Lack of trusted diagnostic tools for undetermined male infertility

Graphical abstract

Semen analysis is the cornerstone of evaluating male infertility, but it is imperfect and insufficient to diagnose male infertility. As a result, about 20% of infertile males have undetermined infertility, a term encompassing male infertility with an unknown underlying cause. Undetermined male infertility includes two categories: (i) idiopathic male infertility—infertile males with abnormal semen analyses with an unknown cause for that abnormality and (ii) unexplained male infertility—males with “normal” semen analyses who are unable to impregnate due to unknown causes. The treatment of males with undetermined infertility is limited due to a lack of understanding the frequency of general sperm defects (e.g., number, motility, shape, viability). Furthermore, there is a lack of trusted, quantitative, and predictive diagnostic tests that look inside the sperm to quantify defects such as DNA damage, RNA abnormalities, centriole dysfunction, or reactive oxygen species to discover the underlying cause. To better treat undetermined male infertility, further research is needed on the frequency of sperm defects and reliable diagnostic tools that assess intracellular sperm components must be developed. The purpose of this review is to uniquely create a paradigm of thought regarding categories of male infertility based on intracellular and extracellular features of semen and sperm, explore the prevalence of the various categories of male factor infertility, call attention to the lack of standardization and universal application of advanced sperm testing techniques beyond semen analysis, and clarify the limitations of standard semen analysis. We also call attention to the variability in definitions and consider the benefits towards undetermined male infertility if these gaps in research are filled.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-020-02037-5.

Introduction

Infertility, the inability to achieve pregnancy after 1 year of regular unprotected sexual intercourse, is an extremely emotional, physical, and financially stressful condition for patients to experience [1]. Approximately 12–15% of couples in the USA are infertile [2–4]. Infertility might be considered a public health problem when the prevalence reaches 15%, so the current situation should fall under this classification [5]. Societal pressures and blame have typically been focused on the females in these circumstances; however, data has consistently showed that males are the cause in up to 50% of infertile couples (Fig. 1 and Table 1) [6–12]. This amounts to about 7% of all married couples in the USA being unable to conceive due to male infertility, which is over eight million people.

Fig. 1.

About $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$3$}\right.$ of infertility is due to male infertility (dark blue, male factor infertility). Since infertility has similar contributions from males and females, we estimated that half of unexplained couple infertility (seemingly fertile couples who are not conceiving for unknown causes or combined male and female) is due to unexplained male infertility ($\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$6$}\right.$) (light blue) (Table 1). Altogether, males cause about 50% of infertility [6–11]

Table 1.

Male infertility plays a role in about 50% of infertility [6–11]

Origin of Infertility	Thonnea 1991	Razza 2002	Bayasgal 2004	Ekwere 2007	Farhi 2011	Oztekin 2019	Average (% ± SD)
Male-only infertility	20%	37%	26%	30%	29%	46%	31 ± 8
Female-only infertility	33%	46%	46%	43%	31%	8%	35 ± 13
Combined male and female	39%	2%	19%	12%	19%	6%	16 ± 12
Unexplained couple	8%	15%	10%	15%	21%	41%	18 ± 11
Patient number	1686	250	430	750	1992	406	Total of 5514

Male infertility is not only a males’ health issue but also a females’ health issue, as the current burden of reproductive treatment for undiagnosed male infertility falls on females [13]. Since male infertility is a significant cause of infertility, this review focuses mainly on male infertility; other reviews on female infertility are available [14, 15]. The purpose of this review is to uniquely categorize male infertility based on intracellular and extracellular causes. Although many reviews on male infertility have been published, this review uniquely combines two categories of male infertility based on their commonality of having unknown causes. We include two categories in undetermined male infertility: (i) idiopathic male infertility—infertile males with abnormal semen analyses who present with no known cause for these abnormalities and (ii) unexplained male infertility—males with “normal” semen analyses who are unable to impregnate due to unknown causes. This categorization is then used to call attention to pitfalls in current diagnostic techniques. A glossary is included to help clarify terminology. This review is meant for patients, clinicians, and scientists.

A deficient semen analysis currently categorizes male infertility

Semen analysis is considered the foundation of the male fertility evaluation [16, 17]. Evaluating infertility in males through semen analysis has been a clinical standard since the 1930s and became more widely accepted when the World Health Organization (WHO) adopted it in 1980 [18–20]. It includes both quantitative measures (e.g., semen volume, sperm count in semen) and qualitative measures (e.g., semen viscosity, sperm morphology) [21]. To improve semen analysis, its standards have evolved over the years and the updated 2010 WHO manual includes major changes, such as the reference values for sperm motility and sperm count [20]. As a result of these changes, fewer males are classified as infertile [22]. Although semen analysis has become the cornerstone of defining male infertility, it has many flaws and should not be used as the sole evaluator [23]. Its standards were determined by examining only a fertile population [20]. It has large inter-laboratory and intra-individual variability [24–29]. It is an indirect measure of fertility that looks at semen’s properties (e.g., low sperm count) rather than sperm’s ability to conceive a healthy child, and it is not an absolute predictive measure (unless a male has no sperm such as in azoospermia) [30–32]. And most significantly, it only examines a few essential features of the sperm such as sperm count, motility, and morphology rather than intracellular sperm components such as DNA and centrioles [30–32]. Indeed, a recent consensus group discussed the issue of semen analysis at length and agreed that basic semen analyses only provide diagnostic clues and very little functional information, and that progress in the assessment of sperm function over the last 25 years has been very limited and disappointing [33]. Therefore, many infertile males can be overlooked if their semen analyses are “normal” and be diagnosed with unexplained infertility. With better analysis, we may discover that many of these couples actually have undiagnosed male factors (Fig. 1).

Exploring the prevalence of the various causes (etiology) of male factor infertility is one purpose of this review. However, the word “cause” is somewhat vague since a cause may have an underlying deeper cause, and the terms “proximate” and “ultimate” causation have been used to make this distinction [34]. For example, semen analysis may reveal that a patient has oligospermia or low sperm count (proximate causation). Additional testing may show the presence of a hormonal imbalance (another proximate causation), which may be the result of a genetic mutation (ultimate causation). Such a detailed level of understanding of causation is rare in the current clinical environment, so to keep this concept simple, we defined cause as the condition that can be treated. Therefore, in the above example, the cause of this patient’s infertility is the hormonal imbalance, since that might be treated with hormone replacement therapy [35]. If this treatment did not solve the infertility, then the cause is still unknown, and he is classified under idiopathic male infertility.

To provide future direction for male infertility research, we organized male infertility by causes, giving attention to whether the cause rises from within the sperm or its surroundings, to show where research needs to be prioritized. Male infertility causes can be divided into two main domains (Fig. 2). The domain of Male factor infertility is where the male partner is known to be infertile by current diagnostic tools (e.g., they have abnormal semen analyses) (dark blue in Fig. 1 and Fig. 2). The infertility in this domain can be divided into two categories (Fig. 2, gray background): Explained male factor infertility, males with a known cause of their abnormal semen analyses, and Idiopathic male infertility, males with an unknown cause of their abnormal semen analyses. Explained male factor infertility has three main subcategories: anatomical, hormonal, and genetics and environmental (Table 2). Since explained male factor infertility has been more thoroughly researched, this review focuses on idiopathic male infertility [39, 40].

Fig. 2.

Male infertility causes can be divided into two main domains: Male factor infertility (infertile males with abnormal semen analyses) and Unexplained male infertility (infertile males with “normal” semen analyses). The Male factor infertility domain (dark blue background) can be divided into two categories (light gray boxes): Explained male factor infertility, males with a known cause of their abnormal semen analyses, and Idiopathic male infertility, males with an unknown cause of their abnormal semen analyses. Explained male factor infertility has three main subcategories: anatomical, hormonal, and genetics and environmental (Table 2). Idiopathic male infertility can be broken down based on the location of the abnormality to two locations: Extracellular sperm defects (e.g., semen viscosity, volume, pH) and General sperm defects (e.g., viability, count, motility, shape) (dark gray boxes, red text). Many of these general sperm defects are associated with intracellular sperm defects (yellow arrow). The Unexplained male infertility domain (light blue background) is likely caused by an intracellular sperm defect, resulting in a normal semen analysis (yellow boxes). Together, unexplained male infertility and idiopathic male infertility are named Undetermined male infertility (green line). The prevalence of categories and types is reported as well (Table 2, Online Resource 1 and 2) [5–7, 16, 36–38]

Table 2.

Categories of male factor infertility and prevalence: 23% of infertile males have idiopathic causes (Online Resource 1) [5–7, 16, 36–38]

Categories	Definition	Examples of causes	Estimated prevalence N = 8895
Anatomical	Any defect in or within the anatomy that affects mechanical processes that can lead to male infertility	Varicocele, ductal or vasal obstruction, testicular failure, ejaculatory dysfunction, cryptorchidism, testicular torsion, surgical injury, obstructive azoospermia, testicular damage	55%
Genetics and environmental	Genetic abnormalities and/or external biological, chemical and other external factors intertwining to affect processes that can lead to male infertility	Genital infections, drugs, radiation, cancer, systemic disease or infection, immunologic conditions, sickle cell disease, chromosomal aberrations, Sertoli cell syndrome, male accessory gland infection, congenital conditions	17%
Hormonal	Any hormonal imbalance leading to processes affecting sperm production, sexual desire, or any other hormonal process that can lead to male infertility	Hypoandrogenism, decreased or increased hormone levels, psychological factors	4%
Other	Causes stated in studies, so not idiopathic, but provided with minimal information so cannot be accurately categorized	Sexual problems or dysfunction, coital failure, heat atrophy, maturation arrest, ultrastructural	2%
Idiopathic	Abnormal semen analysis with no known cause for that abnormality; broken down by location into males with extracellular sperm defects and general sperm defects	- Extracellular sperm defects: semen volume, semen pH, semen viscosity, sperm agglutination, pyospermia - General sperm defects: oligospermia, azoospermia, asthenozoospermia, teratospermia, necrospermia, hyperspermia	- 23% of infertile males have idiopathic male infertility; 38% of idiopathic males have extracellular sperm defects; 62% of idiopathic males have general sperm defects

Idiopathic male infertility consists of infertile males with abnormal semen analyses but an unknown cause for that abnormality. We compartmentalized this category based on the presence of the abnormality at two locations: Extracellular sperm defects (e.g., semen viscosity, volume, pH) and General sperm defects (e.g., viability, count, motility, shape) (Fig. 2, red highlight). As will be discussed, many of these general sperm defects are expected to be associated with intracellular sperm defects (Fig. 2, yellow arrow), and knowledge of the precise intracellular defect is critical for selecting the best reproductive treatment.

The second domain is Unexplained male infertility; this encompasses males from couples with unexplained infertility (both partners have no findings in their fertility evaluation that explains their infertility) that are predicted to be infertile but are undiagnosed due to a lack of clinical diagnostic tests (light blue in Fig. 1 and Fig. 2). The likely explanation for this hidden male infertility is a defect in an intracellular sperm component that did not affect sperm viability, motility, number, or shape, resulting in a normal semen analysis (Fig. 2, yellow highlight). For example, DNA fragmentation or centriole dysfunction could be mechanisms of unexplained male infertility [41, 42]. Therefore, developing trusted intracellular sperm analysis is critical for identifying these defects and correctly diagnosing males with undetermined male infertility (Fig. 2, green line).

Idiopathic male infertility is the 2nd most prevalent category within male factor infertility

Categories of male factor infertility included anatomical, hormonal, genetics and environmental, and idiopathic. To determine the prevalence of the various causes of infertility, we performed a systematic literature search on Google Scholar using the following keywords: “male infertility + causes + prevalence” and “male infertility + etiology + prevalence”. Studies that clearly reported the prevalence of multiple male infertility causes were included, while studies that reported on one cause were excluded. A total of seven studies were reviewed and data was abstracted to provide a pooled estimate of the prevalence (Table 2) [5–7, 14, 36–38].

Anatomical causes were found to be the most frequent explanation for male factor infertility, 55% (Fig. 2) [5–7, 14, 36–38]. Idiopathic male infertility was the next largest category, 23%, and an important category in which research is lacking: about a quarter of infertile males have an unknown cause for their abnormal semen analyses [5–7, 14, 36–38]. Genetics and environmental and hormonal causes followed, at 17% and 4% respectively. Some studies reported vaguely described causes, such as “coital failure,” that we categorized as “other causes” (Table 2). These studies included less than 9000 infertile males in total; therefore, more comprehensive studies are needed for a more reliable estimate.

Males with idiopathic infertility have an abnormal semen analysis, and some of the reviewed studies reported the specific semen parameter that was abnormal in these males [5, 6, 36, 38]. Among the idiopathic male infertility subtypes, general sperm defects (defects in number, motility, shape or viability) were shown to have the highest frequency, 62%, when compared to extracellular sperm defects (Table 2). Within general sperm defects, abnormal sperm count was most frequent, 74% (Fig. 2). However, the true frequency of sperm defects in males with idiopathic infertility is very unclear due to (i) small sample sizes (ii) vague terminology and laboratory parameters, and (iii) lack of data on sperm defects occurring in combination [5, 6, 36, 38].

Small sample sizes

Of the seven papers we identified, only four papers reported the prevalence of sperm defects specifically in the males with idiopathic infertility. These papers included a total of 211 males (Online Resource 2). Reliable calculations of the frequency of general sperm defects in idiopathic male infertility require a much larger sample size.

Vague terminology and laboratory parameters

If terminology and laboratory parameters are clearly reported, the frequency of sperm defects in males with idiopathic infertility would be more accurate. For example, oligospermia and azoospermia (no sperm) can be overlapping terms: if the definition of oligospermia is “sperm concentration less than the reference value,” then technically azoospermia would be in this group as well [43]. Therefore, these studies needed to explicitly state definitions and laboratory parameters, and stating that WHO standards were followed is not enough since there can be large inter-laboratory variability [24–27]. It is also important to keep in mind that different versions of the WHO manual may have been used depending on the year of the study.

Lack of data on sperm defects occurring in combination

An important limitation of these studies is that they only reported one cause per patient. Sperm defects may occur in combination in about 20% of infertile males, but most studies only reported one sperm defect for each idiopathic infertile male, which can greatly alter the reported frequency [5, 44–46]. It is unclear whether their semen analyses only showed one defect or if only one defect was reported [5, 6, 36, 38]. Distinguishing single versus multiple defects is important because multiple sperm defects may be a stronger indicator of infertility prognosis than a single sperm defect [33]. For example, having asthenoteratospermia or oligoasthenoteratospermia is associated with an increased incidence of chromosomal abnormalities and lower success with in vitro fertilization (IVF) [47–49]. Therefore, studies must clarify how they report sperm defects.

An accurate estimation of general sperm defect frequency and additional research is necessary to determine the cause of altered semen parameters in males with idiopathic infertility. For example, associations have previously been discussed between teratospermia (morphology defects) and DNA fragmentation, asthenospermia (low motility) and mitochondrial depletion, asthenospermia and centriole defects, and teratospermia and centriole defects [50–53]. Additional studies are required to confirm these findings. Also, there is a need to develop clinical tests that assess some intracellular components of the sperm, since they lack widely available clinical tests (e.g., centrioles and RNA). Due to lack of data evaluating clinical outcomes and variability in testing, diagnostic tests beyond semen analyses are not routinely recommended [54].

Intracellular sperm analysis can complement semen analysis to resolve unexplained male infertility

We propose that one solution to overcoming the deficiencies in semen analysis is to use intracellular sperm analysis, a term we coined to encompass diagnostic tools that are able to look for defects inside of the sperm [13]. Semen analysis looks at the general properties of the sperm and the seminal fluids [55]. On the other hand, intracellular sperm analysis looks at the intracellular components of the sperm. Some of the defects that intracellular sperm analysis would detect include DNA fragmentation, abnormal RNA, defective centrioles, sperm aneuploidy, or an excess of reactive oxygen species (ROS) [13, 56, 57].

Research on intracellular sperm defects and diagnostic tools, with the exception of DNA, is scarce in general, and particularly in undetermined male infertility [58–60]. For example, none of the reviewed studies assessed idiopathic infertile males for intracellular sperm defects as their cause of infertility [5–7, 14, 36–38]. In order for clinicians to trust and utilize intracellular sperm analysis, much more research needs to be done in the field of intracellular sperm defects and diagnostic tools. We must first develop sensitive and specific diagnostic tests that can quantitatively analyze multiple types of intracellular sperm defects. Then, rigorous studies need to be conducted that analyze the predictive value of the diagnostic tools towards infertility. If successful, clinicians can use intracellular sperm analysis routinely or before assisted reproductive treatment (ART) to precisely analyze the individual sperm being injected (without killing it or affecting its integrity) to maximize the chance of successful fertilization. Ideally, the combination of results from semen analysis and intracellular sperm analysis can provide patients an individualized percentage of estimated success of ART.

Taking all of that into consideration, we considered three main reasons to pursue intracellular sperm analysis:

• i.Reduce the prevalence of idiopathic male infertility as no demonstrable cause of an idiopathic male’s abnormal semen analysis may simply mean that the cause lies inside the sperm
• ii.Reduce the prevalence of unexplained couple infertility as intracellular sperm defects may not always manifest through an observable sperm defect, thus resulting in a normal semen analysis
• iii.Provide couples a realistic idea of their chances of a successful pregnancy, so that couples have all the necessary information before making difficult decisions regarding their reproductive treatment [61–63]

Intracellular sperm analysis is vital but under-researched and not currently trusted by clinicians

Intracellular sperm components are essential for male fertility because they allow sperm to serve their functions of navigating within the female reproductive tract to the correct location, and then fusing and depositing life-sustaining substance into the egg. The intracellular components that allow these vital functions include DNA, RNA, centrioles, ROS, and egg activation factors, which are essential for sustaining the life of the embryo, and chemokine receptors and their signal transduction machinery, which help navigate the sperm. However, this list may be incomplete, and we expect that further research will identify additional essential intracellular sperm components.

DNA

Most intracellular sperm defects are extremely under-researched, but significant research has been done in the field of DNA integrity. A well-established mechanism of DNA dependent intracellular sperm infertility is sperm aneuploidy, which can be tested by multiple methods including karyotyping [64, 65]. Sperm aneuploidy has been observed in unexplained recurrent pregnancy loss, which might help determine that the male is the underlying factor for this occurrence [64, 65]. Since sperm aneuploidy has been shown to contribute to recurrent pregnancy loss and implantation failure, genetic counselors can advise the couple on how to continue their fertility journey [66].

DNA fragmentation is another well-studied intracellular sperm mechanism. However, the use of fragmentation testing as part of the clinical investigation is controversial [67, 68]. About twelve assays to test DNA integrity exist, but only sperm chromatin structure assay (SCSA) has demonstrated some clinical effectiveness to complement semen analysis [69–73]. Even though it is the most validated, it is not an exemplary test and SCSA is not commonly used in andrology labs since it is not thoroughly researched, trusted, or predictive [33, 74, 75]. SCSA uses an indirect method of assessing how susceptible chromatin is to DNA damage, while other tests such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and single-cell gel electrophoresis assay (Comet) can detect actual DNA breaks, and SCSA has been found to have the worst predictive capacity for pregnancy conception and live births after ART [33]. Therefore, testing the DNA of all infertile males is currently recommended against by the American Society of Reproductive Medicine, since current diagnostic tests are unpredictive or unreliable [76].

A few studies have discussed the role of DNA fragmentation in undetermined male infertility, and these studies have demonstrated the need for these assays to be further developed. About 26–40% of males with undetermined male infertility have DNA fragmentation at levels associated with decreased fertility naturally and with ICSI [77–80]. Since providing DNA is the fundamental role of sperm, further development of reliable and predictive tests that detect DNA defects should be a priority. Such an assay should be used as soon as a male is diagnosed with undetermined infertility and before ART, since the likelihood of successful pregnancy with ICSI may be 3.5 times lower with sperm DNA fragmentation [81, 82]. With the expedited development in DNA technology, adapting it to male infertility may be relatively inexpensive and can save couples a large financial burden of a failed ART [74, 83, 84].

Another aspect of DNA that is under investigation as a potential cause of male infertility is its packaging into chromatin. This includes (1) histone modification and (2) protamine packaging. For example, one study found that 71% of infertile males had histone retention throughout their genome, and other studies have discussed histone retention and reduced reproductive potential [85–88]. Another study found that 54% of infertile males have abnormal protamine ratios, which other studies have found to be associated with worse semen parameters, DNA fragmentation, lower fertilization rates, and poorer embryo implantation [89–92]. However, sperm assays for histone and protamine are not yet validated, trusted, or routinely implemented in the clinic.

RNA

Traditionally, RNA in sperm was thought to have no biological function [93]. However, recent studies have found that altered RNA expression and regulation may be a useful marker for male infertility [93–97]. The absence of sperm RNA elements from males diagnosed with idiopathic infertility was found to decrease the chance of having a live birth when timed intercourse and IUI were used as the conception methods [98]. Also, the sperm of asthenozoospermic patients contained a modified amount of various RNAs compared to the sperm of fertile males [95]. Similarly, normozoospermic infertile males had different miRNAs expressed in their sperm compared to normozoospermic fertile males [97]. Therefore, it is worth further exploring the role of RNA in male infertility.

Centrioles

Abnormal centrioles may reduce the chances of successful fertilization [50, 99–103]. Abnormal centrioles can form an abnormal sperm tail (although it may not always manifest through a sperm morphological defect) and reduce the chances of successful fertilization, but currently there are no diagnostic tools available to detect centriole abnormalities in the clinic [42, 50, 99–102, 104, 105]. Developing these tools is important because if a sperm contributes its centrioles to the embryo, a defect in the centrioles of the sperm would impact embryo development [50]. So even if a male appears to have a normal semen evaluation, the centriole defect could cause infertility and prevent a successful pregnancy [53]. Recent studies in bovine embryos have implicated the first two centrosomes in redistributing the chromosomes in the pronuclei and participating information of two spindles in the zygotes. These unexpected observations demonstrate the need for more basic research on this subject [106, 107].

If intracellular sperm analysis of the male were to show that his sperm have centriole defects, then he and his partner may avoid undergoing expensive procedures like intracytoplasmic sperm injection (ICSI) with his sperm. Alternatively, the presence of a centriole defect may indicate the need to perform a genetic diagnostic test of the early developing embryo prior to transferring to the uterus (i.e., the use of pre-implantation genetic testing, PGE) [108]. However, this test would increase costs, which are not usually covered by insurance [109]. Although centriole defects may currently not be a treatable cause for unexplained infertility, futile and expensive procedures can be avoided if chosen and more effort can be placed exploring safe and practical options for their situation.

Reactive oxygen species

Excess of ROS is a mechanism of intracellular sperm infertility, and 30 to 80% of infertile males may have elevated ROS levels [110, 111]. Increased ROS has been linked with asthenospermia and DNA damage, but there is still no consensus regarding which infertile males should be tested for this defect [112]. Furthermore, empirical medical treatment (EMT) is a non-specific treatment used in idiopathic males and it includes antioxidant supplementation [113]. There is much controversy regarding whether these treatment protocols for infertility are efficacious [110, 112–117]. Nonetheless, we consider excess ROS a cause since it has a proposed treatment that might improve the semen parameters and sperm function of infertile males [114]. Therefore, assessing for and diagnosing idiopathic infertile males with intracellular sperm defects, such as excess ROS, via intracellular sperm analysis might decrease the prevalence of idiopathic male infertility.

Other intracellular sperm components

The hyaluronan-binding (HA binding) ability of sperm can potentially predict the success of oocyte fertilization [118]. Sperm that have good HA binding capacity are more likely to have a mature sperm membrane, no DNA fragmentation, and a normal nucleus [119]. In male factor infertility, physiological intra-cytoplasmic sperm injection (PICSI) using a HA binding system can be utilized in order to select the best sperm for ICSI, which have been seen to increase pregnancy rates and decrease pregnancy loss [120, 121]. However, other research has indicated that analyzing HA binding does not enhance live birth rate or pregnancies in ICSI, IVF, or IUI [122, 123]. Selecting sperm through this system might result in positive outcomes for couples undergoing fertility treatment, so more research needs to be done in this field to further understand its impact [123].

Apoptosis also plays a role in male infertility through increasing the expression of apoptotic markers found in sperm [124]. Higher levels of apoptotic markers, such as active caspase, annexin V binding, and DNA fragmentation, were found in the sperm of infertile males when compared to fertile males, leading to inept sperm. The sperm of infertile males also had more caspase activation with a higher degree of DNA fragmentation than the sperm of fertile males [125]. In addition, about 20% of sperm collected from infertile males were found to be apoptotic, and the sperm concentration was lower in males with more apoptotic sperm [126]. Therefore, it is worth exploring the option of testing for apoptotic sperm in infertile males.

Phospholipase C zeta deficiency is another cause of undetermined male infertility. It is an enzyme that activates the egg after fertilization to start embryo development [127–132]. Supplementing this enzyme during ICSI may help treat unexplained couple infertility.

Chemokines released from the female play an important role in accurately guiding the sperm; however, genetic defects can impact chemokine receptors on sperm and hinder this crucial step in fertilization [133–138]. Overall, these intracellular sperm components need much more research in developing diagnostic tools and therapeutics.

The difficulty in developing a diagnostic test that predicts sperm quality

A test that provides information on sperm quality needs to address the multitude of sperm functions, starting from translocation to the egg via the complex female reproductive tract, fusion with the egg, and then supporting new life that is generated through the contribution of intracellular structures and molecules [139]. A single test that can examine all these functions is currently unavailable, but it can be achieved once we understand all the sperm functions and develop a system that can mimic them.

An alternative approach is to generate multiple tests for each of the essential functions of the sperm, and then compile them together to determine the overall sperm quality [140]. This strategy requires knowledge of all the essential functions of sperm, which would require more research. This approach would include multiparametric analysis that includes semen analysis and advanced sperm analysis for sperm DNA, RNA, proteins, and centrioles. Creation of different technologies will hopefully drive the ability to properly identify the probability that a sperm has to successfully fertilize an egg [141].

Concluding remarks

We estimated that 20% of infertile couples have undetermined male infertility (quarter of male infertility + half of unexplained couple infertility) (Fig. 1 and Fig. 2). Without medical intervention, about 75% of infertile couples will never have biological children [16]. Couples spend a significant amount of time and money at infertility clinics. For example, the chance of a successful live birth after IVF is 30% or lower if the female is older than 35, according to the Society of Assisted Reproduction’s 2018 National Summary Report. These couples typically have to undergo many cycles of IVF, and each cycle costs about $10,000–$15,000, which insurance does not typically cover in the USA [109, 142, 143]. An infertile male with asthenospermia may undergo this expensive procedure with his wife. But if his asthenospermia is accompanied with DNA defects, this procedure may still lead to no success. A closer look at undetermined infertile males can prevent financially and emotionally devastating situations such as this.

Funding

We would like to thank the University of Toledo for sponsoring S.P. for this project through the Medical Student Research Program. This work was supported by Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) grant number R03 HD098314.