Abstract
The evaluation of sperm morphology as a standard procedure to be included in routine semen analysis is under persistent pressure due to several factors relating inter alia to the changing definition and description for a morphological normal spermatozoon. In addition, the description of different methods for the morphology evaluation process [1], the different ways in which the results are reported by different laboratories [2], and the continued lowering of the cut-off, or lower reference value, in the consecutive World Health Organization (WHO) semen analysis manuals published between 1980 and 2010 changed. An additional factor in the demise of sperm morphology evaluation in the routine Andrology laboratory is the continuing trend of non-adherence in many laboratories to the guideline given in the WHO manual(s) [3].
5.1 Introduction
The evaluation of sperm morphology as a standard procedure to be included in routine semen analysis is under persistent pressure due to several factors relating inter alia to the changing definition and description for a morphological normal spermatozoon. In addition, the description of different methods for the morphology evaluation process [1], the different ways in which the results are reported by different laboratories [2], and the continued lowering of the cut-off, or lower reference value, in the consecutive World Health Organization (WHO) semen analysis manuals published between 1980 and 2010 changed. An additional factor in the demise of sperm morphology evaluation in the routine Andrology laboratory is the continuing trend of non-adherence in many laboratories to the guideline given in the WHO manual(s) [3].
The current 2010 manual [1] provides detailed descriptions for the preparation of the semen smear, the staining of the smears and the methodology for the evaluation process itself. An important aspect for the morphological evaluation of spermatozoa is the criteria for a morphologically normal spermatozoon. In this regard, the 2010 WHO manual now recommends the use of a strict approach based on the (Tygerberg) strict criteria [4]. Due to the stricter criteria and a worldwide decrease in the percentage of morphologically normal spermatozoa, the 2010 WHO manual now proposes a lower reference value of only 4 percent morphologically normal spermatozoa. In a currently published French survey, it was found that many clinicians are regarding sperm morphology of little clinical importance and that even laboratory persons performing sperm morphology assessments regard the procedure as unreliable or the results not very reliable in analytic terms [3]. However, with the stringent application of the current WHO guidelines, sperm morphology can still play a role in the evaluation of a male’s fertility potential and clinical treatment.
5.2 Principle of Sperm Morphology Evaluation
The principle of sperm morphology evaluation can be divided into two stages or periods. The first is the early approach, later called the liberal approach [5], which was also the method described in the early WHO manuals (1980–1992). The second is the strict (criteria) approach as introduced by the Tygerberg Hospital team in 1986 and is now the method advocated in the 2010 WHO manual as the method to be followed [6].
5.2.1 Liberal Approach
In contrast to the sperm morphology of domestic animals, showing a very homogeneous picture with very little variation in shape and form, the human sperm morphology is quite different with a very heterogeneous picture showing a wide range of morphological abnormalities. Thus, while in domestic animals it was easy to describe what is a normal spermatozoon, application of this principle was not possible for the human male. Therefore, normality of sperm morphology was reached by describing all types of sperm forms regarded as abnormal. Some investigators identified more than fifty abnormal classes. Sperm normality was therefore determined indirectly or by default. MacLeod made an important contribution by reducing all the different classes of abnormalities to only seven head abnormality classes [7]. In 1971, another important contribution towards standardization of human sperm morphology evaluation was provided by Eliasson, who proposed standard measurements to identify sperm head abnormalities as too small or too large or to be within the normal range [8]. Eliasson also introduced the principle that a spermatozoon should only be regarded as morphologically normal, if the whole spermatozoon was normal, thus including midpiece and tail defects. The spermatozoon should be regarded as normal, unless it fits into an abnormal category.
5.2.2 The Strict Criteria Approach
According to Sigman [6], a major paradigm shift occurred in the late 1980s and early 1990s when a group from South Africa reversed the long-held concept that sperm were normal unless they fit an abnormal category. The Tygerberg Hospital group used the homogeneous appearence of sperm present in the upper endocervical mucus to define morphological normal spermatozoa with precise criteria [6]. Several articles published previously on different aspects of sperm-cervical mucus interaction in vivo and in vitro all reported that there was a strong selection for morphologically normal appearing spermatozoa. However, Menkveld et al. [4] used this observation to describe the appearance of what could be regarded as a morphological normal spermatozoon based on the biological principle of the specific morphological shape being able to penetrate good ovulatory cervical mucus. The spermatozoa population observed in cervical mucus presented a very homologous picture with only small variations in contrast to the very heterogeneous picture seen in the original semen samples.
The basic description for a morphologically normal spermatozoon was primarily based on the work of Eliasson that states the evaluation of the whole spermatozoon and its measurements should be used [8]. However, the strict criteria distinctly differed by stating that the so-called “borderline” normal spermatozoa should be regarded as abnormal. The reasoning behind this was to keep the variations allowed for normality as small as possible. Menkveld et al. [4] also stressed the importance of the WHO manual’s guidelines with regard to the making of semen smears, staining of these smears, and the methodology for evaluating the spermatozoa on these smears. Due to these principles, the description by Menkveld [9] and Menkveld et al. [4] is now referred to as the (Tygerberg) strict criteria approach [5].
5.2.3 Classification Criteria for Normal Sperm Morphology
Spermatozoa consist of a head, neck and tail region, with the tail subdivided into the middle, principal and end pieces. For classification purposes, the neck and middle pieces of the spermatozoon are classified together. For a spermatozoon to be considered as morphologically normal, all of the head, neck and tailpieces must be normal. All borderline forms should be considered as abnormal [1, 4].
The head of the spermatozoon must have an oval form with smooth contours. A clearly visible and well-defined acrosome must be present and should cover about 30–60 percent of the anterior part of the sperm head and exhibit a homogeneous light-blue staining indicating an intact acrosome. The generally given measurements for a normal sized spermatozoon of between 3–5 µm and 2–3 µm for length and width, respectively, is probably too long and wide for general purposes as sperm head measurements are staining specific. Several head measurements are available for Papanicolaou stained spermatozoa:
The WHO 2010 manual [1] provided the following measurements as obtained by a computerized sperm morphology analysis system (CAMA); median length 4.1 µm (95 percent CI 3.7–4.7 µm) and median width 2.8 µm (95 percent CI 2.5–3.2 µm).
Normal CAMA head measurements (mean and SD) as published by Maree et al. in 2010 [10] are 4.28(0.27) µm for length and 2.65(0.19) µm for width.
Manual measurement (mean and SD) as published by Menkveld in 2013 [11] are 4.07(0.19) µm for length and 2.98(0.14) µm for width.
The principal piece of the tail should have a uniform calibre along its length, be thinner than the midpiece and should be about 45 µm to 50 µm long and without any sharp bends. A tail looped on itself is regarded as normal. The tail tip is very seldom visible.
No cytoplasmic residues may be present at the neck/midpiece region or on the tail.
5.2.4 Identification Criteria of the Four Abnormal Sperm Morphology Classes
In the literature, four main abnormal sperm morphology classes are distinguished viz. head, neck and midpiece defects, tail defects and the presence of cytoplasmic residues, sometimes also called cytoplasmic droplets [11].
5.2.4.1 Head Defects
5.2.4.1.1 Size
A head is said to be classified as having size defect where the head is too large or too small based on the actual staining method used, but still presenting with an overall oval form.
5.2.4.1.2 Form
This is where the spermatozoa do not present with the classical oval from. This category includes spermatozoa described as elongated forms, tapering forms, and which according to Eliasson (1971) may be smaller or larger than the normal oval form [8], and the so-called pear-shape or pyriform which are usually larger than the normal oval from. Spermatozoa with a V-shaped posterior (post-acrosomal) ends are classified as morphological normal forms on the condition that no elongation is present.
5.2.4.1.3 Acrosomal Defects
Acrosomal defects include size defects, staining defects and structural defects. Size defects are present when an acrosome covers >60 percent or <30 percent of a normal sized head. Acrosome defects can also include staining defects, where the acrosomes do not show the homogeneous light-blue staining when stained according to the Papanicolaou method, or where the acrosome staining in certain areas is absent and the acrosomes show white (patchy) areas. This should not include the presence of (large) vacuoles or cysts. Vacuoles are deemed present when a lighter stained area(s) with well-defined round borders is observed. A cyst is deemed to be present when a lighter stained area(s) with well-defined round borders is present on the outer acrosome area, giving an extruding impression.
5.2.4.1.4 Duplications
Duplications are deemed to be present when usually, two heads are joint together at the neck/midpiece area, but this may also occur at any other part of the sperm structure. When a head duplication is present, this is primarily classified as head abnormality.
5.2.4.1.5 Amorphous
The term amorphous heads includes all other head forms not classified under any of the named abnormal sperm head categories. Spermatozoa with a slight, but definite alternation from the morphological normal forms are considered as borderline and must be classified as an abnormal head form.
5.2.4.2 Neck/Midpiece Defects
As specific neck defects per se are difficult to identify, neck and midpiece defects are considered as one defect type. This includes bend necks, where the neck/midpiece forms a definite angle with the sperm head, an asymmetrical implantation of the neck/midpiece to the posterior region of the sperm head, a thickened neck/midpiece as well as asymmetric bend midpieces. Cases where the mitochondrial material has shifted to the neck or towards the principal tail region showing a very thin midpiece. In these cases, the mitochondrial material present at the neck region must not to be confused with the presence of excessive cytoplasmic material.
5.2.4.3 Tail Defects
Tail defects are present where a definite bend (some definitions specify >90°) is observed at any part of the principal tail piece. Other tail defects are double tails, two or more, extruding from a single sperm head or midpiece, coiled tails or irregular tails or combinations. A bend between the end of the midpiece and beginning of the principal piece of the tail is classified as a tail abnormality and not a neck/midpiece abnormality. A bent tail should not to be confused with a curved tail. Short tails are shorter than tails of the normal specified length and must not be confused with abnormal tails due to the short tail syndrome. Coiled tails are in most cases not due to artifacts, but due to definite abnormalities, as the tails are enclosed in a membrane structure.
5.2.4.4 Cytoplasmic Residues
Although no cytoplasmic material should be present, a small amount of cytoplasmic material, <30 percent of a normal sized sperm head, sometimes occurs and can be regarded as normal. Cytoplasmic material on spermatozoa in stained semen smears are not common and, when present, are usually associated with the occurrence of other sperm defects such as bent necks and elongated spermatozoa. The presence of cytoplasmic material on spermatozoa is associated with the excessive production of reactive oxygen species (ROS).
5.3 Methods for Preparation and Examination of Semen Smears
As part of the standard semen analysis, semen smears are prepared, stained with the Papanicolaou method, and examined for the presence of spermatozoa and round cells. Spermatozoa are then assessed under oil magnification to determine the percentage of morphologically normal spermatozoa for the specific sample.
5.3.1 Preparation of Semen Smears
The identification (ID) information of the semen sample must be noted with pencil on the frosted part of the slide in such a way that, if the slide is kept up-right the frosted end is on top and the ID information is upright and readable for easy recovery when stored. Do not use paper labels as the labels may become contaminated when placed in the staining solutions and ID information may therefore not be visible.
Place a drop of 5–15 µL of liquefied semen, depending on the sperm concentration, just under the frosted part of the slide. Another slide is then placed in front of the semen drop, slightly pulled back towards the frosted part so that the semen can spread over the entire width of the slide and the slide is then moved towards the end of the slide, on which the semen is deposited. The speed of the movement and the angle at which the slide is held to draw the semen drop will determine the thickness and number of sperm on the slide. A smaller drop of semen and slow forward movement will provide a thinner smear and less sperm per visual microscopic field, while a bigger angle and a faster forward movement will deposit more sperm in a smaller part of the slide. The smears are slightly air-dried and fixed for at least 10 minutes in an appropriate fixative.
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