In humans, infertility affects around 15% of the couples trying to conceive, and a male factor is present in up to 50% of these cases (reviewed by Evers 1). Male infertility can be produced by a wide variety of physiological, environmental, and genetic factors 2 although a high percentage of cases remain with an idiopathic origin (around 25%; reviewed by Roy et al.) 3.
In this sense, one of the most significant handicaps in the investigation of the spermatogenic function is the accessibility to the target tissue. The obtainment of a small portion testicular biopsy in those patients that seek for a reproductive advice requires the use of painful methods which involve invasive surgery and anesthesia 4. Moreover, the amount of data obtained from these samples is reduced and the consequences for the reproductive fitness of the individual can be detrimental.
To overcome this hindrance, spermatozoa have been proposed as a surrogate tissue for the assessment of human male reproductive disorders 5. These cells represent the latest differentiation stage of the spermatogenic process and their ultimate function is to be a "vehicle" for transmitting the amassed genomic, transcriptomic and proteomic paternal content to the embryo. In this context, the establishment of specific sperm biomarkers for the assessment of the human male infertility becomes an important goal either for clinicians and researchers.
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Nowadays, the study of the fertilizing competences of the spermatozoa can be performed from a physiologic point of view by using the classical practices of the semen analysis, or using other more recently developed molecular approximations in where several aspects of their cargo can be evaluated. Although the seminal analysis are the most commonly used tests in the clinical practice, the information obtained through alternative molecular analysis brings the opportunity to obtain information beyond the specific physical status of the analyzed cells. These data can be related to previous spermatogenic events and also be considered regarding the implications for the future embryo development.
In addition, the use of sperm biomarkers can be of great help for monitoring the conditions of the spermatogenic process in males that have been exposed to specific environmental or occupational risk factors during a certain period of time. The gamete production in human males is a continuous process that allows to cyclically renewing the cells produced every 74 days (for a review see Amann, 2008) 6. Male fertility can be impaired after exposure to toxic agents or harmful environmental factors but it can also be recovered in further waves of spermatogenesis. Therefore, the availability of specific indicators that can be used to evaluate the testis function in a time frame would be of great interest for the reproductive advice of these individuals.
In the next lines, there will be outlined the most significant sperm biomarkers that can be taken into account in the assessment of human male infertility.
Semen analyses are the most widespread and common methods used in the andrological centers to assess male fertility. These studies include the evaluation of several biological aspects related with the components of the ejaculate and just require the use of basic laboratory equipment.
Among them motility, morphology, vitality and sperm concentration are the parameters most frequently included in basic semen analyses. Figures obtained in these screenings must be always considered in relation to previously established standardized reference ranges (Table 1). The cut-off values that define normal conditions versus poor conditions are described and periodically revised in the manuals of examination published by the World Health Organization 7-11.
Table 1. Cut-off values for semen variables as published in the fifth edition of the World Health Organization (WHO) manual 11
â‰¥15 million spermatozoa/mL
Total sperm number
â‰¥39 million spermatozoa/ejaculate
Total (progressive + non-progressive) motility
â‰¥4.0% normal forms
â‰¥ 58% live
White blood cells
â‰¤ 1.0 million spermatozoa/mL
This parameter refers to the percentage of sperm movement but also to the characteristics of their progression. Moving and nonmoving spermatozoa are clearly distinguishable at the microscope but not all the moving sperm in a given specimen act uniformly. To account for these differences, the WHO classifies sperm in four categories: Category A (rapid progressive motility), B (slow but progressive motility), C (non-progressive motility), and D (no motility). Category A plus B is reported as progressive motility and A plus B plus C is total motility.
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Sperm motility has been considered one of the most powerful discriminators of fertility for some authors 12, but there are also other that found this test of limited value 13.
Currently, there are more two main classification systems to determine normal versus abnormal spermatozoa: the early or liberal approach and the Kruger or Tygerberg strict criteria 14. In the first one, observers would describe different evident abnormalities regarding the shape of the sperm head, the midpiece, and tail of the spermatozoa. In these analysis normal spermatozoa are usually identified by default (spermatozoa without obvious abnormal features are classified as normal). On the other hand, the Tygerberg criteria analysis identify normal sperm using exact numbers for the measurement single ellipse-shaped head, the percentage of the head occupied by the acrosome, the width and length of the midpiece, the tail length and appearance, and the amount and location of cytoplasmic droplets. This approach was adopted by the WHO in the 4th edition of the manual 10.
Regarding to this parameter, several authors agree in the fact that sperm morphology could be the best predictor for poor fertility but other authors have found contradictory results 12.
This parameter evaluates the percentage of viable spermatozoa based on the measurement of the integrity of the sperm membrane by dye exclusion. In this analysis, sperm are exposed to a dye (commonly eosin-based stains) and only those that are able to exclude the stain from their intracytoplasmic environment are reported as viable. This test is commonly used in combination with a motility test to differentiate necrospermia from non-motile but viable.
Total Sperm Number and Sperm Concentration
These two parameters can be assessed by dispensing the samples on a grid, examination under the microscope, and extrapolating the number of sperm to the total sample or their concentration. Although total numbers of sperm mostly correlate with pregnancy rates, this correlation has a loose significance when this factor is taken as a single indicator. When combined with the rest of seminal parameters, this correlation is much improved 17. On the other hand, it should be taken into account that a certain production of spermatozoa/ml over 20 million is not always extrapolable to a normal condition: a male with a potential production of 60x106 spermatozoa/ml could have a 50% of his reproductive competence and still be qualified as normozoospermic.
Overall, the real prognostic value of all these traditional semen analysis in couples with male factor subfertility has been a topic of debate for several years . Most of the criticisms are referred to the fact that these parameters overlap between fertile and infertile men. Therefore the absence of altered seminal parameters (normozoospermia) does not discard infertility as other male factors can be present. Moreover, the affectation of each one of these parameters can be unequal in infertile individuals but still there is no agreement regarding the relative significance of each of them. Finally, it must be also taken into account that human semen samples display a high heterogeneity -even between consecutive ejaculates from a same individual-.
For these reasons, many authors consider that other biomarkers and more robust testsÂ to evaluate sperm function should be required in order to accurately diagnose male infertility (for a review see Lefièvre et a. 2007) 19.
The delivery of the proper chromosome complement by a spermatozoon to the oocite is fundamental. The assessment of this aspect can represent an important biomarker not only about the reproductive competence of an individual but also about the potential genetic risks for the offspring.
The use of fluorescence in situ hybridization on decondensed sperm nuclei provides a useful technique to test the incidence of spermatozoa with chromosomal aneuploidies as well as diploidies in a given sample 20. In this methodology, fluorescent labeled probes are used to identify the presence of specific chromosomes in a representative amount of sperm cells. From the combination and number of colors obtained, percentages of disomic/nullisomic and diploid spermatozoa can be inferred (Figure 1).
Figure 1. Tri-color FISH on decondensed sperm nuclei using centromeric probes for chromosomes X (SpectrumGreen), Y (SpectrumOrange) and 18 (SpectrumAqua).
XY disomic spermatozoa
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Diploid spermatozoa cannot produce viable embryos but represent a reduction of the percentage of normal sperm. On the other hand, disomic or nullisomic spermatozoa for a particular chromosome can give rise to the production of trisomic or monosomic embryos respectively. Although most of these aneuploidies will be unviable, some of them can lead to the production of several genetic syndromes (e.g. Down, Klinefelter, Patau, Edwards or Turner syndromes). In this sense, it has been described that fathers of individuals with a Down Syndrome of paternal origin produce increased amounts of spermatozoa with chromosome 21 disomies 21. This association has also been observed in fathers of girls with chromosome X monosomy (Turner Syndrome). These men also presented increased percentages of spermatozoa with chromosome X nulisomies when were compared with a control population 22.
Regarding infertile individuals, It has been widely reported that they constitute a population with higher incidences of chromosomal aneuploidies (reviewed by Egozcue et al. 23). These increased taxes have also been associated with recurrent pregnancy loss in the couples affected 24.
Interestingly, it is worth to mention that the percentages of chromosomal numerical abnormalities found in infertile males have been correlated with low sperm counts from seminal analyses 25.
According to that, the analysis of chromosomal aneuploidies using sperm FISH represent a useful tool for clinicians in order to improve the genetic reproductive advice to be offered at these patients.
Sperm DNA integrity testing
Reactive oxygen species (ROS) are products of normal cellular metabolism. During spermatogenesis, oxidative stress resulting from excess of ROS can result in DNA damage. The persistence of these lesions in spermatozoa is related with the down-regulation of DNA repair systems during the late stages of spermatogenesis. Sperm DNA damage has been described to be associated with poor semen quality, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring (reviewed by Lewis and Aitken, 2005)26. Thus the increasing interest for assessing the presence of DNA damage in spermatozoa has resulted in the developing of several methodologies to evaluate this parameter. The tests that are most frequently used are described below:
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick-end labeling assay (TUNEL)
This assay is based in the detection of DNA strand breaks by using the enzyme Terminal deoxynucleotidyl transferase, a specialized DNA polymerase which catalyzes the addition of labeled dUTPs to the 3' terminus of a DNA molecule. Results can be analyzed at the microscope or using ï¬‚ow cytometry. The principal handicap of its application in the assessment of male infertility is the lack of standardized thresholds that delimit specific populations. Nevertheless, some laboratories have start to work in the proposal of reference ranges of DNA damage in normal and infertile men 27.
Sperm chromatin structure assay (SCSA)
It measures the susceptibility to in situ DNA denaturation in sperm exposed to acid. Following this exposure, samples are stained by acridine orange (AO) and thus, the fluorescence intensity emitted can be measured by ï¬‚ow cytometry: AO produces green fluorescence when binding to native DNA and red fluorescence when binds to the fragmented DNA. The ratio yields the percentage of DNA fragmentation which can be used as a diagnostic tool in the fertility assessment 28
Acridine orange assay (AOA)
This is a simplified method based on the same principle as the SCSA. In this case, the fluorescence emitted by the spermatozoa are analyzed by visual interpretation through the microscope avoiding the use of expensive flow cytometry equipment 29. Nevertheless, the subjectivity in the analysis of the observer has been considered a disadvantage for the predictive value of this test.
In this assay spermatozoa are embedded in agarose and put on a glass slide. The application of electrophoresis produces the migration of the DNA molecules causing the formation of structures that reproduce comet tails. These results can be evaluated using specific software. The major problem with COMET assay is that it is a labor intensive test.
Sperm chromatin dispersion assay (SCD)
It consists in denaturing the sperm DNA and evaluate the fragment patterns resulting of this procedure. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA after acid denaturation and removal of nuclear proteins 30
With the exception of the acridine orange test, the results obtained by most of these methodologies correlate with each other even despite the elevated heterogeneity of the protocols 31. Data previously published indicates that men with higher levels of sperm DNA damage display a reduction in their natural fertility 32 and have poorer outcomes after intrauterineÂ insemination (IUI) 33. Nevertheless DNA damage does not seems to have a significant impact the fertilization rate or pregnancy outcome after Intra-Cytoplasmic Sperm Injection (ICSI) 34.
The principal weakness of these approaches is that they do not allow to differentiate between relevant DNA fragmentation and irrelevant fragmentation from a clinically point of view. In fact, all these techniques consider any DNA nicking or fragmentation as a pathologic event. Moreover, it could also be argued that a certain small amount of damage affecting genomic areas with crucial genes could be more deleterious than higher levels of damage in inactive regions of the genome. Therefore, at the moment the clinical value of the sperm DNA damage as a fertility biomarker stills remains to be firmly confirmed.
Spermatozoal RNA profiles
Although spermatozoa do not have translational activity 35 these cells are carriers of a wide variety of RNAs 36. The synthesis of these transcripts takes place prior the transcriptional arrest that occurs at the late stages of spermatogenesis. From our observations, approximately, a single human spermatozoon carries an average of 20 fg of RNA 37 which will delivered to the oocite during fertilization.
The functions of these transcripts are still a focus of study of several groups, but there is a general agreement about the fact that they represent a transcriptomic fingerprint of earlier spermatogenic events and they may have a potential role in the early embryonic development 38.
The profiling of spermatozoa RNAs as clinical biomarkers of human male infertility has been investigated by several groups. Initially, specific sets of transcript levels were evaluated by RT-PCR and ISH (for a review see Dadoune 2009)39. More recently, the use of high-throughput evaluation techniques have allowed to have a wide perspective of the RNA content.
According to this, these technologies start to be proposed as potential clinical diagnostic tools for enabling a better understanding of the pathogenesis of male infertility. They provide a high amount of precise data with a high resolution that can be used to provide a very accurate reproductive advice to those couples with some underlying spermatozoa dysfunction.
Gene expression microarrays
The microarray technology has allowed to screen the expression of thousands of mRNAs in a single experiment 40. Several authors have get use of the microarrays technology to assess the RNA content of human spermatozoa 41-45.
Some groups have been working in determining the existence of differences in the transcriptomic profile between different populations of fertile and infertile males. Platts et al. 46 first described the existence of differences in the RNA profile between fertile and infertile males with teratozoospermia. This study allowed to link the generation of morphological abnormalities during meiosis to an altered gene expression. More recently, other studies have revealed differences in the transcriptomes obtained from pooled samples of fertile men and infertile individuals with normal sperm count and motility 47, as well as between fertile and infertile males that achieve or not pregnancy after IUI 48 or ICSI 49.
Therefore, these data present the sperm transcriptomic profiles as really interesting biomarkers for the human male fertility assessment. However, it may be taken into account that most of the published studies 47-49 have been performed without a previous selection of the spermatic fraction which cannot guarantee the complete absence of transcripts from somatic cells in the analyzed transcriptomes. On the other hand, these studies have also been carried out in pooled semen samples from several individuals Thus, it would be still necessary to assess if these differences can be detected at an individual level.
Around 2004, next-generation sequencing (NGS) technologies emerged 50 and allowed to generate massive amounts of sequence data in a rapid and cost-effective mode. These NGS platforms have been applied to many genomic contexts like the generation of genome-wide profiles of immunoprecipitated DNA-protein complexes (ChIP-seq), genome wide profiling of epigenetic marks (methyl-seq) and DNase I hypersensitivity sites (DNase-seq). Among them, the massively parallel sequencing of cDNA has been named RNA-seq and has revolutionized transcriptomics. This methodology requires the isolation of the RNA samples and the preparation of libraries of cDNA which can be sequenced by specific work flows to obtain relatively short sequence reads. These reads can be assembled and aligned to the genome and analyzed by bioinformatic approaches (for a review see Metzker, 2010 51).Â
Microarrays and NGS differ from each other in two main aspects. First, microarrays technology depends on prior knowledge of the genes that will be detected (the probes included in the array must be previously designed) whereas NGS brings access to all the transcripts of a cell. Second, NGS have a dynamic range of detection much wide than microarrays: they do not fail to detect low expressed genes 52.
According to these advantages, the use of this methodology for the analysis of the sperm transcritome content will offer a new point of view for the assessment of reproductive competence of human male. Until now, only one study has been performed using RNA-seq to assess the RNA content of human sperm samples 53. This report characterizes the content of 15-36nt small non-coding RNAs (sncRNAs) in spermatozoa from three human fertile males. Their findings allow to specifically defining the complete population of RNA transcripts present in these cells. Among them, the authors report the presence of 274 different microRNAs (miRNAs), 58 of those were predicate miRNA described for the first time in the mature male gamete. They also describe a population of 9,759 Piwi-interacting RNAs (piRNAs) and a smaller fraction of other sncRNAs such are small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), Y RNA, Telomerase RNA, Vault and 7SK.
It is really plausible that knowing the complete sperm RNA content carried and delivered to the oocite would have a huge potential value for the assessment of the human male infertility. Nevertheless, still no data is available regarding the differences of the complete sperm transcriptomes between fertile and infertile men and how to use this huge amount of information. Certainly, further studies are needed to explore the value of full transcriptome analysis as clinical biomarkers of the reproductive competence of a specific individual.
Spermatozoal proteomic profiles
The proteome accounts for the set ofÂ proteinsÂ expressed by a genome. The knowledge of the sperm cell proteome is also relevant for the understanding the structural andÂ functionalÂ features of these cells. Spermatozoa display several specific properties that make them appropriate cell types for proteomic analyses: they can be obtained in extremely high purity and large concentrations, they have a reduced content of proteins compared with many other cell types and lack in new protein synthesis 54. Nevertheless, studies examining theÂ proteomic content of spermatozoa were started few years ago. These analysis report the identification of a wide catalog of proteins using either 2D gel-based approaches 55-58 or liquid chromatography-mass spectrometry assays (LC-MS/MS) 59 which include nuclear proteins, proteins required for sperm motility, and membrane proteins required for capacitation, egg interaction and fertilization among others.
In the search for identifying potential infertility biomarkers in spermatozoa, the presence of an altered proteomic profile in infertile patients in relation to sperm samples from fertile donors has also been a recent topic of study of several groups. Pixton et al. 60 identified twenty different proteins in the proteomic mapping of a patient who experienced a failure after a in-vitro fertilization (IVF) cycle when was compared with controls samples. Motility alterations in spermatozoa have also been associated with different protein profiles in asthenozoospermic males 61-63. And interestingly, a proteomic study performed in 57 sperm samples (47 infertile individuals and 10 control donors) reported a high correlation between the presence of specific proteins and the results of DNA integrity (using the TUNEL assay) in these individuals 58.
Although the advances in the knowledge of the sperm proteomic content, the use of mass spectrometry based protein identification for the reproductive advice still represents a complex mission. This is due mainly because this technology requires an expensive set of instruments and specialized personnel for operation which makes it a challenging task.
The entire comprehension of the etiology of the human male infertility has promoted the development of many applications from disparate technologies. Nowadays we dispose of a wide range of tests and biomarkers that can be used by researchers and clinicians to properly understand the mechanisms that negatively affect the fertility. Nevertheless, despite it is widely accepted that the information provided by conventional semen analysis is limited, its use in the clinical evaluation of male fertility still represents a mandatory first step.
In this sense, although none of the other approaches described have been revealed to be the perfect alternative by themselves, it will be necessary to keep searching the usefulness of these tests for determining which factors truly participate in the reduction of the fertility potential. This would allow not only to improve the reproductive advice that can be offered to these individuals, but also to elucidate the etiology of the unexplained infertility processes.