2. Semen collection and analysis

The sample of semen should be collected after 2–7 days of sexual abstinence. The first ejaculate gives a correct conclusion in at least 85% of cases. It is helpful to examine two or three samples to obtain more precise data [2, 3].

Semen analysis includes:


#### 2.1. Evaluation of the spermatozoa morphology

#### 2.1.1. Light microscopy of stained spermatozoa

The light microscopy of stained spermatozoa is the fundamental and commonly used method for evaluation of spermatozoa morphology. Two techniques can be used for this evaluation: (1) the microscopic analysis of stained samples as visual observations of spermatozoa (manual method) and (2) computerized analysis. Primarily for both methods, the stained smears of semen must be prepared. The droplet of the semen sample are smeared on a glass slide, dried in the air and fixed. The smears must be stained for providing sharp contrast for defining the spermatozoa outline and cell details. There are a lot of methods for the staining of human and animal spermatozoa. World Health Organization recommends three routine staining methods for the evaluation of morphology of human spermatozoa: Papanicolaou, Shorr or Diff-Quik (Rapidiff) [3, 4]. Some laboratories use a new stain Sperm-Blue® successfully [5, 6].

The stained smears are analyzed by the magnification 1000 with oil immersion. Using a manual method, the laboratory technician examines 200 spermatozoa and categorizes each spermatozoon as normal or abnormal. Subsequently, the anomalies are classified using strictly defined criteria. The measurement of spermatozoa can be performed using the ocular micrometer. By computerized analysis method, various computer analyzing programs, systems and modules categorize and measure automatically different morphological features of each selected spermatozoon [5, 7–11].

A few classifications of human spermatozoa morphology have been originated and used worldwide: MacLeod's [12], David's [13, 14], Dusseldorf [15, 16], Strict (Tygerberg's) criteria [17, 18] and others. The World Health Organization (WHO) confirmed the Strict (Tygerberg's) criteria as conventional standard for spermatozoa morphology [4].

#### 2.1.2. Motile sperm organelle morphology examination (MSOME)

The aim of this work was to review the methods of spermatozoa morphology assessment,

The sample of semen should be collected after 2–7 days of sexual abstinence. The first ejaculate gives a correct conclusion in at least 85% of cases. It is helpful to examine two or three samples

The light microscopy of stained spermatozoa is the fundamental and commonly used method for evaluation of spermatozoa morphology. Two techniques can be used for this evaluation: (1) the microscopic analysis of stained samples as visual observations of spermatozoa (manual method) and (2) computerized analysis. Primarily for both methods, the stained smears of semen must be prepared. The droplet of the semen sample are smeared on a glass slide, dried in the air and fixed. The smears must be stained for providing sharp contrast for defining the spermatozoa outline and cell details. There are a lot of methods for the staining of human and animal spermatozoa. World Health Organization recommends three routine staining methods for the evaluation of morphology of human spermatozoa: Papanicolaou, Shorr or Diff-Quik (Rapidiff) [3, 4]. Some laboratories use a new stain Sperm-

The stained smears are analyzed by the magnification 1000 with oil immersion. Using a manual method, the laboratory technician examines 200 spermatozoa and categorizes each spermatozoon as normal or abnormal. Subsequently, the anomalies are classified using strictly defined criteria. The measurement of spermatozoa can be performed using the ocular micrometer. By computerized analysis method, various computer analyzing programs, systems and modules categorize and measure automatically different morphological features of each

A few classifications of human spermatozoa morphology have been originated and used worldwide: MacLeod's [12], David's [13, 14], Dusseldorf [15, 16], Strict (Tygerberg's) criteria [17, 18] and others. The World Health Organization (WHO) confirmed the Strict (Tygerberg's)

criteria as conventional standard for spermatozoa morphology [4].

features of the normal spermatozoa and the reasons of their various abnormalities.

• Seminal fluid analysis (biochemical markers of accessory glands secretions) [3].

• Spermatozoa analysis (number, vitality, motility, morphology), • Immunological analysis (anti-spermatozoa antibodies detection),

2. Semen collection and analysis

2.1. Evaluation of the spermatozoa morphology

2.1.1. Light microscopy of stained spermatozoa

to obtain more precise data [2, 3].

Semen analysis includes:

52 Spermatozoa - Facts and Perspectives

Blue® successfully [5, 6].

selected spermatozoon [5, 7–11].

This method is used for evaluating the morphology of the live (non-fixed, non-stained) spermatozoa before the ICSI. Using the high magnification (6000 and more) by the inverted computerized microscope, it is possible to observe the morphological abnormalities in spermatozoa, which are not visible with magnification 400 (Figure 1). The neck, tail, midpiece, mitochondria, acrosome and post-acrosomal lamina and the nucleus of spermatozoon are morphologically examined in live motile spermatozoa [19–25].

Figure 1. Spermatozoa morphology using MSOME ([19], open access). (A) Normal spermatozoa observed at high magnification (8400); (B) spermatozoa with large nuclear vacuoles observed at high magnification (8400).

Figure 2. The birefringence of human spermatozoa ([26], with permission).

#### 2.1.3. Polarized light microscopy

An estimation of head birefringence by polarized light using polarized microscope method is used for analysis of live spermatozoa morphological quality. This birefringence is associated with sub-acrosomal protein filaments and nucleoprotein filaments (Figure 2). The presence of birefringence in the head indicates the good morphology of a spermatozoon [26–31].
