Bruce R. Gilbert, M.D., Ph.D.
George W. Cooper, Ph.D.
Marc Goldstein, M.D.
Department of Urology - The New York Hospital-Cornell Medical Center
and the Department of OB-GYN and Urology - North Shore University Hospital
15% of couples have difficulty conceiving, and in 50-70% of these there is either a primary male factor or an impaired semen quality sufficient to reduce the probability of pregnancy in the subfertile female (1, 2). Accordingly, the first and the easiest test in the evaluation of the subfertile couple should be the semen analysis.
Although "seminal antimolecules" or spermatozoa were first described in the human semen by Van Leeuwenhoek in 1677 (3), their morphologic evaluation did not begin in earnest until the 1900's when normal and 'pathologic' forms were found to co-exist in semen (4). To this day there is still controversy as to what constitutes the "normal" spermatozoa in semen and the genetic constitution of the "abnormal" forms. Three recent guidelines for evaluation of human semen are described in the World Health Organization laboratory manual for the examination of semen and semen-cervical mucus interaction (5), in Keel (6), and in the atlas of Menkveld et al. (7).
Unlike routine serum biochemical tests, information obtained from the macroscopic and microscopic examination of semen can be completely altered by events preceding these examinations. Therefore, we will detail the collection protocols prior to discussing the parameters for macroscopic examination, as well as microscopic and biochemical tests. Particular emphasis will be given to sperm morphology, since recent results of IVF have provided insight as to which morphologic characteristics correlate with fertilization in vitro (8, 9). We will also discuss when and how to test for antisperm antibodies, as well as the relative advantages, and disadvantages, of computer assisted semen analysis. For the purposes of uniformity, the WHO values (5) will be used as the normal semen values (Table 1), unless otherwise stated. The nomenclature used for some semen variables is listed in Table 2.
It is important to provide patients with clearly written instructions on how to produce a specimen and as to the type of container to use. Routinely we ask for 48-72 hours of abstinence. Longer periods usually produce a larger volume, but often poorer motility quality. Occasionally when the percent motility is reduced to 20% or less, we ask patients to produce a second sample within 1-2 hours after the first collection to rule out male tract storage problems. This often results in a specimen with much improved motility, albeit with a decreased total number of sperm though this can be within the normal range. We also ask patients to evacuate their bladder prior to ejaculation and routinely ask for a post-ejaculate urine to rule out retrograde ejaculation and/or possible retention of semen in the urethra secondary to a noncompliant urethra.
The type of container used has always been of some concern in the past. Glass containers were used for their supposed inertness, but frequent breakage has led most semen analysis labs to use wide mouth polypropylene containers with a screw top cap. Some plastics, such as polystyrene, have been found to cause an increase in semen viscosity, and in fact, may be toxic to sperm. We ask for the specimen to be delivered to the lab within an hour to 1 1/2 hours and to be kept as close as possible to body temperature (preferably in a jacket pocket). When received in the laboratory, we note the time of collection, the time that we evaluate it and whether it is a complete sample or whether some loss occurred during collection. We also ask for a post-ejaculate urine to be given at this time. A sample of our work sheet is shown in Figure 1.
Liquefaction usually occurs within 10-20 minutes of collection. On a scale of 0-4 (4 being the normal value for a well liquefied sample), the failure to liquefy is usually a sign that there is inadequate secretion by the prostate of the proteolytic enzymes fibrinolysin, fibrinoginase and aminopeptidase (10). Importantly, liquefaction should be differentiated from viscosity, as abnormalities in viscosity can be the result of abnormal prostate function and/or the use of an unsuitable type of plastic container. High Viscosity can usually be reduced by repeated aspiration through an 18 or 19 gauge needle before or after diluting the semen with a suitable buffer or culture medium to reduce the amount of shear during physical disruption of the seminal gel. Volume is measured with 2 to 5mL serologic pipettes; normal volume being between 2-6 mL. Color is normally opaque or opalescent with translucent semen usually being oligospermic. The presence of a yellow hue or a very milky turbidity is associated with pyospermia while a rust colored or reddish semen is indicative of hematospermia. pH (normal 7.2 - 7.8) is also measured with colorpHast pH 6.5-10.0 indicator strips or a standard pH meter. In acute infection (prostate, seminal vesicles or epididymis) the pH of semen will be greater than 8.0 when measured soon after liquefaction. In cases of obstruction of the ejaculatory ducts or when only prostatic fluids are secreted the pH is usually less than 7.0.
Sperm concentration (normal > 20 million/mL) is usually measured either with a Makler chamber (Sefi Medical Industries, Haifa, Israel)(11, 12), a Neubauer hemocytometer (American Optical Company, Buffalo, N.,Y.) or a disposable device (Micro-Cell; Fertility Technologies, Inc., 215 Oak Street, Natick, MA.). Various investigators have their preferences and certainly for normospermic samples, each of these devices yields consistent results. However, controversy exists as to which is the most accurate measurement device (13). The advantages of the Makler chamber and Micro-Cell are that sperm counts can be done directly on an undiluted semen after immobilization of the spermatozoa and evaluation of the percent of motile sperm can be directly quantified. Use of the hemocytometer requires dilution of the semen sample, due to chamber depth which can be done with accuracy using volumetric pipettes with the diluent containing a fixative. No matter which method is used, several measurements on a well mixed specimen should be done and the mean of the results recorded. Adequate sampling is particularly important in severely oligozoospermic semen. When fewer than 10 million sperm/mL are present, we concentrate the specimen and re-suspend in a constant volume to increase the sensitivity of our measurements. Total sperm count (normal > 50 million) is then calculated by multiplying concentration times volume. Motility (normal > 50%) is expressed as the percent of spermatozoa that have motion and the forward progression (normal > 3) is noted. A forward progression of 4 denotes spermatozoa rapidly moving in a straight line with no yaw or lateral movement; unfortunately, the rapidity (or linear velocity) of forward progression has not yet been standardized. A forward progression of 3 denotes spermatozoa similarly moving linearly but at a slower velocity. Sperm movement with a forward progression of 2 often exhibits angular displacement or yaw to varying degrees while a progression of 1 denotes only tail motion without progression. Zero progression being no movement at all. The measurement of the duration of motility in seminal fluid is controversial. MacLeod (14) has pointed out that the seminal plasma is only a temporary transport medium and that evaluation of the duration of sperm motility in vitro in semen, is probably not physiologically significant as sperm rapidly lose their motility in the specimen container but remain motile for days in the mid cycle cervical mucus.
If motility is less than 50% a viability stain is done using Eosin Y with Nigrosin as a counterstain. Greater then 50% of the sperm should be viable (i.e., non-stained). This is seen by bright field microscopy as red dye (Eosin Y) taken up by the head of most non-motile sperm. Agglutination is also noted if present (Figure 2). Though clumping of spermatozoa can mean sperm antibodies are present, clumping usually only occurs around debris in the sample. However, if motile dimers, of either head to head or tail to tail associations are seen (Figure 2B), this is diagnostic for the presence of antisperm antibodies in semen bound to the sperm.
Other than the spermatozoa, the presence of round cells should be noted and when a count is made of spermatozoa, quantification of round cells by cell type should also be made. Round cells should be differentiated between immature germinal cells (Figure 3A), which usually have a single or double round nucleus, are variable in size with a decreased nuclear to cytoplasmic ratio as compared to polymorphonuclear leukocytes (PMN; should be 1 x 106 /mL) (Figure 3B), which are more constant in size, smaller than the germinal cells, have interconnected lobes to their nucleus and have an increased nuclear to cytoplasmic ratio. The classic method of distinguishing PMN's from immature germinal forms is the peroxidase stain (15). PMN's are peroxidase-positive while degranulated PMN's, lymphocytes and 'immature' germ cells are peroxidase negative. The quantification of red blood cells, if present, should also be made. In addition, bacterial contamination and presence or absence of epithelial cells should be noted. If no sperm are seen, and/or the volume is less than 1.5 cc, a fructose test should be done to confirm the presence of seminal vesicle fluid, as well as a post ejaculate urine, to rule out retrograde ejaculation.
MORPHOLOGY (Figure 4)
Sperm have a head, a mid-piece and a tail, each component of which has particular morphologic characteristics. Figure 5 shows a comparison between the strict morphologic criteria of Kruger, et al (9) and that of the WHO (5). The head of the normal human spermatozoa is ovoid in the frontal view and pyriform in the lateral view. Fixed and stained (Papanicolaou) sperm heads measure approximately 3-5 microns in length and 2-3 microns in width. The acrosome should make up somewhere between 40-70% of the normal sperm head. The mid-piece has a mitochondrial sheath and often excess cytoplasmic material from the developing spermatid. The tail principal piece is approximately 50-55 micra in length and varies in thickness from about 1 micron near the base to 0.1 microns at the tip of the end-piece as shown by electron microscopy. The tail is composed of an axial core consisting of two central singlet microtubules surrounded by nine pairs of doublet microtubules , an outer ring of nine dense fibers surrounded by the fibrous sheath which define the principal piece of the tail (Figure 6a,b). The various structural alterations that can occur in each region of the spermatozoa are shown in Figure 7. Menkveld et al. (7) have described an association between the "strict" morphologic evaluation of spermatozoa and the results of in vitro fertilization. Of particular note is the direct relationship between acrosome size and the frequency of both pregnancy and fertilization (Figure 8).
WHO TO TEST FOR ANTI-SPERM ANTIBODIES
In recent years, immunologic causes of fertility have been more frequently recognized. As mentioned above, the presence of agglutination and in particular motile dimers, should lead to investigations for anti-sperm antibodies, as well as the standard indications of idiopathic infertility or abnormal post coital tests. Multiple risk factors have been implicated with increased risk of anti-sperm antibodies (Table 3). There are several ways to test for antisperm antibodies, (Table 4). Good tests detect and define binding sites to sperm surface antigens on motile, viable spermatozoa. Non-motile and/or non-viable sperm are not normally involved in sperm/egg interactions and it is the internal and subsurface antigens of these cells which have too often been studied and which are not of clinical relevance. Thus, tests that rely on sperm whose subsurface antigens are reveled as the consequence of chemical fixation or freezing or air-drying all yield both false positive and false negative results (16). In addition, assays that only evaluate the antisperm antibodies in seminal fluid residual after removal of sperm or in serum by passive transfer to donor third party sperm are of questionable clinical significance.
Two current methods of detecting antibodies bound to the surface of motile sperm are the mixed agglutination reaction assay (MAR test; only for IgG's) and the immunobead binding assay (for IgA, IgG and IgM's). Both tests require motile sperm, the MAR test (SpermMar: Fertility Technologies, Inc., 215 Oak Street, Natick, MA.;(17)) is a simple test that can be done with a light microscope on unprocessed semen. It is highly sensitive and IgG specific and is performed by mixing fresh, untreated semen with latex particles coated with human IgG. To this mixture a monospecific antihuman IgG antiserum is added. The formation of mixed agglutinates between latex particles and motile spermatozoa indicates the presence of IgG antibodies on the spermatozoa. The binding to motile sperm and can be easily seen by light microscopy and localized as binding to different regions of the head, mid-piece or tail. The % of motile sperm, which are head , mid-piece or tail bound or not bound are recorded. Greater than 40% binding is considered to be significant. In the indirect SpermMar test, washed motile donor spermatozoa are incubated with diluted decomplemented patient serum of male or female origin. If the serum contains antisperm antibodies, these will bind to the donor spermatozoa which will react with latex particles in the SpermMar test. To-date the kit is available only for IgG antibodies. A similar test for IgA, probably a clinically more important antibody, is presently under production.
The immunobead assay (18) has the advantage of being extremely sensitive and specific and can evaluate binding in three immunoglobulin classes ( IgG, IgA and IgM). The immunobead assay also can be performed as a direct test on sperm isolated from semen as well as an indirect test of immunoglobulin binding following incubation in seminal fluid or semen. The procedure requires one additional step, washing sperm prior to incubation with the polyacrylamide microspheres which also have been bound with anti-human IgG, IgA, or IgM antiserum. The immunobead study requires a phase contrast microscope for evaluation.
TESTS OF SPERM FUNCTION
No review of semen analysis can be complete without mentioning the tests of sperm function. Although spermatozoa may look normal, move with good forward progression and lack surface antibodies, this is no assurance that they perform properly. Of the many tests of sperm function available (Table 5), none unequivably identify functional spermatozoa. (1) The spermatozoa penetration assay (SPA) utilizes the golden hamster egg which is unusual in that removal of it's zona pellucida results in loss of all species specificity to egg penetration. The SPA can therefore be used to yield information regarding the fertilizing capacity of human spermatozoa. The concept of the sperm penetration assay was introduced by Yanagamachi in 1972 and modified for use in humans in 1976 (19, 20). The spermatozoan must undergo several changes prior to penetrating a zona pellucida-free hamster egg. These are capacitation and the acrosome reaction which require still poorly understood biophysical and biochemical changes induced both in vitro and in vivo in the presence of mature oocytes. Thus the zona-less egg penetration assay measures sperm capacitation, the acrosome reaction, sperm/oolemma fusion, sperm incorporation into the ooplasm and the decondensation of the sperm chromatin. However, penetration of the zona pellucida and normal embryonic development are not tested. Thus, a positive SPA does not guarantee fertilization of intact human eggs nor their embryonic development while conversely, a negative SPA has not been found to correlate with poor fertilization in human IVF. (2) The acrosin assay measures acrosin, a trypsin like serine proteinase found in the acrosome which may be responsible for penetration of the zona pellucida and also triggering the acrosome reaction (21). Measurement of acrosin is thought to be correlated with physiologic facilitation of the acrosome reaction and sperm binding to and penetration of the zona pellucida. (3) The hemizona assay (22) measures the binding of sperm to the zona pellucida of human eggs. Human spermatozoa will bind firmly to only the human zona pellucida and that of closely related higher primates (23). For ethical reasons one does not perform diagnostic "functional" assessments of binding of human spermatozoa to intact viable human oocytes. In the human hemizona assay, unfertilized oocytes obtained through donation are bisected and the number of sperm tightly bound to the outer surface counted. The major advantages of the hemizona assay is that: 1) the two halves of the hemizona are functionally (qualitatively), equivalent surfaces allowing controlled comparison of binding, and 2) the limited number of available human oocytes is amplified. The results of this test show a good correlation with "strict" morphology (9) and IVF fertilization rate (24). (4) The hypoosmotic swelling test (HOS) (25) measures the membrane integrity of sperm, important not only for metabolism, but successful union of male and female gametes. A critical property of all cell membranes is their ability to selectively transport molecules. Therefore, when viable spermatozoa are exposed to hypoosmotic conditions, water enters the sperm resulting in swelling. This places the tail fibers under tension causing curling of the tails, which can be readily visualized by phase contrast microscopy. The HOS test correlates highly (r=0.9), with the outcome of zona free hamster penetration assay when normal ejaculates are used (25). A distinct advantage of this test is that it is simple, cheap and readily applicable clinically. (5) The cervical mucus penetration assay quantitates the sperm-mucus interaction and yields information regarding the motility of the spermatozoa. Although millions of sperm are deposited in the vagina, only hundreds ever reach the ampulla of the fallopian tubes. A major factor in this dramatic reduction is the cervical mucus. After the estrogen surge, the cervical mucus is about 98% water, compared to its composition at times under control of progesterones when the water content falls to about 90%. At midcycle, during the more aqueous phase, cervical mucus is less of a barrier to sperm and it is at this point that the post coital test (PCT) is performed. Alliquots of cervical mucus are collected within 12 hours after coitus at midcycle. Good PCT being defined as greater than 10 motile sperm/high powered field. Fewer sperm may indicate collection of non midcycle mucus or poor mucus sometimes referred to as hostile cervical factors. The presence of antibodies, oligozoospermia or sperm with poor motility also yield poor test results . Female factors that contribute to a poor PCT include inadequate estrogen priming, deficient endocervical tissue or previous surgery to the cervix. There are also other in vitro tests, using either human or bovine cervical mucus, again to test the penetration by motile sperm. These are subject to variable mucus quality and are difficult to standardize and interpret.
COMPUTER AUTOMATED ANALYSIS OF SEMEN
Automated semen analysis is increasingly being used for the routine evaluation of semen quality, as well as for assessing the role motility plays in fertilizing potential in vivo and in vitro. To meet these goals an automated analyzer must accurately count sperm, and percent motility, evaluate motion characteristics based on their ability to quantitate both the angular and linear motion (26, 27) of sperm populations and analyze sperm morphology. However, the present 'gold standard' remains manual evaluation performed by trained technicians. Automation of this seeks to increase not only the rapidity of measurement but also the accuracy allowing standardization of semen parameters between laboratories by eliminating inter-technician biases.
Automated semen analyzers, although doing a superb job with motion evaluation, suffer from many technical problems (28) including their inability to adequately differentiate small particles ('debris') from both sperm or round cells (29-32). In addition, the evaluation of sperm morphology, is still not well developed for automated analyzers (33). Although, the hope was to standardize the measurement of semen parameters by automated devices, this has not yet been obtained. Jasko et al. (34) found when comparing two automated semen analyzers that the results were often dissimilar making direct comparisons of results difficult. Lastly, even functional assessment is questioned since the need for 'human' intervention in the analysis (31) has made inter and intra laboratory comparisons of motion related phenomena difficult.
Therefore, although the potential for automated semen analysis is great. It has not yet reached its goals of providing a highly reliable method to evaluate a semen specimen. Until the technique becomes further developed it remains primarily a research tool.
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