used as a predictor of pregnancy outcome in patients undergoing controlled ovarian hyper stimulation (COH) and intrauterine insemination (IUI) and thus help in the counselling of couples before any treatment is advised. Patients having HZI < 30 had a lower pregnancy rate as compared to patients with HZI ≥ 30. HZA can act as a predictor of fertilization in IVF but it can also help in the prediction of pregnancy in IUI. The threshold value HZI < 30 for poor fertilization established previously for IVF was also statistically valid in the IUI setting [87].

The assessment of sperm chromatin is essentially important and helps in the prediction of fertility. SCSA assesses the sperm nuclear chromatin integrity. Studies on the assessment of SCSA as a predictor of fertility/pregnancy outcome have shown varied results. Bungum et al. 2004 [22] correlated the results of SCSA with the outcome of IVF, IUI, and ICSI in 306 couples undergoing ART. The assay showed that pregnancy is possible despite of high DFI because oocyte and the embryo can repair the DNA damage to a certain extent. The chances of pregnancy/delivery were higher in those groups were the DFI ≤ 27% and HDS ≤ 10% in all the 3 ART groups. Interestingly, the pregnancy outcomes in the group with DFI >27% were better in ICSI cases than IVF group [22]. Study by Lin et al. (2008) [95] has shown that men with high sperm DFI (> 27%) and HDS >15% could achieve successful pregnancy and delivery after either IVF or ICSI; however, the abortion rates were increased in IVF cases suggesting that ICSI could be recommended in such cases. On the other hand, Virro et al. (2004) [104] have shown that men with high levels of DNA fragmentation (> or =30% DFI) had low blastocyst rates and pregnancy rate but the fertilization rate was not affected [95]. Nijs et al. [105] also confirmed the threshold of < 15% HDS for obtaining successful fertilization and pregnancy in IVF; however, ICSI outcome was not affected by any conventional or functional sperm parameters as it avoids the biological anomalies in sperm [105]. Meseguer et al. (2011) [96] later showed that the effect of SDF on pregnancy was independent of the sperm origin (fresh or thawed) and the fertilization procedure. For every 10% increase in DFI there was a decrease in the chances of achieving pregnancy by 1.31, if oocytes from fertile women were used. The investigators attributed the heterogeneity in the results of DNA fragmentation in predicting pregnancy outcomes to the DNA damage repair capacity of the oocyte. A systematic review and meta-analysis suggested that the recent SDF tests are not sufficient to discriminate couples who have a low chance to conceive from those who have a greater chance of conceiving after medically assisted reproduction (MAR) [106]. HOST being an easy to conduct test can be used as an alternative. Stanger et al. (2010) [107] have established that low HOST values in neat semen samples were significantly associated with increased DNA damage (p < 0.01) evaluated by both the TUNEL and SCSA assay. Esteves et al. (2022) [91] assessed the reliability of the SCD assay to evaluate sperm DNA damage in infertile men (n = 219). They classified the infertile men in normal, intermediate, and high SDF categories and evaluated the reliability of the assay by re-testing SDF within a 3-month interval under similar conditions. Their observations overall support SCD test for patient classification using predefined SDF thresholds.

Recurrent pregnancy loss is a devastating problem which affects couple worldwide and standard semen parameters are not sufficient enough to predict this recurrent pregnancy loss (RPL). An assessment of the sperm DNA integrity may act as a prognostic factor for the determination of idiopathic recurrent pregnancy loss (iRPL) following spontaneous conception. A systematic review and meta-analysis by Mcqueen et al. (2019) [108] suggested that there is an association between RPL and DNA fragmentation and there is a requirement for further studies to correlate the DNA fragmentation rate with miscarriage frequency, live birth rates, etc.

Hervás et al. (2022) [93] studied the effect of SDF (TUNEL assay) on ICSI outcomes by evaluating the per embryo transfers (ETs) collective live birth rates (CLBRs), embryos replaced (EmbR), and metaphase II (MII) oocytes required in successive treatments to attain the first newborn. They concluded that high SDF does not affect the LBR. Also, the cumulative chance to have a child in couples undergoing ICSI cycles is not affected per ET, per EmbR, and per donated MII oocyte used.

Generation of short-lived reactive oxygen radicals has a detrimental effect on various sperm functions and may cause premature acrosome reaction, lipid peroxidation, which can induce the apoptotic cascade causing DNA fragmentation and can cause a decrease in the mitochondrial membrane potential. Hammedah et al. (2006) [109] showed that there is a negative correlation between the production of ROS and fertilization rate in IVF and ICSI programs. Zorn et al. (2003) [100] also found that ROS level had a negative correlation with pronuclear formation and sperm MMP is a negative correlation between ROS production and embryo development. In ICSI patients, the sperm MMP was affected by both H2O2 and O2- but H2O2 exclusively has a role in the prevention of pronucleus formation [110]. The reason for the poor pronuclear formation in the presence of intracellular ROS is attributed to the destruction of microtubules and cytoskeletal structures by the high ROS levels.

Progesterone-induced acrosome reaction is an extremely important event that dictates the success of fertilization, and hence more research in this area guarantees better treatment for male infertility [111]. Throughout the literature there has been conflicting evidence regarding the use of acrosome reaction tests in the prediction of fertilization rates with some studies claiming that with a decrease in the acrosome reacted sperm, there is a decrease in the fertilization rate in IVF and ICSI [88, 89, 99, 112], and some studies claim no correlation at all between the status of acrosome reaction and the fertilizing ability [113]. Acrosome reaction (AR) and sperm DNA fragmentation are good predictors of fertilization and blastocyst rate, respectively. Recently, an equation combining sperm chromatin integrity and AR developed by investigators can work in the individual level and predict fertilization and blastocyst rates [114].

Antisperm antibodies are known to hamper the process of fertilization by hindering the passage of the sperm through the female genital tract. ASAs interfere by binding to specific antigens present on the sperm membrane, preventing the interaction of the sperm with the oocyte and also preventing the acrosome reaction to occur. A study by Janssen et al. (1992) [94] concluded that IVF provides an equivalent conception chance in couples with antisperm antibodies provided all the other sperm functions are normal. The results obtained by Vujisic et al. (2005) [115] and N. Sukcharoen et al. (1995) [116] further stated that the presence of antisperm antibodies in the semen or the follicular fluid does not have an impact on the IVF outcome. Esteves et al. (2007) [90] have shown in their study that ICSI outcomes are also not influenced by ASA.

Discussion

Conventional semen analysis has been an integral part of clinical andrology and male fertility evaluation. While the test gives a fair idea of the seminal characteristics, it cannot be used as an accurate predictor of male fertility potential and fecundity [8, 11]. This notion comes from the fact that around 10% of men with normal semen analysis are infertile and similar percentage of men with abnormal semen analysis is fertile. Hence, the low predictive capacity of conventional semen analysis has necessitated the quest to develop more sensitive and specific sperm function tests on which clinicians can base their preliminary diagnosis [11, 117]. However, it may not be necessary to go for specialized sperm function tests for every individual presenting to andrology clinic. Men with fairly good semen parameters may not require to be tested. But for those with borderline semen parameters or with history of infertility, it becomes necessary to perform a battery of tests to evaluate sperm quality [11, 118].

Over the past two decades, a number of sperm function tests have been developed to determine the quality of spermatozoa. These tests basically evaluate the intact sperm plasma membrane and acrosome, acrosome reaction, capacitation, sperm–zona pellucida interactions, chromatin quality, nuclear and mitochondrial sperm DNA integrity, mitochondrial functions, etc [118]. These SFTs can help clinicians to make more precise diagnosis of male factor contributing to infertility. This may also help in treating the underlying conditions and may in turn, help in improving sperm quality/functions and pregnancy outcomes. ART is the ultimate disease management option for infertility [119]. These tests could also help in better patient counselling and more tailored and effective ART.

However, majority of these sperm function tests singly or together do not have proven clinical value in predicting fertility and have not been adopted into routine clinical use as yet. The basic limitation of these sperm function tests to reach the clinics is unavailability of high-quality clinical data correlating the sperm function to fertilization or pregnancy outcome [11]. Any test to be clinically relevant, it necessarily has strong positive predictive value for fertilization capacity and pregnancy outcome as well as have small overlap between fertile and infertile samples. Also, there is a need to undertake multi-centric trials which can establish the robust thresholds for these SFTs. The other inherent technical issues limit these tests to reach to the routine clinical practice are complexities to set up these tests and equipment costs. Requirement of more robust, simplified, reliable, cheaper, and reproducible SFTs in male andrology work flow is evident.

So far, growing literature on sperm OS and SDF measurement has highlighted its clinical utility [100, 109, 120]. The condition of oxidative stress is present in 30% to 80% of cases and sperm DNA damage is observed in approximately 5% to 25% of infertile men with normal and/or abnormal semen analysis [22, 95, 104, 105]. Hence, including both these sperm function tests in to male infertility diagnostic workup could be relevant.

Conclusion

Conventional semen analysis has low prognostic value in predicting pregnancy outcomes. Therefore, advance sperm functional tests are much needed in the workup of the infertility clinics. This could be helpful to guide the clinician to make more precise clinical decisions. Various attributes together contribute to sperm function and fertilization capability. Hence, any single sperm function test may not be able to predict its quality and functions adequately. An array of high-sensitivity tests with high pregnancy predictive value and low false positive rates are required to be introduced to the andrology clinical practice. Currently, unavailability of the high-quality clinical data, robust thresholds, complex protocols, and high cost, etc. are the limiting factors prohibiting current sperm function tests to reach to clinics. Further multi-centric research efforts are required to fulfil the existing lacunas and pave the way for these tests to be introduced into the clinics. Including OS and SDF tests for male fertility evaluation could be clinically useful.