Abstract
The investigation of the male fertility potential starts with the analysis of seminal fluid. The seminal fluid or ejaculate is composed of a heterogeneous water-based solution (seminal plasma) deriving from secretions of prostate, testes, seminal vesicles and bulbourethral glands, and cellular components that include mature spermatozoa and epithelial cells derived from the genitourinary tract as well as the generically defined “round cells” (i.e. leukocytes, Sertoli cells and germ cells) [1]. Hence, the standard semen analysis provides insight into the testicular production of spermatozoa as well as the functionality and secretory activity of the associated sex glands [2]. Moreover, it permits the identification of genetic conditions associated with male infertility, such as azoospermia or globozoospermia, and orientates the choice of treatments or the necessity for further tests and investigations. Currently, semen analysis is performed according to the most recent WHO guidelines [2], which provide instructions for the evaluation of macroscopic (liquefaction, viscosity, appearance, volume, pH) and microscopic (sperm concentration, motility, morphology, vitality, presence of round cells and agglutination zones) seminal characteristics. The lower reference value for each parameter is represented by the fifth percentile, calculated based on a selected population of 1953 recent fathers [2]. However, it should be noted that men having seminal parameters below the reference values provided can still be fertile. On the other hand, men showing seminal parameters above the lower reference values are not necessarily fertile as about 15 percent of the men are reported to be infertile despite having normal semen parameters according to World Health Organization (WHO) criteria [3].
4.1 Introduction
4.1.1 Semen Analysis
The investigation of the male fertility potential starts with the analysis of seminal fluid. The seminal fluid or ejaculate is composed of a heterogeneous water-based solution (seminal plasma) deriving from secretions of prostate, testes, seminal vesicles and bulbourethral glands, and cellular components that include mature spermatozoa and epithelial cells derived from the genitourinary tract as well as the generically defined “round cells” (i.e. leukocytes, Sertoli cells and germ cells) [1]. Hence, the standard semen analysis provides insight into the testicular production of spermatozoa as well as the functionality and secretory activity of the associated sex glands [2]. Moreover, it permits the identification of genetic conditions associated with male infertility, such as azoospermia or globozoospermia, and orientates the choice of treatments or the necessity for further tests and investigations. Currently, semen analysis is performed according to the most recent WHO guidelines [2], which provide instructions for the evaluation of macroscopic (liquefaction, viscosity, appearance, volume, pH) and microscopic (sperm concentration, motility, morphology, vitality, presence of round cells and agglutination zones) seminal characteristics. The lower reference value for each parameter is represented by the fifth percentile, calculated based on a selected population of 1953 recent fathers [2]. However, it should be noted that men having seminal parameters below the reference values provided can still be fertile. On the other hand, men showing seminal parameters above the lower reference values are not necessarily fertile as about 15 percent of the men are reported to be infertile despite having normal semen parameters according to World Health Organization (WHO) criteria [3].
4.1.2 Leukocytes in the Ejaculate
Leukocytes are physiologically present in seminal fluid of all men. Polymorphonuclear (PMN) granulocytes represent 50–60 percent of circulated leukocytes in semen. These cells mainly originate from the prostate and seminal vesicles and have a diameter of 14 µm [1]. In addition to PMN granulocytes, macrophages (20–30 percent) and T-lymphocytes (2–5 percent) are less represented in semen and originate from the epididymis and rete testis, and have a cellular diameter of 16–20 µm and 8–12 µm, respectively [1]. Leukocytes can be differentiated from other round cells through microscopic evaluation at x1000 magnification, as nuclear size and shape can help identifying specific cellular types. More sophisticated techniques are based on an immunocytochemical approach based on the detection of leukocyte antigens such as CD45. However, the most common assay performed in clinics for neutrophils’ discrimination is the Endtz test [4], described in detail in the further sections.
4.1.2.1 Function of Leukocytes
Leukocytes are physiologically present in seminal fluid of fertile and infertile men. Their concentration can increase in case of genital tract infection and inflammations and are therefore considered a diagnostic parameter [5, 6]. Generally, leukocytes play an important role in the removal of pathogens, or inflammation. Furthermore, leukocytes are involved in the phagocytosis of defective sperm, reducing the ejaculation of morphologically abnormal and immature sperm. In addition to this elimination of sperm in the male genital tract, leukocytes are also involved in their elimination and selection in the female genital tract. Since spermatozoa have antigenic properties, leukocytes will infiltrate the female genital tract and eliminate defective and moribund spermatozoa from the cervix and uterus. This in turn contributes to the selection of the most competent sperm to further ascent the female genital tract into the fallopian tubes. Different processes characterize this “silent” phagocytosis of male germ cells in the male and female genital tracts where reactive oxygen species (ROS) and pro-inflammatory cytokines play no role [7, 8]. Instead, an intrinsic apoptotic process is triggered by translocating phosphatidylserine from the inner leaflet of the plasma membrane to the out leaflet. Since the male germ cells are transcriptionally and translationally silent, this process needs to be called a regulated cell death rather than programed cell death [9].
By using electron microscopy, it has been observed that the process of the spermatozoa phagocytosis includes a) an increased volume of leukocytes, b) the development of cellular projections to contact the sperm cell, c) the formation of tight adhesions between leukocytes and spermatozoa, followed by d) the engulfment of the cell to the leukocytes’ cytoplasm [10]. Simultaneously, leukocytes can also remove damaged and/or immature sperm by forming extracellular structures, which act like a trap. The fusion of extracellular traps formed by different leukocytes has also been observed when leukocytes act close to each other [10]. Once sperm have been phagocytized, ROS are generated by the enzyme peroxidase in the phagosomes and literally destroys the pathogens [11].
4.1.2.2 Occurrence of Leukocytes in the Semen
4.1.2.2.1 Infections
As important components of the immune system, leukocytes are activated in case of infections and involved in the removal of pathogens. The most common pathogens originate from the urinary tract or are sexually transmitted, and include Escherichia coli, Proteus spec., Klebsiella spec., Streptococcus spec. and Chlamydia trachomatis [11]. Several infections can be asymptomatic, such as urethritis and orchitis, while the symptomatology of prostatitis can vary significantly between patients [12]. Pathogens are eliminated by PMN granulocytes and macrophages by the generation of ROS as well as through phagocytosis. The entire process is orchestrated by cytokines, small (about 5–20 kDa) proteins secreted by leukocytes having pro- or anti-inflammatory effects. These molecules regulate the communication between cells and are further subdivided into interleukins (ILs), chemokines, interferons and tumor necrosis factors (TNFs) [13]. The secretion of cytokines regulates the activation of an inflammatory response as well as its inactivation [11]. The infection and the related tissue damage induce the synthesis of interleukin-1, which, in turn, activates neutrophils and macrophages. In a complex network of cell-to-cell interactions, these leukocytes secrete other molecules (i.e. IL-8, ROS, IL-6 and hepatocyte growth factor – HGF), which regulate their own function, the inflammatory response and aim to remove pathogens [13]. Although the function of cytokines is to control the infection, as a side effect, they can affect the male fertility potential. A negative association between high levels of cytokines (IL-6, IL-8 and TNF) and poor semen quality has been reported in the literature [11, 14, 15]. IL-6 secreted by PMN granulocytes and macrophages is able to induce the synthesis of antibodies by B-lymphocytes, which can have a negative impact on sperm functions [14]. On the other hand, IL-8 and TNF-α increase the level of ROS and lipid peroxidation and negatively affect sperm membranes, sperm morphology, metabolism and thus male reproductive functions [16–18].
4.1.2.2.2 Lifestyle Factors
Besides infections, other conditions related to lifestyle can induce leukocytospermia and/or increase the concentration of ROS released into the semen. Cigarette smoking represents a well-known cell mutagen and carcinogenic factor and its impact on male infertility has been widely investigated. A positive association between smoking habit and seminal leukocytes and ROS concentrations has been reported, with an increase of 48 percent and 107 percent, respectively, observed in smokers in comparison with nonsmoker patients [19]. In addition, the increase in oxidative stress leads to higher levels of 8-oxo-2’-deoxyguanosine, a validated marker of oxidative stress and sperm DNA damage [19]. Moreover, in patients abusing alcohol and/or drugs, poor semen quality has been reported as well as a significant higher percentage of seminal leukocytospermia and oxidative stress [20]. Eating habits also determine the establishment of an oxidative microenvironment in semen as a diet rich in sugars and lipids determines obesity [21], which is characterized by a systemic inflammatory response with high seminal ROS levels [22]. The increased visceral fat causes the systemic release of inflammatory mediators with repercussion on seminal quality and redox balance [23]. Furthermore, semen quality is altered after exposure to air and environmental pollution such as pesticides, plasticizers or heavy metal, as well as radiation. All this is leading to a shift of the redox balance towards an oxidative condition, the increase of inflammatory markers and reduced semen quality [24].
4.1.2.3 Reactive Oxygen Species Production of Leukocytes
Leukocytes produce ROS as a mechanism of defense against the presence of pathogens. ROS are oxygen-based molecules which are, due to the presence of one or more unpaired electrons in the outer orbit, highly reactive, with extremely short half-life times. Molecules exhibiting such unpaired electrons are called radicals and include the superoxide anion (O2–.), hydroxyl radical (OH.), peroxyl radicals (ROO.) and alkoxyl radicals (RO.). On the other hand, ROS also include non-radical oxygen derivatives such as organic hydroperoxides (ROOH) and hydrogen peroxide (H2O2) [25]. Due to their electronic instability, ROS can interact with lipids, proteins, or the DNA and thereby cause lipid peroxidation of membrane lipids, inactivation of enzymes and sperm DNA damage. In this context, it is important to realize that H2O2, compared to other ROS, is relatively stable. However, H2O2 is a reactive component of the Fenton and Haber-Weiss reactions where H2O2 reacts with ferrous (Fe2+) and ferric (Fe3+) ions to produce OH. and dismutate to superoxide, respectively [26]. In addition, considering that H2O2 is not charged, it can penetrate plasma membranes just like water and then react with proteins and DNA inside spermatozoa.
When they are activated, leukocytes can produce 1000 times more the amount of ROS produced by sperm [27]. The generation of ROS by leukocytes starts with the enzyme glucose-6-phosphate dehydrogenase, involved in the hexose monophosphate pathway and responsible for the synthesis of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH, in turn, is an electron donor for the enzyme NADPH oxidase, which catalyzes the conversion of oxygen into O2-. [21].
Generally, there are a number of tests available to identify leukocytes. According to the WHO manual [2], leukocytes should either be determined by their cellular peroxidase content by means of the use of ortho-toluidine [28] or with a more time-consuming and more expensive immunocytochemical test against CD45 [29]. Alternatively, the peroxidase stain using benzidine, the Endtz test [4], is commonly used.
4.2 Principle of the Peroxidase Stain Tests
Leukocytes are selectively differentiated from other round cells on the basis of their enzymatic peroxidase content as tested by the ortho-toluidine and Endtz tests. These tests are based on the histochemical staining of cellular granules containing myeloperoxidase, an enzyme characteristically expressed by granulocytes. Other leukocytes such as lymphocytes, macrophages and monocytes as well as immature germ cells are unstained as they do not express the enzymatic peroxidase activity. This allows the discrimination between granulocytes and other round cells. However, activated granulocytes are also unstained when they have already undergone the exocytosis of their granules before the staining.
4.2.1 Protocol of the Ortho-Toluidine Test
4.2.1.1 Preparation of Stock and Working Solutions
A 67 mmol/L phosphate buffer, pH 6.0, is prepared by dissolving 9.47 g Na2HPO4 in 1 L distilled water. In addition, another solution with 9.08 g KH2PO4 is also prepared in 1 L distilled water. Then, add 12 mL of the Na2HPO4 solution to 88 mL of the KH2PO4 solution until the pH is 6.0. Further, a saturated (250 g dissolved in 1 L distilled water) solution of NH4Cl and a solution of 148 mmol/L of disodium ethylenediamine tetra-acetic acid (Na2EDTA) in phosphate buffer as well as the ortho-toluidine solution (2.5 mg in 10 mL 0.9 percent saline) are prepared.
In order to prepare the working solution, 1 mL of the NH4Cl solution, 1 mL of Na2EDTA and 10 µL of 30 percent H2O2 are added to 9 mL ortho-toluidine substrate and vortexed. This solution can be used up to 24 hours after preparation
4.2.1.2 Cellular Staining
After liquefaction, the semen sample is mixed well and 0.1 mL liquefied semen is then mixed with 0.9 mL of the working solution and incubated for 20–30 minutes at room temperature. Following another mixing, an aliquot of this suspension is loaded on a hemocytometer, incubated in a humid chamber for 4 minutes and analyzed under phase contrast at ×200 or ×400 magnification.
4.2.1.3 Evaluation of Stained Leukocytes
At least 200 brown (peroxidase-positive) leukocytes in a replicate are counted (peroxidase-negative cells are not stained) and the concentration of peroxidase-positive leukocytes (106 cells/mL) is calculated.
4.2.2 Protocol of the Endtz Test
4.2.2.1 Preparation of Stock and Working Endtz Solution
The Endtz stock solution is prepared with benzidine (0.125 g), 96 percent ethanol (50 mL) and sterile water (50 mL). It should appear clear and yellow and it is stable for six months after preparation. From this stock solution, the working solution is prepared by mixing 2.0 mL stock solution and 25 µL 3 percent hydrogen peroxide. The working solution should be freshly prepared on a weekly basis. Both solutions should be stored in the dark.
4.2.2.2 Cellular Staining
When liquefaction of the ejaculate is complete, an aliquot of the semen sample is mixed with phosphate buffered saline (PBS) and working Endtz solution (1 : 1 : 2 vol./ vol.) in an amber centrifuge tube. The solution is properly mixed and incubated for five minutes, at room temperature in the dark.
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