Semen Analysis

13 Semen Analysis

Bengt Fredricsson KarolinskaInstitute, Huddinge University Hospital, Stockholm, Sweden

The analysis of semen gives limited but useful information about the hormonal condition of the individual. Many steps are involved between the endocrine prerequisites and the end product, and these steps may be disturbed by several conditions (e.g., anatomical, developmental, infectious, and environmental). Nevertheless, patients with a normal sperm density seldom have disorders of the hypothalamic-pituitary-gonadal axis and a simple semen analysis is often easier to accomplish than more sophisticated endocrine tests. This is not the place to give a detailed description of the endocrinology and hormonal regulation of the functions of the testes and the accessory genital organs. The reader is referred to the chapter on gonadal disorders and to appropriate surveys (e.g., Refs. 1,2). In summary, the Sertoli cell is stimulated by follicle stimulating hormone (FSH) and the Ley dig cell by luteinizing hormone (LH). Inhibin and testosterone are responsible for the respective negative feedback control loops. Testicular steroidogenesis is not only quantitatively but also qualitatively under gonadotrophic regulation (3). Although FSH stimulates the seminiferous tubules, the direct effect of this hormone seems to be restricted to the Sertoli cells. The germinative cells do not themselves produce any known substance exerting feedback on FSH production. High FSH levels characteristic of absence of germinative cells may be due to concurrent damage to the Sertoli cells. However, high intratesticular levels of testosterone are needed for spermatogenesis, in which prolactin also may play a role. Testosterone is transported to the seminiferous epithelium via the Sertoli cells. Damage to the blood-testis barrier seems to involve damage also to the Sertoli cells and hence results in disturbed testicular testosterone transport. A 133

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defective blood-testis barrier may also result in the formation of antispermatozoal antibodies. The constituents of semen are for the major part derived from the accessory sex organs, mainly the seminal vesicles, which contribute about two-thirds. About one-fifth, in general the first part of the ejaculate, is derived from the epididymis. Hence the content of spermatozoa differs considerably between different portions of the ejaculate, in general being highest in the first. The process from the first meiotic division to the appearance of the resulting spermatozoa in the ejaculate may take 10 weeks or more, a period that includes morphologic and functional maturation and storage within the epididymis. An unsatisfactory semen sample may therefore be due to interference from disease or other events during this period. This must be considered by the clinician and multiple samples over a prolonged period may be required. Semen analysis is most often requested because of infertility. Although selfevident, it should be stressed that the result pertains to the particular sample and does not implicate the retrospective or prospective fertility of the couple or the man and is not a diagnosis in itself. A poor result should be explained by further investigation if possible. A proper evaluation of the results needs sampling and analysis under standardized and well-defined conditions. A manual for the examination of human semen has recently been elaborated within the WHO (4). Some additional comments will be given here for the benefit of the clinician. I. COLLECTION OF SAMPLE A. Abstinence

The period of abstinence before sampling should correspond to the coital activity of the couple (i.e., in general 3-5 days). Long periods of abstinence may impose a higher degree of uncertainty to the evaluation than short periods. B. Collection of the Semen Sample

The sample should preferably be produced by masturbation and the complete ejaculate collected. The different portions of the ejaculate differ in composition. Collection by coitus interruptus may be incomplete and is therefore inappropriate. The use of condoms is condemned as they may affect the spermatozoa, in particular their motility. C. Transport to the Laboratory

For the study of sperm count, motility, and morphology the ejaculate may be produced at home and delivered to the laboratory within a few hours. For

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special studies the patient may have to produce the sample at the laboratory. The sample should be protected against cooling below room temperature at least before assessment of sperm motility. Patients living far away from the laboratory may deliver the sample to the nearby clinic, where motility and concentration is assessed and permanent preparations for the study of morphology and vitality can be made. The sample may be centrifuged and the seminal plasma frozen and transported when special tests are requested. In a small number of cases the patient is not able or willing to produce a sample and a postcoital test will then give indirect information which may be of value. In cases with aspermia (no semen), retrograde ejaculation may be the cause. The bladder contents should then be examined after orgasm has been achieved. Spermatozoa are as a rule immobilized in the urine unless this has been made alkaline (e.g., by previous oral intake of sodium bicarbonate). II. ANALYSIS A. Physical Characteristics

The volume of the sample is recorded. The presence of particulate material, inhomogeneity, abnormal color, incomplete liquefaction, or increased viscosity should be recorded. There are no simple methods to measure these variables objectively. Complete liquefaction should normally be achieved within 20 min from ejaculation. B. Motility

The relative number of motile spermatozoa is evaluated in the microscope as is progressive motility. The evaluation is subjective and therefore often inaccurate (5, 6). The problem with the subjective evaluation can be overcome by employing photographic or other objective methods (7-9). The presence and type of spermatozoal agglutination (head to head, tail to tail, or mixed) should be recorded. C. Sperm Concentration

Sperm concentration is determined in a hemocytometer after appropriate dilution in an immobilizing medium such as 0.4% formaldehyde (1 part 40% formalin in 100 parts distilled water), preferably brought to a neutral pH. D. Morphology

Seminal smears are prepared. After fixation in ethanol, spray-cyte, or equivalent they can be stored and later stained using the method of Papanicolaou or similar

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staining technique. A differential morphologic count at high magnification (oil immersion lens) implies a subjective evaluation, and both normal range and clinical interpretation show local variation. The sperm head, midpiece, and tail should be examined. Different taxonomies are employed. In general practice, abnormalities of the head are allowed to "cover" abnormalities within midpieces and tails. The sum of the frequencies of the different abnormalities and normal spermatozoa is then 100%. It has been advocated, however, that the frequency of each type of abnormality is more of interest and therefore should be recorded irrespective of other abnormalities simultaneously present. In this way the sum of frequencies will exceed 100% (10). E. Leukocytes

Leukocytes are frequently present. Their occurrence is best determined by counting their number per 100 spermatozoa in the stained smear. Knowing the sperm concentration, the number per cubic millimeter is easily calculated. F. Vitality

Determination of sperm vitality may sometimes be of interest. This depends on the fact that all immotile spermatozoa may not be dead. Discrepancy between the figures of motility and vitality may have biochemical or immunological reasons. Vitality is easily determined after supravital staining with eosin in a buffered solution (11). Only dead spermatozoa take up the stain. G. Biochemical Tests

Biochemical tests may be of value in selected cases. Fructose and prostaglandins are excreted from the seminal vesicles, acid phosphatase, citric acid, Mg and Zn from the prostate, glycerolphosphorylcholine, and carnitine from the epididymis. Although the secretory functions of these organs are hormonally controlled, interference from irregular emissions, infection, and benign prostatic hyperplasia renders their analysis of no value in the endocrine evaluation of the individual. III. INTERPRETATION A. General Comments

It should be remembered that the values of analysis relate to a particular sample and not necessarily to the individual's reproductive capability. The implication of normal or abnormal values in the strict sense may therefore be misleading. Normal does not mean fertile. The reference ranges given here (Table 1) are

Semen Analysis /137 Table 1 Evaluation of Semen Findings

Normal Volume (ml) Sperm concentration (millions/ml)

Abnormal

2-6

<1.5

20-250

Total sperm number (millions)

>80

<10 <20

Motile spermatozoa exhibiting good progression Morphology (spermatozoa of normal configuration, %) Acid phosphatasea (mkat/liter)

>50

<30

>40

<30

>6.9

<4.2

a

Zinc (mmol/liter) Magnesiuma (mmol/liter) Fructosea (mmol/liter)

1.2-3.8 2.9-10.3 6.7-33

<0.8 <2.1 <4.4

a

Biochemical borders are valid only if volume is normal. Source: Modified from Ref. 10.

those generally employed. They are based on studies of samples delivered within a reasonable time of conception. However, the results in infertile males extend very much into the fertile reference range. This means that discrimination between fertile and infertile patients is often not possible by the variables of semen analysis. The reference ranges given indicate instead the cases in which further investigation or particular consideration may be worthwhile. Findings in the interval between the normal and abnormal values of the table are often difficult to explain clinically. If a reproductive problem is present, in general more than one semen sample should be analyzed covering a period of at least 10 weeks, whereby proper consideration is given to the length of the spermatogenic cycle. Additional information of considerable clinical value is gained by penetration tests in vivo (the postcoital test) and in vitro (see below). B. Volume

The major part of the ejaculate is derived from the seminal vesicles, and from the prostate gland to a lesser degree. Small volumes may indicate disorders of these glands but seldom have reproductive significance. However, in a few cases with retroflection of the uterus or a wide, voluminous vagina (e.g., after surgical removal of the rectum), a small volume may become crucial. A normal postcoital test will rule out this problem. If this test shows no penetration of spermatozoa

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into the cervical secretion, treatment by insemination with husband's semen is logical. C. Sperm Concentration

There are indications of a decline of the sperm counts of fertile men during the past decades (12). However, sperm density shows very considerable intraindividual and interindividual variation (4, 13). Paternity has been proven in occasional cases with sperm counts persistently in the order of 1 million per milliliter. D. Total Sperm Number

The total number of spermatozoa may be of greater clinical significance than the concentration, although proper documentation of this statement seems to be lacking. E. Motility

Poor motility reduce the chances of conception significantly. The cause may be extrinsic or intrinsic. Varicocele, prostatovesiculitis, and environmental factors should be ruled out. Agglutinating or immobilizing antibodies may be present, particularly if progressive motility is poor or if vitality is much better than motility. No motile spermatozoa in spite of normal degree of vitality may indicate the presence of the immotile cilia syndrome (see below). F. Morphology

The study of sperm morphology shows considerable local variation due to the subjective evaluation. Studies of sperm ultrastructure indicate that aberrations are more frequent than previously anticipated (14). If spermatozoa of normal appearance are few, the supravitally stained preparation can be used to assess the frequency of normal and abnormal spermatozoa within the live sperm population (15, 16). The photomicrographic technic of Makler may be used for the same purpose (17). In general, the clinical significance of the different types of abnormality is not known. The presence of increased numbers of tapered sperm heads is considered to indicate stress to spermatogenesis (e.g., caused by infections, or associated with varicocele). Round sperm heads affecting all spermatozoa indicates a constitutional abnormality which imports sterility and cannot be treated (18). Increased numbers of coiled tails also reduces the fertilizing capacity of the semen (19). If all spermatozoa are immotile although the cells are alive, this may indicate the presence of the immotile cilia syndrome. This condition cannot be treated.

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It is due to absence of the dynein arms of the cilia and the diagnosis can only be settled by electron microscopy (20, 21). The condition also results in difficulties of clearing the bronchial tree, resulting in chronic bronchitis and bronchiectasis. About 50% of the cases also have situs inversus. IV. COMMENTS A. Aspermia

Failure to deliver a semen sample may indicate cohabitation difficulties but may also be due to side effects of medical treatment, or endocrine or metabolic abnormality (e.g., hyperprolactinemia may disturb male sexual potency). Aspermia, no visible ejaculate in spite of orgasm, may indicate retrograde ejaculation, which in turn may have very different causes which merit thorough investigation. Motile spermatozoa may be recovered from the bladder as described above. These spermatozoa may then be used for insemination (22, 23). B. Azoospermia

Azoospermia means complete absence of spermatozoa in the ejaculate. A congenital or acquired block of the excretory duct system is not uncommon. The endocrine balance is not disturbed and a testicular biopsy will reveal normal spermatogenesis. Defects within the pelvis affecting the ejaculatory duct and associated structures are as a rule characterized by the absence of fructose (derived from the seminal vesicles) in the seminal fluid. High serum levels of FSH indicate the absence of seminiferous epithelium either due to secondary damage (e.g., previous mumps orchitis or congenital germ cell aplasia such as in Klinefelter's syndrome). This syndrome is frequently diagnosed first after the finding of azoospermia in the infertility investigation. C. Low Sperm Counts (Oligozoospermia)

Patients with very low counts should be clinically examined and subjected to endocrine investigation. However, hormone estimations seem not to be helpful in patients with a sperm density exceeding 5 million per milliliter (24). The value of testicular biopsy remains to be proven, but it offers the possibility of studying the pattern of testicular steroidogenesis, which may be of interest not only in hypogonadal states (3, 25). D. Normal Sperm Count

Moderate disturbances of thyroid and adrenal function, of glucose metabolism (diabetes), and of liver function may interfere with testicular function to a

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degree which may not be clinically overt or even indicated by commonly employed laboratory tests. E. Functional Tests

There has been some concern about the significance of the apparently excessive numbers of spermatozoa in the ejaculate. No doubt, a significant number of spermatozoa are either immotile or dead, or anatomically or functionally defective. The crucial point is that no reliable criterion can be ascribed to the spermatozoon capable of fertilization. Functional tests have therefore been elaborated, of these the postcoital test and in vitro penetration tests are more or less indispensable in the infertility investigation. No penetration of spermatozoa into a favorable cervical secretion suggests a poor prognosis for fertility and may be due to antispermatozoal antibodies within either man or woman. Good morphology of those spermatozoa which are found in the cervical secretion close to the external os speaks against a male cause of infertility (16, 26). In vitro penetration into cervical mucus may be assessed quantitatively under standardized conditions (27). Cross-checking employing, in addition, donor semen and donor mucus will rule out if a poor result is due to a male or female factor. If bovine mucus (27, 28) or an artificial medium (30) is used, the test will be exclusively focused on spermatozoal function. However, these media are still under study and not yet recommended for general use. Human spermatozoa are under certain conditions capable of penetrating zonafree hamster eggs, and it has been suggested that such a test can be used to characterize the fertilizing capacity of human spermatozoa (31-35). This test may be a valuable preliminary before attempting human in vitro fertilization. REFERENCES

1. 2. 3. 4.

Burger HG, deKretser D, Hudson B. Spermatogenesis and its endocrine control. In Human Semen and Fertility Regulation in Men, Hafez ESE (ed). Mosby, St Louis, 1976, pp 3-16. diZerega GS, Sherins RJ. Endocrine control of adult testicular function. In Comprehensive Endocrinology, The Testis, Burger H, deKretser D (eds). Raven, New York, 1981, pp 127-140. Berg AA, Kjessler B. Effect of long term treatment with gonadotrophic hormones on in vitro metabolism of (3H) progesterone in human testicular tissue. Int J Androl 1:53-60, 1978. Belsey MA, Eliasson R, Gallegos AJ, Mogjiissi KS, Paulsen CA, Prasad MRN. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. WHO, Geneva/Press Concern, Singapore, 1980.

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Wu, FCW, Edmond P, Raab G, Hunter WM. Endocrine assessment of the sub fertile male. Clin Endocrinol 1981; 14:493-507. hredncsson B, Carlstrom K. Human testicular disorders: serum and urinary hormone patterns and in vitro conversion of (21-14C,7-3H)progesterone. Int J Androl 1979; 2:432-448. Fredricsson B, Bjork G. Morphology of postcoital spermatozoa in the cervical secretion and its clinical significance. Fertil Steril 1977; 28:841-845. Ulstein M, Fjallbrant B. In vitro tests of sperm penetration in cervical mucus. In Human Semen and Fertility Regulation in Men, Hafez ESE (ed). Mosby, St Louis, 1976, pp 383-388. Gaddum-Rosse P, Blandau RJ, Lee WI. Sperm penetration into cervical mucus in vitro. II. Human spermatozoa in bovine mucus. Fertil Steril 1980;33:644-648. Mangione CM, Medley NE, Menge AC. Studies on the use of estrous bovine cervical mucus in the human sperm-cervical mucus penetration technique. Int J Fertil 1981;26:20-24. Lorton SP, Kummerfeld HL, Foote RH. Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil Steril 1981; 35:222-225. Barros C, Gonzales J, Herrera L, Bustos-Obregon E. Human sperm penetration into zona-free hamster oocytes as a test to evaluate the sperm fertilizing ability. Andrologia 1979; 11:197-210. Binor Z, Sokoloski JE, Wolf DP. Penetration of the zona-free hamster egg by human sperm. Fertil Steril 1980; 33:321-327. Overstreet JW, Yanagamachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human zona pellucida and the zonafree hamster egg: a study of fertile donors and infertile patients. Fertil Steril 1980;33:534-542. Hall JL. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 1980; 35:457463. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SE, Smith WD. Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 1981; 57:620-623.