**Molecular Markers in Sperm Analysis**

Rita Payan-Carreira, Paulo Borges, Fernando Mir and Alain Fontbonne

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52231

**1. Introduction**

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92 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

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of Animal Science 2002;80 1-10.

In mammals, the success of fertilization largely depends on gamete fertility potential and consequently on what concerns sperm and oocyte quality they are both equally important.

Sperm contribution to fertilization is usually estimated through evaluation of semen param‐ eters. A loss of fertility potential associated to manipulation and preservation techniques is usually calculated based on the semen characteristics at collection and on the knowledge of the damages associated with the technique to be implemented.

Assessment of sperm quality conventionally relies on microscopic evaluation of sperm pa‐ rameters including total sperm count, sperm concentration, percentage of motile sperm and percentage of normal sperm morphology. Some of these parameters are correlated with fer‐ tility though it does not truthfully predict male fertility [1-3]. Concentration and morpholo‐ gy are considered to be important to evaluate the fertilizing ability of sperm cells, as well as motility and the acrosome status, which are critical elements regarding fertilization. These parameters are currently analysed under light microscopy. Computer-assisted semen analy‐ sis (CASA) increases the reliability and the accuracy of the analysis with the increase of cell counting [4,5]. Results of the functional testing (such as the *zona pellucida* binding assay, the hemi-zona essay or the hypoosmotic swelling test) are better correlated with the AI outcome than the results of conventional semen evaluation [1,2].

Nevertheless, these methods have limited prognostic value for the reproductive success of the donor male [6,7]. Discrete and unclear sperm abnormalities impairing the reproductive success of sperm and egg interaction often remain undiagnosed. This is the major limitation for the most conservational *in vitro* methodologies of sperm evaluation, either in humans or animals. Inability of the *in vitro* assessment methods to accurately predict spermatozoa fer‐ tility may be attributed to the complexity and multifactorial nature of male fertility.

© 2013 Payan-Carreira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the past decades, attempts to escape these limits led to the introduction, in the laboratori‐ al panel, of some sophisticated analyses. Those included the use of fluorescent markers to assess the acrosomal status, the use of vital staining for mitochondrial activity, the use of particular fluorochromes to detect altered sperm chromatin or DNA integrity along with several molecular regulators of thermal and oxidative stress. Proteomic, biochemical, and immunocytochemical approaches are now starting to highlight some key events that may determine the success of the sperm function. Existing functional tests were also retained, such as the hypoosmotic swelling test and the hemi-zone assay, to assess membrane func‐ tional integrity and sperm ability to interplay with the oocyte.

The search for effective predictors of spermatozoa fertility is now on the table, and the iden‐ tification of suitable molecules would greatly benefit the semen industry and would strengthen the proposal of new therapies for infertility, in both man and animal. Further‐ more, it would allow a better understanding of the side effects of technology (such as freez‐ ing/thawing or sex-sorting procedures) upon the sperm integrity and functionality, as well as to evaluate the reasons of some undesirable responses of exotic or endangered species'

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 95

A large number of factors and molecules have been proposed to be of interest or tested as putative predictors for sperm fertility. Before playing their role in fertilization, spermatozoa are required to survive in the female genitalia, accomplish to reach the place for fertilization and to acquire competence to fertilize the oocyte (Figure 1). This is true, for both the natural mating and the artificial insemination. These are important actions, which reflect a multi‐ tude of complex and specialised functions that, in brief, result in sperm survival and fertili‐ ty. Yet, all these functions would hardly be evaluated together through a sole molecule.

In this review it is the intent to present and discuss the use of new methods for sperm as‐

sperm to preservation.

sessment and estimation of spermatozoa fertility.

**Figure 1.** Major cellular mechanisms associated with main roles of the spermatozoon.

Understanding the main determinants of sperm fertility and knowing how fertility changes or is influenced by sperm manipulation (such as cryopreservation and sperm-sorting) would allow to enhance the knowledge on extender design, to accurately estimate sperm fertility and to predict sperm survival after processing. The knowledge to adequately extend the lifespan of cryopreserved sperm would also be improved, in particular on what con‐ cerns the programs for genetic biodiversity preservation. Nowadays, the lack of reliable methods allowing the accurate *in vitro* assessment of semen quality, limits our capacity to properly monitor semen freezing-thawing damages and to predict its performance at in‐ semination [8].

Though extensively used in domestic species (such as bovine, pigs and dogs), it is well known and accepted that cryopreservation damages the sperm, with a large number of cells losing their fertility potential after freezing/thawing. Further, it is also common knowledge that individual variations exist on sperm resistance to cell damage during these procedures, justifying why some males are "better freezers" than others, even if no differences are found in fresh semen quality assessment [9, 10].

Determination of additional markers for semen quality is now being explored either as a complementary assessment of sperm quality or as an additional way to study in more detail the side effects of extenders or molecules associated to infertility. Seminal markers reveal molecular pathways that could be suppressed or stimulated by *in vitro* sperm manipulation. Moreover, it may be of utmost importance when considering the development of protocols for sperm cryopreservation of wild and endangered species. Up to now, the extender selec‐ tion in those species is mainly based on phylogenetic or physiological resemblances and on the trial-and-error approach.

Another issue strengthening the need for additional tests in laboratory assessment of sperm quality relates to the fact that standard seminal parameters (motility, concentration and morphology) currently used for all the species are insufficient to predict fertility and to de‐ tect sub-fertile males. In addition, sperm samples are very heterogeneous and although spermatozoa may look the same on traditional semen analysis, more sophisticated methods allow identifying different spermatozoa subpopulations with distinct biochemical and phys‐ iological characteristics. It is the combination of sperm cells of different functional compe‐ tences that largely determines the fertility potential of a specific male.

The search for effective predictors of spermatozoa fertility is now on the table, and the iden‐ tification of suitable molecules would greatly benefit the semen industry and would strengthen the proposal of new therapies for infertility, in both man and animal. Further‐ more, it would allow a better understanding of the side effects of technology (such as freez‐ ing/thawing or sex-sorting procedures) upon the sperm integrity and functionality, as well as to evaluate the reasons of some undesirable responses of exotic or endangered species' sperm to preservation.

In the past decades, attempts to escape these limits led to the introduction, in the laboratori‐ al panel, of some sophisticated analyses. Those included the use of fluorescent markers to assess the acrosomal status, the use of vital staining for mitochondrial activity, the use of particular fluorochromes to detect altered sperm chromatin or DNA integrity along with several molecular regulators of thermal and oxidative stress. Proteomic, biochemical, and immunocytochemical approaches are now starting to highlight some key events that may determine the success of the sperm function. Existing functional tests were also retained, such as the hypoosmotic swelling test and the hemi-zone assay, to assess membrane func‐

Understanding the main determinants of sperm fertility and knowing how fertility changes or is influenced by sperm manipulation (such as cryopreservation and sperm-sorting) would allow to enhance the knowledge on extender design, to accurately estimate sperm fertility and to predict sperm survival after processing. The knowledge to adequately extend the lifespan of cryopreserved sperm would also be improved, in particular on what con‐ cerns the programs for genetic biodiversity preservation. Nowadays, the lack of reliable methods allowing the accurate *in vitro* assessment of semen quality, limits our capacity to properly monitor semen freezing-thawing damages and to predict its performance at in‐

Though extensively used in domestic species (such as bovine, pigs and dogs), it is well known and accepted that cryopreservation damages the sperm, with a large number of cells losing their fertility potential after freezing/thawing. Further, it is also common knowledge that individual variations exist on sperm resistance to cell damage during these procedures, justifying why some males are "better freezers" than others, even if no differences are found

Determination of additional markers for semen quality is now being explored either as a complementary assessment of sperm quality or as an additional way to study in more detail the side effects of extenders or molecules associated to infertility. Seminal markers reveal molecular pathways that could be suppressed or stimulated by *in vitro* sperm manipulation. Moreover, it may be of utmost importance when considering the development of protocols for sperm cryopreservation of wild and endangered species. Up to now, the extender selec‐ tion in those species is mainly based on phylogenetic or physiological resemblances and on

Another issue strengthening the need for additional tests in laboratory assessment of sperm quality relates to the fact that standard seminal parameters (motility, concentration and morphology) currently used for all the species are insufficient to predict fertility and to de‐ tect sub-fertile males. In addition, sperm samples are very heterogeneous and although spermatozoa may look the same on traditional semen analysis, more sophisticated methods allow identifying different spermatozoa subpopulations with distinct biochemical and phys‐ iological characteristics. It is the combination of sperm cells of different functional compe‐

tences that largely determines the fertility potential of a specific male.

tional integrity and sperm ability to interplay with the oocyte.

94 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

semination [8].

in fresh semen quality assessment [9, 10].

the trial-and-error approach.

A large number of factors and molecules have been proposed to be of interest or tested as putative predictors for sperm fertility. Before playing their role in fertilization, spermatozoa are required to survive in the female genitalia, accomplish to reach the place for fertilization and to acquire competence to fertilize the oocyte (Figure 1). This is true, for both the natural mating and the artificial insemination. These are important actions, which reflect a multi‐ tude of complex and specialised functions that, in brief, result in sperm survival and fertili‐ ty. Yet, all these functions would hardly be evaluated together through a sole molecule.

In this review it is the intent to present and discuss the use of new methods for sperm as‐ sessment and estimation of spermatozoa fertility.

**Figure 1.** Major cellular mechanisms associated with main roles of the spermatozoon.
