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SEMEN ANALYSIS AND OTHER INVESTIGATIONS

Semen Analysis

The most important laboratory investigation in male infertility is semen analysis. The variables assessed and the methods are in the WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction.[73] Automation of semen analysis is in progress and should be used in most specialized laboratories soon.

It is crucial that the laboratory is experienced in the performance of semen analyses, participates in quality assurance activities and has a room nearby for the collection of semen. Semen may be obtained by masturbation or coitus using a special nontoxic condom. Ordinary latex contraceptive condoms are unsatisfactory because the rubber usually immobilizes the sperm.[74] If these methods of collection are not possible, postcoital examination of midcycle cervical mucus may give some information about the likelihood of adequate semen quality if many motile sperm are found. In contrast, a negative postcoital test on its own is of little diagnostic value as conception can occur in the same cycle.[75]

The man should be provided with a wide-mouth, sterile and not toxic collection jar and written instructions about collection and delivery to the laboratory specifying a period of abstinence from ejaculation from two to five days, delivery of the sample to the laboratory within one hour of collection and avoidance of exposure to lubricants or extremes of temperature.
To check for retrograde ejaculation, urine collected immediately after ejaculation is centrifuged and the pellet examined for sperm.

Several semen analyses at intervals of two or more weeks are necessary in a man with an abnormality in the first test because of the variability of results (Fig. 2). Even with complete collection of samples there is variability caused by counting error, other technical errors and differences in the ejaculate from day to day.[73, 76] These large variations need to be remembered when interpreting results of semen analysis.

Figure 2. Variability of semen analysis results in a fertile sperm donor. C, sperm concentration; V, semen volume; M, total motility; MI, motility index-product of grade and percentage of sperm with progressive motility graded 0 to 3. (Mallidis C et al: Variation of semen quality in normal men. Int J Androl 14:99-107, 1991. Used by permission Blackwell Scientific Publications.)

Assays of semen constituents from the accessory glands and testis are available: zinc and acid phosphatase from the prostate, fructose from the seminal vesicles, neutral a-glucosidase, glycerophosphocholine and L-carnitine from the epididymis and inhibin B from the Sertoli cells.79 [77, 78] Prostatic fluid is acid (pH about 6.0), but the ejaculate is alkaline because of the admixture with seminal vesicle fluid. Semen biochemistry is of limited usefulness in clinical practice. Some examples are given in Table 3.

Immunobead test
Tests for sperm antibodies should be done routinely on all men being evaluated for infertility because no semen analysis pattern is characteristic of sperm autoimmunity.79 The immunobead test (IBT) with beads binding to more than 50 per cent of motile sperm is regarded as positive, but there usually is more than 70 to 80 per cent IgA binding with clinically significant sperm autoimmunity. Tail-tip only IBT binding is not significant.[73] The indirect IBT in which normal donor sperm are exposed to test serum or seminal plasma can be used to test men with too few motile sperm for the direct IBT. An alternative screening method for men with sperm in the semen would be to perform a sperm-mucus penetration test.[73] The mixed antiglobulin reaction test is an alternative to the IBT.[73]

Sperm-mucus penetration tests can be performed by postcoital examination of sperm in cervical mucus collected at midcycle or after estrogen treatment (ethinyl estradiol, 50 mg twice daily for four days) to produce mucus of equivalent quality.[73] In vitro capillary mucus penetration (Kremer) tests are particularly important for evaluating the significance of sperm autoantibodies; failure of sperm to penetrate more than 2 cm in one hour indicates severe sperm autoimmunity with a poor prognosis if untreated.[22]

Sperm function tests
A number of tests of sperm function are available to examine the human fertilization process (Fig. 3). These are only performed in specialist laboratories. If simpler approaches or active preparations of zona pellucida (ZP) or sperm receptor proteins become available they will be widely used to improve the assessment of human sperm. IVF has permitted many conventional and new tests of sperm function to be examined. Groups of sperm variables that are independently significantly related to the proportion of oocytes that fertilize in vitro can be determined by regression analysis.[79] This approach has confirmed the importance of sperm morphology and the ability of sperm to interact with the coverings of the oocyte.

Human Sperm-Oocyte Interaction

Figure 3. Stages of human fertilization. Spermatozoa swim through the surrounding medium and cumulus mass (not shown) and bind to the surface of the zona pellucida. The acrosome reaction is stimulated by zona proteins and the acrosome reacted sperm penetrates the zona, enters the perivitelline space and binds to the oolemma via the equatorial segment. Oocyte processes surround the sperm head and it enters the ooplasm and decondenses. Infertility could result from defects of any of these processes. For example, abnormal sperm particularly with defective head morphology bind poorly to the zona.

HUMAN SPERM-ZP BINDING RATIO TEST. Because the number of sperm bound to the ZP is strongly related to the fertilization rate, human sperm-ZP interaction tests have been developed using oocytes that failed to fertilize in vitro.[73, 79, 80]These oocytes can be used either fresh or after storage in concentrated salt solutions.[80] Because the ZP binding capacity is variable, control (fertile donor) and test sperm are labeled with different fluorochromes (fluorescein and rhodamine). After incubation with equal numbers of control and test sperm, the oocytes are aspirated through a wide bore pipet to dislodge loosely adherent sperm and the numbers of sperm tightly bound to the ZP are counted with a fluorescence microscope. Results are expressed as a ratio of binding to the ZP of test and control sperm for four oocytes.[79] An alternative method is to cut the zonae in half and expose each to test and control sperm (Hemizona assay).[80]

HUMAN SPERM-ZP PENETRATION TEST. It is difficult to determine the number of sperm penetrating into the ZP when many sperm are bound to the surface. The sperm bound to the surface of the ZP can be sheared off by repeatedly aspirating the oocyte with a pipet with internal diameter less than the diameter of the oocyte (120 ?m). The sperm penetrating into the ZP or perivitelline space can be then counted easily and the results of this test are the most predictive of fertilization rates with standard IVF.[81]

ZP-INDUCED ACROSOME REACTION TEST. Sperm dislodged from the ZP can be stained with a fluorescein labeled lectin such as pisum sativum agglutinin or an antibody specific for the acrosomal contents to determine the proportion that are acrosome reacted. This test is useful for diagnosing disordered ZP induced acrosome reaction.[82]

HUMAN SPERM-OOLEMMA BINDING RATIO TEST. Sperm-oolemma binding has been studied in the same way as the sperm-ZP binding test but using oocytes that have had the ZP removed.[79]

ZONA FREE HAMSTER OOCYTE PENETRATION TEST. In a number of countries human sperm-zona free hamster oocyte penetration tests are performed to assess the ability of sperm to capacitate, acrosome react, fuse with the oolemma, and undergo nuclear decondensation in the ooplasm.[73] This test does not evaluate sperm interaction with the ZP.

Interpretation of Semen Analysis Results
Table 3 shows various patterns of abnormality of semen quality and their common causes. It is always important to ask the question, is the result spurious? Were there any problems with collection? Repeated tests are necessary to establish an average and to determine the variability within an individual man (Fig. 2).

VARIATIONS IN SEMEN VOLUME AND APPEARANCE. Low semen volume suggests incomplete collection, short duration of abstinence from ejaculation before the test, absence or obstruction of the seminal vesicles or androgen deficiency. High semen volume (>8 ml) may be seen in association with oligospermia but is of little practical significance. Hemospermia is usually is the result of minor bleeding from the urethra but serious conditions, such as genital tract tumors must be excluded. Other discoloration of the semen may indicate inflammation of accessory sex organs. The semen may be yellow with jaundice or salazopyrine administration. Defects of liquefaction and viscosity are relatively common and presumably result from malfunction of the accessory sex organs.[5] Although these may cause problems with semen analysis and preparation of sperm for ART, they are probably of little relevance to fertility. Sperm agglutination is common with sperm autoimmunity but can also occur for other reasons.

AZOOSPERMIA. The total absence of sperm from the semen needs to be confirmed in repeated tests with vigorous centrifugation of the semen and careful examination of the pellet.[73] Rarely an illness or difficulty with collection will cause transient azoospermia. With severe spermatogenic disorders and some obstructions sperm may be present in the semen intermittently. If any live sperm can be found, these can be cryopreserved for intracytoplasmic sperm injection (ICSI).

OLIGOSPERMIA. Sperm concentrations of less than 20 million/ml are classified as oligospermic.[73] This figure probably derives mainly from the work of MacLeod, who found that only 5 per cent of fertile men had sperm concentrations less than 20 million/ml.[83] There is a correlation between sperm concentration and other aspects of semen quality, such as percentage motility and normal morphology. Both motility and morphology are usually poor with oligospermia.

ASTHENOSPERMIA. Asthenospermia is defined as a sperm motility of less than 50 per cent total or less than 25 per cent with rapid progressive motility.[73] Spurious asthenospermia because of exposure of sperm to rubber (particularly condoms), spermicides, extremes of temperature, or long delays between collection and examination should be excluded before accepting that sperm motility is poor. Low sperm motility is a frequent accompaniment of oligospermia, and there usually is a mixed picture of morphological defects. It presumably arises because of defective spermiogenesis.

Severe asthenospermia requires evaluation by electron microscopy.[33, 34, 84] Specific ultrastructural defects of the axoneme are associated with zero sperm motility or extreme asthenospermia (less than 5 per cent motile sperm) and sterility. Absent dynein arms, other less common axonemal defects, mitochondrial abnormalities, disorganized fibrous sheath or outer dense fibres with stumpy tails, or normal ultrastructure may be found.[33, 34, 84] The gene defects in some of these disorders are being discovered.[85] Standard semen analyses of these patients usually show normal sperm concentrations and normal sperm morphology, although some have tail abnormalities at the light microscopic level-short, straight, or thick tails or mid-piece defects.

Viability tests help to distinguish this group of patients from those with necrospermia.[86] Patients with structural defects in the sperm that cause complete immotility are untreatable and sterile except for ICSI.

Asthenospermia may also be associated with sperm autoimmunity. The causes of other motility defects of moderate degree are unidentified. Some suggested abnormalities of protein carboxylmethylase, lactate dehydrogenase C4, and energy generation are unconfirmed and contentious.[87, 88]

NECROSPERMIA. Necrospermia is important to distinguish from other types of severe asthenospermia because some patients produce pregnancies despite the low sperm motility.[86]

Necrospermia is characterised by usually less than 20-30 per cent total motility, less than 5 per cent progressive motility and a viability test less than 30-40 per cent indicating a high proportion of dead sperm. The condition may fluctuate in severity, particularly with changes in coital frequency.

Necrospermia may be caused by defective storage of sperm in the tails of the epididymides or stasis in the genital tract.[89] There are ultrastructural features of degeneration in the ejaculated sperm but normal structure of late spermatids in testicular biopsies.[86, 89] Characteristic of necrospermia is an improvement of sperm motility with increased frequency of ejaculation. Treatment with antibiotics may have a beneficial effect, but this is not proved. The couple should have intercourse once or twice every day for three to four days up to the time of ovulation.

TERATOSPERMIA. If there is a reduced percentage of sperm with normal morphology assessed by light microscopy, the classification of teratospermia is used.[73] The microscopic assessment of sperm morphology is highly subjective and difficult to standardize between laboratories. Various approaches to morphology are used. The simplest is to assign defects with a priority in order head, mid-piece, and tail, and to record as normal only those that conform to ideal shape with no defects in any region. In the Strict morphology approach, marginally abnormal sperm are assigned abnormal. Differential counts give the proportions of abnormal sperm with large, small, tapered, pyriform, or amorphous heads; normal heads but mid-piece defects; or normal heads and mid-pieces but abnormal tails.[73] Indices based on the average number of defects per spermatozoon are also used (teratospermia index).[73] Automated methods involving image analysis by computer are being developed which promise to overcome the between-laboratory variability and greatly improve the predictive value of semen analysis for natural conception.[90]

The percentage of sperm with normal morphology assessed by strict criteria after washing the sperm and adjusting the concentration to 80 million/ml provides one of the most useful predictors of IVF rates. There is a progressive reduction in fertilization rates from 60 per cent to 20 per cent as abnormal morphology increases from less than 70 per cent to more than 95 per cent.[79] Patients with high proportions of sperm with abnormal morphology should have ICSI because of the risk of failure of fertilization with standard IVF. ICSI results are independent of sperm morphology.
It is important to distinguish mixed abnormalities of sperm morphology from those in which all or the majority of sperm show a single uniform defect, such as spherical heads with absence of the acrosomes and pin head sperm, which are produced by defective spermiogenesis. Pin-head sperm result when the centrioles from which the sperm tails develop are not correctly aligned opposite the developing acrosome so that the sperm heads are lost and absorbed during epididymal transit.[84] Both these conditions are extremely rare.

Hormone Assessment

It is not necessary to perform hormone measurements routinely. Follicle-stimulating hormone (FSH) levels in patients with azoospermia, normal testicular volume and normal virilization may help distinguish genital tract obstruction from a spermatogenic disorder. But some men with primary seminiferous tubal failure have normal FSH levels. Normal FSH is common with germ cell arrest at the primary spermatocyte stage. Rarely, high FSH levels are seen with normal spermatogenesis.[91, 92] Measurement of FSH, luteinizing hormone (LH), and testosterone is useful in men with reduced testicular volume and signs of androgen deficiency to distinguish primary from secondary hypogonadism. Inhibin B measurement may provide additional information about the state of spermatogenesis.[93, 94] Isolated FSH deficiency due to mutations in the FSH b gene have been described.[95, 96]

Prolactin should be measured in men with galactorrhea or androgen deficiency and loss of libido.[67, 68] Other hormone investigations occasionally are required, for example, thyroid function tests with hyperprolactinemia, 17-hydroxyprogesterone measurements with congenital adrenal hyperplasia, estradiol with liver disease or tumors, human chorionic gonadotropin (hCG) with tumors and estrogen excess, and pituitary function tests for panhypopituitarism.[25-27]

Genetic Studies

Karyotypes are performed in men with clinical evidence of primary testicular failure and small testes to confirm a clinical diagnosis of Klinefelter syndrome, in which the karyotype usually is 47,XXY, but there may be higher numbers of X chromosomes or a sex-reversal 46,XX karyotype.[97-101] While most men with Klinefelter syndrome produce no sperm some are oligospermic and very rarely, fertile.[101] Also sperm for ICSI may be obtained by testicular biopsy.[100] Defective spermatogenesis may occur with 47,XYY but the clinical picture is much less uniform than it is for Klinefelter syndrome.[98, 99] It appears that the extra sex chromosome is deleted early in gametogenesis as the embryos and children generally have normal karyotypes.[99-101] However an increased rate of sex chromosomal aneuploidy has been noted in some studies of sperm from XXY and XYY men.[99, 101] Because of the increased frequency of autosomal abnormalities: reciprocal and Robertsonian translocations and inversions, in patients with defective spermatogenesis and the risk of transmitting these in the unbalanced form in offspring, karyotypes should be performed before treatment in all men with moderate to severe oligospermia (for example average sperm concentrations less than 5 million per milliliter) of primary testicular origin.[99]

Cystic fibrosis gene studies are important for evaluation of patients with congenital absence of the vas and their partners.[8, 15-18] If the woman has a CF gene mutation, preimplantation genetic diagnosis of their embryos can be offered. [17] Microdeletions in the long arm of the Y chromosome have been found in 5-15% of men with severe primary spermatogenic disorders.[8, 19-21] Sons of men with these microdeletions have been found to have the same microdeletions.[8] Androgen receptor defects have also been found in some men with unexplained primary spermatogenic disorders.[8] Mutations in the gene impairing androgen receptor activity produce androgen insensitivity which has a variable phenotypic expression from testicular feminization to otherwise normal males with gynecomastia or hypospermatogenesis and oligospermia. [102, 103] Increases in the number of CAG repeats in exon 1 over about 40 are associated with Kennedy disease - progressive spinobulbar atrophy and men with this condition may be infertile. It is considered that the number of CAG repeats has an inverse effect on the activity of the androgen receptor. Several studies of the CAG repeat numbers in men with primary spermatogenic defects have indicated significant increases in repeat number compared with those in controls although the numbers of repeats are within the normal range.[104, 105] However this has not been confirmed in all studies.[106] A relationship between lower sperm concentration and longer CAG tracts in normal men has also been reported.[107] Whether increasing intratesticular testosterone levels would increase spermatogenesis in men with underactive androgen receptors remains to be determined.
Other specific genetic tests and family studies may be indicated on clinical grounds (Table 2) for example: Kallmann syndrome, FSH and FSH receptor mutations, myotonic dystrophy, mitochondrial gene mutations and defects of sperm ultrastructure.[8-10, 25, 26, 31, 33, 34, 85, 108, 109] At present it is not clear what genetic disorders should be screened for in infertile men. Patients should be counseled about the possibility of transmitting known and unknown genetic defects.

Testicular Biopsy

Testicular biopsies are necessary to assess spermatogenesis in men with presumed genital tract obstruction. A significant proportion of men with azoospermia, normal testicular size, and normal FSH, are found to have severe spermatogenic disorders.[7] Some severe spermatogenic defects may be incomplete and as ICSI can be performed with a few sperm obtained from the testes, diagnostic testicular biopsies can be offered to men with severe primary testicular failure with persistent azoospermia. If any tubules containing elongated spermatids can be found it should be possible to perform ICSI. However, if no areas of spermatogenesis to this stage are seen in the diagnostic biopsies there is less chance that more extensive removal of testicular tissue for sperm retrieval from the testes will be successful.[110]

Open biopsies may be performed under local or general anesthesia. It is most important that the tissue be removed from the testes with minimal damage and placed in a suitable fixative, such as Bouin or Steive's solution. Standard formalin fixatives destroy the cytoachitecture. Needle biopsy procedures have become popular, and although many obtain only isolated cells, these cells may be sufficient for diagnosis on the basis of cytology or for flow cytometry assessment. A technique for obtaining small amounts of tissue by needle aspiration biopsy under local anesthesia has been developed (Fig. 4) that usually provides sufficient material for a histological diagnosis of the state of the seminiferous epithelium.[111] The aspiration biopsy techniques do produce some deformation artefacts in the tissue. Provided there is not a severe spermatogenic defect, needle aspiration biopsy is useful for obtaining testicular sperm for ICSI.[112] Complications of this procedure include failure to obtain tissue particularly with very small or fibrosed testes (<5%), minor bleeding in the skin and testis rarely causing swelling. Some men may faint from anxiety about the procedure. Reactions to the local anesthetic are rare.

Figure 4. (A) Fine-needle tissue aspiration biopsy of the testis. Local anesthetic is injected around the vas to block testicular sensation. (B) Fine-needle tissue aspiration biopsy of the testis A 21 gauge butterfly needle is inserted into the testis. An assistant applies suction to the needle tubing via a 10mL syringe and the operator makes thrusting movements of the needle into the substance of the testis. (C) Fine-needle tissue aspiration biopsy of the testis While maintaining the suction the needle is removed carefully and any seminiferous tubules protruding from the needle are grasped with fine forceps to avoid them falling back into the puncture hole. With this technique seminiferous tubule sections are sucked into the needle and these are expelled into some culture medium. Portions can be sent for histology and the remainder used for extraction of sperm in the IVF laboratory by stripping the seminiferous tissue out of the connective tissue membrane of the seminiferous tubule.

For clinical purposes, testicular histology is classified as follows: normal or hypospermatogenesis (all the cellular elements of spermatogenesis are present but in reduced numbers), germ cell arrest (the initial cellular elements of spermatogenesis are present but at a certain stage the process stops, most often at the primary spermatocytes), Sertoli cell-only syndrome or germ cell aplasia (the tubules contain Sertoli cells but no germ cells), hyalinization (the cellular elements have disappeared leaving only thickened seminiferous tubules walls as in Klinefelter syndrome), and immature testis (no gonadotropin stimulation, prepubertal appearance).[113] Examples are shown in Fig. 5.

Figure 5A. Testicular histology from fine-needle aspiration samples. Normal.

Figure 5B. Testicular histology from fine-needle aspiration samples. Left mild hypospermatogenesis with elongated spermatids with poor head morphology: right normal.

Figure 5C. Testicular histology from fine-needle aspiration samples. Mild-moderate hypospermatogenesis.

Figure 5D. Testicular histology from fine-needle aspiration samples. Moderate-severe hypospermatogenesis.

Figure 5E. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the primary spermatocyte stage.

Figure 5F. Testicular histology from fine-needle aspiration samples. Sertoli cell-only syndrome: low and high power.

Figure 5G. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the spermatogonial stage from gonadotropin deficiency. Upper panel atrophic Leydig cell stained with antitestosterone antibody.

Figure 5H. Testicular histology from fine-needle aspiration samples. Carcinoma in situ, only transformed spermatogonia and Sertoli cells present.

Other Investigations

Ultrasonography is useful for checking for tumors in the testes, particularly when the testes are difficult to palpate because of a tense hydrocele. It can also be used to measure testicular size and confirm the presence and nature of cysts or other abnormalities in the scrotum.[114] Doppler blood flow assessment is valuable in assessing a painful swollen testis for torsion or inflammation and for evaluating varicoceles. Other tests of a varicocele, including thermography, technetium scans, and venography, may be performed but as pointed out below the value of treating varicoceles to improve fertility is uncertain. Rectal ultrasound may demonstrate cysts in the prostate, enlarged seminal vesicles or dilated ejaculatory ducts associated with distal genital tract obstructions.[115] Clinical suspicion of the presence of a pituitary tumor should be followed up by appropriate radiology. Abdominal imaging is necessary to check the position of an impalpable testis.