The Importance of Sperm Morphology in the Evaluation of Male Infertility
The goal of estimating correctly a man’s fertility potential has long been of great interest to researchers. The term ‘male infertility ’ does not constitute a defined clinical syndrome but rather a collection of different conditions exhibiting a variety of etiologies and varying prognoses (1). It is therefore unreasonable to hope for tests that will indicate with absolute certainty that a man will be fertile. On the other hand, when the results of these tests are subnormal, they should not be used as an absolute diagnosis of sterility.
Semen analysis is a keystone in the clinical workup of the infertile man patient. Since first published in 1980, the WHO Manual for Andrology Laboratories (35,36) has gained worldwide acceptance as a source of standard methodology for human semen analysis. In the latest edition (1992) the guidelines for morphology evaluation have been revised significantly.
Sperm morphology is assessed routinely as part of standard laboratory analysis in the diagnosis of human male infertility. This practice has its origins in the work of Mac Leod & Gold (1951) which showed that sperm morphology was significantly different in fertile compared to infertile man.
Despite this standardization, human semen evaluation continues to be influenced by subjectiveness of the investigator and a lack of objective measurements for sperm morphology continues to be a problem.
There is an an ongoing debate on which criteria should be applied to define normal spermatozoa and which classification of abnormal forms is most appropriate.
Studies on sperm morphology should concentrate to obtain measurements and biological data of spermatozoa which are functionally active. Only then the definition of normal can be achieved and clinically useful criteria can be adopted.
However, the definition of a normal spermatozoon as described by WHO in 1992 is different from that used by other authors (17,21,22,31). The evaluation of the morphology of human spermatozoa varies widely between and sometimes even within laboratories. While most investigators agree on the appearance of a normal spermatozoon, standardized analysis is difficult because of the use of different staining techniques which are not always suitable for optimal examination from head to tail (Figure 1). For example, the techniques for preparing morphology specimens have been expanded from three to five.
The difficulty in classifying human sperm morphology is mainly caused by the large variety of abnormal forms found in the semen of infertile men. Only certain types of abnormalities can be analyzed objectively (11).
The definition of ‘morphological normal’ is still discussed, as well as the clinical relevant limits for the rate of pathologic forms.
What is a normal spermatozoon ?
The WHO’s definition of a normal spermatozoon is not based on any biological data. As a consequence, the implementation of new standards has resulted in some controversy. However, sperm morphology was found to correlate more closely with fertilization rates than sperm count and motility (2).
Because it is not possible to determine the fertilizing potential of individual human spermatozoa, physiological endpoints other than fertilization must be studied to obtain insight into the mechanisms by which sperm morphology influences the fertilization process, e.g. examination of morphologic characteristics of spermatozoa recovered from cervical mucus and/or those binding to the zona pellucida.
The strict criteria of sperm morphology (e.g. Kruger’s criteria) use the examination of spermatozoa that had penetrated cervical mucus for the definition of normal spermatozoon. Even by using strict criteria it is difficult to determine if results between studies are really comparable.
In order to avoid subjectivity, over the past 20 years numerous studies describe image analysis techniques in the assessment of sperm morphology. These techniques allow objective characterization of different sperm forms. Automated methods may help, but there remains a lack of biological data to support the use of computer-aided semen analysis in clinical settings.
Recent evidence suggests that sperm morphology assessment by relatively simple and inexpensive methods can provide prognostic information similar to that obtained from some of the more elaborate sperm function tests.
Criteria of the World Health Organization
The WHO manual in 1987 describes a normal spermatozoa: ‘An oval head shape with a regular outline and acrosomal cap covering more than one-third of the head surface. The head: length : 3-5 µm, width : 2-3 µm; lenght/width ratio : 1,5-2. The midpiece: 7-8 µm, long,straight and regular in outline, slender, less than 1/3 of width of the head. The tail: at least 45 µm in lenght, slender, uncoiled and regular in outline.’
Spermatozoa were classified into normal, having head defects (amorphous, small,large,pyriform,tapering), midpiece defects (including cytoplasmic droplets) and tail defects (Table 1). At least 200 spermatozoa need to be examined in an attempt to reduce technical variation.
Sperm morphology is determined using brightfield illumination at x1000 magnification, after preparing air-dried Papanicolaou-stained smears. All slides were read blind by an experienced highly trained technician who produced consistent and reliable results.
In keeping with the move to a more strict definition of sperm morphology, WHO has redefined what they consider to be a normal spermatozoon and subsequently set an ‘empirical reference value of 30% normal forms and above as normal’ (36).
A normal spermatozoon has an oval head shape with regular outline and a well-defined acrosomal region covering 40-70 % of head; vacuoles occupy less than 20% of the head area. The head: length : 4-5,5 µm, width : 2,5-3,5 µm and lenght/width ratio : 1,5-1,75; no cytoplasmic droplets more than 1/3 of the size of a normal sperm head. No dimensions and no description of a normal midpiece are mentioned. Defects are described, e.g. insertion of the tail in more than 90% of the head’s longitudinal axis is abnormal. No dimensions and no description of a normal tail are mentioned, only defects are described.
Using this classification scheme, all borderline forms are considered abnormal.
Head defects are: large, small, tapering, amorphous, pyriform, vacuolated or double heads or any combination of these.
Neck or midpiece defects are: bent or abnormal thin.
Tail deffects include short, multiple hairpin, broken, irregular width or coiled tails, tails with terminal droplets or any combination of these.
A minimum of 200 spermatozoa is counted and a stage micrometer is used to aid interpretation.
The teratozoospermia index is a measure of the average number of defects per spermatozoa, which could be used to improve the correlation between sperm morphology and fertility.
Basic semen evaluation is performed after liquefaction of the specimen. Two morphology slides are prepared for each patient and are stained by the quick-stain technique ( Diff-Quick solution 1 and 2). Special care is taken to clean the slides thoroughly with 70% ethyl alcohol before use and not more than 5 ul of semen is used to prepare the slides as thin as possible. The slides are reported on the same day. The morphology is evaluated by two independent observers.
The Strict Criteria
Another important evaluation aiming to clarify male fertility is the strict criteria for sperm morphology (Kruger’s or Tygerberg’s criteria 1986) (17).
Strict criteria of sperm morphology established by Kruger et al. define normal spermatozoa as having an oval configuration with a smooth contour ( Figure 2). The head in lenght is 5-6 µm, the diameter( width) is 2,5-3,5 µm and the width/lenght ratio is 1/2-3/5. The acrosome is well-defined, comprising 40-70% of the distal part of the head. No abnormalities of the neck, midpiece or tail and no cytoplasmic droplets of more than half of the sperm head are accepted. Borderline forms are considered abnormal.
The amorphous-head group is divided into two categories:
Neck defects are also classified in two categories:
Normal and borderline forms grouped together are called ‘the morphology index ’. Patients with a morphology index less than 30% will have a severe reduction in fertilization as compared with patients having an index greater than 30% (17). In Kruger’s practice, the normal forms considered alone are called the ‘percentage of ideal forms’ (PIF). A PIF greater as 4% is considered favorable and less than 4% unfavorable. At least 200 cells per slide are to be evaluated. A micrometer in the eyepiece of the microscope is used for routine measurements.
In order to improve the strict criteria, in 1987 Menkveld defined a normal spermatozoon, based on the appearance of spermatozoa found in good periovulatory cervical mucus of the upper part of the endocervical canal. These spermatozoa are usually in an apparently homogenous population( Figure 3). The morphological classification used by Menkveld is based on a modification of the methods of Mac Leod and Gold (1951) and Eliasson (1971) .
The head must have a smooth oval configuration with a well-defined acrosome comprising approx. 40-70% of it. The normal head has a lenght of 3-5 µm, width of 2-3 µm and a width/lenght ratio of 3/5- 2/3. The range of variation within the normal population is shown in Figure 4. The most important difference between this classification and other methods is that Menkveld regards borderline-normal head forms and/or spermatozoa with nearly oval heads with no gross abnormalities as abnormal. Neck, midpiece or tail defects are considered abnormal. The midpiece lenght is 1,5 times the head lenght, the width is less than 1 µm, slender and axially attached. The tail measures 45 µm in length, appears uniform, uncoiled and is slightly thinner than the midpiece. Cytoplasmic droplets (remnantes) which comprise less than ˝ the size of the sperm head are accepted. These are strict criteria.
Spermatozoa are classified into seven groups (Figure 5): normal (whole sperm), large, small, elongated (tapering), duplicated and amorphous heads, all with or without the presence of a cytoplasmic droplet and/or tail, neck and/or midpiece defects. The seventh group consist of spermatozoa with a normal head but with a tail and/or a neck and/or a midpiece defect and/or the presence of a cytoplasmic droplet.
At least 100 but preferably 200 spermatozoa are evaluated. Inexperienced workers should use a built-in micrometer when they begin with morphology evaluations. The normal dimensions for spermatozoa stained with the Diff-Quik method (used by Kruger) are larger than those based on the Papanicolaou method.
In order to improve the evaluation of sperm morphology , Davis and Gravance (1994) have emphasized the sensitivity of sperm classification methods when only two morphometric variables are used (lenght and width of the head, for example). Based on linear models as an appropriate mean of describing the size relationship between phenotypic characters, they show that small changes can significantly alter the percentage of normal sperm within a specimen.(9).
A new expression of sperm morphology parameters is the sperm deformity index (SDI), described in 1996 (2). This is a method by where the whole spermatozoon is assessed by the strict criteria and classified more than once if more than one deformity exists. Both normal and abnormal sperms are considered and the average number of deformities per sperm is determined to give a value to this index. This index reflects the balance between the prevalence of sperms with multiple structural deformities and the proportion of sperms with normal morphology in a semen sample.
Computer-assisted methods of sperm morphology evaluation
Over the past 20 years, in order to avoid subjectivity, numerous studies that incorporate image analysis techniques in the assessment of sperm morphology have appeared. These techniques allow objective characterization of different sperm forms.
The method of Moruzzi (1988) for quantification and classification of human sperm morphology by computer-assisted image analysis is semi-automated (25). Sperm heads are imaged through a microscope (NA=1.3) sampled at 0,125 um intervals and measured on an image analysing system. Measurements included stain content, lenght, width, perimeter, area, and arithmetically derived combination. Additionally, each sperm image is optically sectioned at right angles to its major axis, to give a measure of lenghtwise heterogeneity of shape. According to this method, the percentage of normal sperm heads can be accurately predicted using just four sperm head measurements. This classification procedure distinguishes normal from abnormal sperm with 95% accuracy and correctly assigned 86% of the sperm to one of 10 shape classes.
The system described by Perez-Sanchez (1994) is based on the evaluation with a video digitizer board, a brightfield microscope with a 100x immersion objective, two monitors and the image analysis software (31)(Figure 6). Cells are displayed live on the video monitor and each sperm head image is processed automatically using a specific image analysis program for image enhancement and thresholding. Analysis of the sperm midpiece and tail is not included in the program. The system detects the boundary of the sperm head and the outline is displayed as white overlays superimposed on the video image. The set of morphometric parameters used by Perez constitutes a set of characteristics which is valid for characterization of the majority of morphological types of spermatozoa.
The association between semen quality and male infertility has been known for more than 40 years.
Having reviewed the literature, it seems clear that strict morphology has a clinical relevance, being an excellent biomarker of sperm fertilizing capacity, in vivo and in vitro, independent of motility and concentration (27).
Sperm morphology evaluation is considered to be a highly subjective procedure because, unlike the haematopoietic cells for example, the difficulty in classifying human sperm morphology is caused by the large variety of abnormal forms found in the semen of infertile and fertile men. Only certain types of abnormality can be quantitated objectively (11).
Normal sperm morphology needs to consider two points. The first one is the proportion of spermatozoa with normal morphology in semen and the second is the definition and the characterization of the normal spermatozoa.
According to WHO criteria, a normal ejaculate must have at least 30% normal sperm.(36). For the stricter criteria, fertile men have > 14% normal forms in their semen and men with < 4% of normal forms are subfertile. According to Kruger’s criteria, IVF outcome was suboptimal when normal sperm morphology was less than 14% and worst if it was under 4%.(17). The sperm deformity index is a more reliable predictor of the outcome of fertilization in vitro than the proportion of normal sperm morphology.(2).
WHO recommends that each laboratory recruits fertile men (a reference population) in order to investigate and determine the real cut-off values for normality in that laboratory (28). These men are very difficult to recruit, therefore only a few laboratories actually perform this analysis (3).
During the past 15 years there has been an increase in total motile sperm count, secondary to an increase in semen volume, and a decline in normal morphology. Both are independent of the age and the duration of abstinence in fertile men (5).
There is a debate on this subject and some of the arguments discussed by different authors are the analize techniques used by laboratories and the influence of environmental factors on sperm morphology.
A number of authors, under in vivo conditions of conception, have demonstrated that the pregnancy rate was significantly higher in cases of better sperm morphology (13). But, particulary under the usual in vivo conditions of conception, pregnancy rates are influenced by a multiplicity of different parameters.
It seems that sperm morphology evaluated for strict criteria has definitive advantages over the other (liberal) criteria evaluation methods in the prediction of in vivo and especially in vitro fertilization rates (22).
Other publications’ unavoidable conclusion is that sperm morphology according to WHO classification can be a vigorous predictor of IVF outcome (24). Therefore, further investigations of semen morphology and male fertility according to WHO may be clinically rewarding.
There are papers which emphasize the importance of inclusion of abnormalities in the acrosomal region during sperm morphology assessments and suggest that previous emphasis placed on sperm head dimensions in the classification of normality is justified only in cases of extreme deviation of the normal (12).
Neither abnormal sperm concentration, nor abnormal sperm morphology are significantly associated with reccurent spontaneous abortion (14).
Kruger’s opinion is that the existing classification of abnormal and normal shaped sperms are in need of revision by those involved in the field. Scanning electron microscope is worth further evaluation as a tool in the accurate scoring of normal sperm morphology.
The advantage of using strict criteria in morphology evaluation is the fact that the measure is reproducible between patients and between different technicians performing the test. It also allows the clinician to classify the patient into one of two specific groups ( < 14% and >14% normal morphology), giving a reliable criteria to plan the approach for future IVF cycles (17).
For clinicians responsible interpreting results to patients, it is important to emphasize that 0% strict criteria does not necessarily implicate that no normal sperm is present: it only signifies that no sperm meeting a set of criteria was observed in the sample (24).
Nevertheless, even in cases of severe teratozoospermia fertilization may be possible; most promising is the development of intracytoplasmic sperm injection ( ICSI ) as the treatement of first choice in these cases.(27).
Another conclusion is that only one standard method should be recommended for the preparation of morphology slides in order to ensure inter-laboratory comparability of results and to enhance the value of sperm morphology analysis for predicting fertility (23). At present there are five techniques of sperm preparation. For practical purposes, the Papanicolaou method seems the most suited, due to its widespread use.
Sperm morphology must only be considered as an indicator of fertilization potential, not as an absolute indicator of sterility. The aim should be to find the minimum sperm parameters associated with specific methods of seminal treatment and with culture techniques which can lead to a single, common stategy for male infertility problems.
The Human Fertilization and Embryology Authority (1991) and WHO (1993) strongly suggest that for the treatment of infertility both partners must be evaluated in order to exclude subtle problems.
Edited by Aldo Campana,