Basic diagnostics in andrology


  • Sonja Grunewald,

    Corresponding author
    • Clinic and Polyclinic for Dermatology, Venereology and Allergology, University Hospital Leipzig AöR and Leipzig Medical School of the University of Leipzig, Germany
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  • Uwe Paasch

    1. Clinic and Polyclinic for Dermatology, Venereology and Allergology, University Hospital Leipzig AöR and Leipzig Medical School of the University of Leipzig, Germany
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  • Conflicts of interest None.

  • Section Editor Prof. Dr. Jan C. Simon, Leipzig

  • Definition of infertility: failure to conceive after 12 months of regular unprotected intercourse.

  • The age of the woman is a key factor in the fertility status of a couple.

  • The patient history and couple's history should be elicited in a quiet atmosphere. The patient's partner should also be present, if possible.

  • Important points to recall when taking the patient's history are: how long the couple has been unable to conceive; any diseases that may affect fertility; previous and current medication use; exposure to toxic substances; and sex life.

  • In men, waist circumference and BMI are clinically relevant to hormonal status and fertility.

  • The testes and epididymis exhibit a homogeneous echo structure. Inhomogeneity is usually a sign of inflammation; in the testes, it is also correlated with a hematoma or an early-stage tumor.

  • Testicular microcalcifications may indicate an early-stage testicular tumor. Frequent clinical controls, and if the patient has additional risk factors, a testicular biopsy, are warranted.

  • Testicular tumors are echo-poor or “mixed echo” lesions in the parenchyma. Staging and therapy should be performed by an urologist.

  • Hydrocele is an accumulation of serous fluid in the tunica vaginalis or in the spermatic cord (funiculocele). Treatment is usually done for cosmetic reasons.

  • Spermatoceles are very common – and usually harmless – retention cysts on the epididymis.

  • Varicocele refers to enlargement of the veins of the pampiniform venous plexus. There are four degrees of severity. A varicocelectomy improves the quality of sperm and may have a positive effect on fertility.

  • A transrectal ultrasound can aid in the diagnosis of prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer. It may also be useful, given detection of prostate gland or seminal vesicle cysts, for the differential diagnosis of occlusive azoospermia.

  • A spermiogram is central to fertility testing as part of andrological diagnosis.

  • In the new WHO laboratory manual from 2010, the reference values for all standard spermiogram parameters were corrected using an evidence-based approach.

  • Normozoospermia: normal ejaculate parameters

  • Oligozoospermia: low sperm concentration

  • Asthenozoospermia: reduced motility

  • Teratozoospermia: reduced percentage of morphologically normal sperm

  • Oligoasthenoteratozoospermia: density, motility, and morphology are pathological

  • Azoospermia: no sperm in the semen

  • Aspermia: no semen

  • The detection of intact, normomorphic, and motile sperm rules out sterility.

  • The liquidation time of semen is about 30 minutes at 37 °C; it is longer in patients with seminal viscopathy.

  • Volume is determined indirectly by measuring the weight of the ejaculate in a collecting tray (ejaculate density of 1 g/ml).

  • Microscopic analysis begins with an unstained specimen.

  • Sperm concentrations are obtained by counting in counting chamber. If no sperm are detected, centrifugation of the entire ejaculate is performed and the pellet examined.

  • The analysis of sperm motility has been simplified in the 2010 WHO laboratory manual. The former categories (“fast” and slow progressive motility”) were combined into a category called “progressive motility.” This results in fewer errors, but the loss of information is controversially discussed.

  • The change in the evaluation method for sperm morphology by adopting “strict criteria” has lowered the threshold to ≥ 4 % normomorphic spermatozoa.

  • Eosin staining is used to determine the percentage of vital sperm.

  • Leukocytes damage the sperm contained in semen through oxidative stress. Treatment of an underlying infection, or if there is lacking evidence of one, empirical treatment with anti-inflammatory drugs such as doxycycline and diclofenac, can significantly increase the fertility chances of affected men.

  • In agglutinations and especially immunological infertility, anti-sperm antibodies may be detected using a MAR or an immunobead test.

  • To test for occlusive azoospermia, the functional markers of the epididymis (α-glucosidase) and seminal vesicles (fructose) are used.

  • Common tests of sperm function include computer-assisted semen analysis (CASA) and an assessment of acrosomal status.

  • The assessment of the DNA fragmentation rate has proved to be a valuable part of routine andrological diagnosis.

  • The causes of an elevated DNA fragmentation rate vary. Improvement may be achieved by changing one's lifestyle to reduce oxidative stress affecting the sperm.

  • In clinical practice, the most commonly used tests are TUNEL, SCSA® (most standardized), and acridine orange staining.

  • Based on currently available data, the Halosperm® kit cannot supplant established methods of sperm DNA fragmentation analysis.

  • The guidelines of the German Medical Association on “Quality Assurance of Laboratory Medical Tests” (Rili-BÄK) mandate performing tests in duplicate for the spermiogram parameters concentration, motility, and morphology (at least 200 sperm). Andrological laboratories must take part regularly in internal and external quality controls.

  • Basic andrological hormonal diagnosis includes determining FSH, LH, testosterone, and inhibin B levels, and sometimes serum prolactin. Together with the spermiogram, this can yield information about the patient's fertility reserve.

  • In infertility patients, the diagnostic biopsy of the testis is usually also the treatment. The higher the FSH concentration, or the lower the inhibin-B concentration in serum, the less likely it is that sperm will be found with a testicular biopsy.

  • Karyotyping, detection of Y-chromosomal microdeletions, and a mutation analysis of the CFTR gene may be used for genetic diagnosis in infertile men.

Correspondence to

Priv.-Doz. Dr. med. habil.

Sonja Grunewald

Klinik für Dermatologie,

Venerologie und Allergologie

Universitätsklinikum Leipzig AöR

Philipp-Rosenthal-Straße 23

04103 Leipzig, Germany



Basic andrological diagnosis consists of taking the patient's medical history and the couple's history as well as performing a physical examination including genital ultrasound, spermiogram, and hormonal analysis. If needed, a testicular biopsy and genetic testing may also be performed. Recent studies have shown the effect of lifestyle factors on male fertility. Thus, the patient history and clinical/andrological examinations have been broadened to include information on metabolic disorders like obesity and diabetes mellitus.

The biggest changes occurred with the publication of the fifth edition of the WHO laboratory manual in 2010 and the introduction of a section on semen analysis in the German Medical Association guidelines (RiliBÄK). The reference values for almost all spermiogram parameters were adapted in an evidence-based approach using worldwide prospective population studies. For central parameters such as sperm motility and morphology, the assessment criteria were changed. New independent markers such as sperm DNA fragmentation rate are now routinely used in clinical diagnosis. For German andrological laboratories, there are now mandatory quality assurance measures for semen analysis (in the German “Rili-BÄK” guidelines). These include duplicate testing of all standard semen parameters and inter-laboratory comparison at regular intervals.


In clinical practice, andrological diagnosis is usually done for patients who wish to have children, or who have erectile dysfunction or ejaculation problems, or for a malignancy (for preparing and performing cryoconservation), or, sometimes, in patients with endocrine disorders or inflammation.

Basic andrological diagnosis consists of taking the patient's medical history and the couple's history, performing a physical examination (including additional studies if needed, such as testicular or transrectal ultrasound or a Doppler ultrasound), and hormonal analysis. In patients with fertility disorders, the most important test is a semen analysis.

Infertility is defined as the failure to conceive after regular unprotected intercourse for 12 months. Prospective studies have shown that pregnancy will occur in more than 90 % of fertile couples within one year [1]. Andrological diagnosis before that time is thus only advisable if there is evidence in the patient's history of reduced fertility (e.g., maldescensus testis) or the examination (e.g., small testes), or if the couple is attempting assisted reproduction for gynecological reasons. The age of the patient's partner also plays a role: from age 35 onward, a woman's fertility declines markedly, and after age 40 it declines rapidly [2].

Patient history

The patient history and physical examination are an essential part of basic andrological diagnosis. They are needed for interpreting the spermiogram results and hormonal tests and for arriving at the correct clinical diagnosis. To help establish patient-physician trust, it is important to ensure a quiet atmosphere during the patient interview, as such topics are often considered taboo.

When eliciting the patient's medical history, the physician should look for potential influences on his reproductive, endocrinological, and sexual health. A checklist is a useful tool for structuring the interview (Table 1); electronic databases are an alternative (Winsperm®, Androbase) [3].

Table 1. Checklist for history
Checklist for the andrological history
▸ Length of time trying to conceive
▸ Close relatives with infertility and hereditary diseases
▸ Beginning of puberty and onset of voice change and growth of facial hair
▸ Positional anomalies of testes and treatment
▸ Surgery on pelvis or genital region (e.g., herniotomy)
▸ Trauma affecting pelvic or genital region (e.g., in soccer players, bicyclists)
▸ Infections and inflammation of the genital tract (e.g., chlamydia, postpubertal mumps orchitis)
▸ Illness with fever in recent months (e.g., flu, impaired spermatogenesis possible for up to 6 months)
▸ General diseases (e.g., arterial hypertension, liver or kidney dysfunction, diabetes mellitus, malignancy, HIV)
▸ Medications
▸ Drugs (alcohol, nicotine, narcotics)
▸ Genital heat exposure (sauna, heated seats, etc.)
▸ Exposure to toxic substances (heavy metals, pesticides, solvents, etc.) and radiation exposure
▸ Partner-related or situation-related disorders affecting libido, erection, ejaculation, or orgasm

Medication may directly or indirectly influence male fertility due to erection or ejaculation disorders. Not every drug is adequately tested for its influence on fertility. The package insert often refers merely to “lacking data.” In such instances, a current literature search should be performed and the potential risk discussed individually with the patient.

Disorders of spermatogenesis are often caused by cytostatic agents or immunosuppressants, anti-epileptic drugs, antibiotics, and anti-emetics [4]. With the exception of germinal cell damage from certain cytostatic drugs (e.g., cyclophosphamide), these effects are usually reversible within 6 months after stopping the drug [5].

A further mechanism that can impair fertility is the interaction with epididymal and sperm functions (e.g., related to calcium antagonists, anti-epileptic agents, sulfasalazine, and colchicine). In addition, various drugs also alter endocrine regulatory mechanisms, especially androgen levels. Typical examples are anabolic steroids, estrogen, gestagen, and androgens such as ketoconazole, cardiac glycosides, spironolactone, and finasteride [6].

Blood pressure medication frequently leads to erectile problems; and antidepressants may lead to ejaculation disorders [6].

If there is suspicion of age-related hypogonadism, the physician should ask about further symptoms of androgen deficiency (Table 2).

Table 2. Symptoms of age depended hypogonadism
Symptoms of age-related hypogonadism
▸ Fatigue and difficulty concentrating
▸ Reduced physical ability
▸ Diminished muscle strength and mass
▸ Bone pain and spontaneous fractures
▸ Change in fat distribution
▸ Mood fluctuations (especially depressive mood)
▸ Diminished libido
▸ Erectile and ejaculation disorders

In patients who are unable to conceive, the couple's history should also be taken. The physician should ask how long they have been trying to conceive, any prior pregnancies (also with other partners), and the outcome of prior pregnancies, as well as the couple's sexual behavior (e.g., frequency of intercourse and awareness of the woman's fertile days).

Physical examination

First, a physical examination of overall health is performed. This includes assessing physical proportions, hair status, pitch of the voice, and whether the patient has gynecomastia or lipomastia. Based on the results of various studies in recent years, waist circumference and body mass index (BMI) should also be measured. On the one hand, an increased waist circumference correlates with diminished serum testosterone levels [7], while on the other obesity may impair sperm quality [8].

Most men find that the subsequent examination of the penis, testes, and prostate causes a sense of shame. The patient should be given the option of undergoing the examination alone, without his partner.

The penis should be inspected for hypospadias or epispadias, phimosis, balanitis, and any changes affecting the cavernous body (e.g., induratio penis plastica). A very short penis (referred to as micropenis) can occur in a number of genetic diseases with hypogonadotropic/hypergonadotropic hypogonadism or androgen resistance. Examples include Klinefelter and Kallmann syndromes.

Both testes should be in the scrotum. If not, the patient has maldescensus testis or cryptorchidism. In normal patients, testicular volume is 12–20 ml [9]. The volume is measured manually using an orchidometer (Figure 1) or ultrasound. The consistency should be firm. Small, soft testes are usually a sign of a disorder of spermatogenesis, often corresponding with high serum FSH levels. If the testes are enlarged, or if there are palpable nodules, further tests are needed to assess the presence of a tumor. Hydrocele causes painless testicular swelling; diagnosis may be confirmed using diaphanoscopy, but is generally done with ultrasound.

Figure 1.

The orchidometer beads are compared with the testicles by palpation and the volume is read off the bead which matches best in size.

The epididymis may be palpated at the upper posterior pole of the testis as a soft mass. An enlarged, painful epididymis usually is correlated with epididymitis. Spermatocele may be evident as painless cysts. Forming the continuation of the epididymis, the vas deferens may be palpated as a firm cord-like structure. In 75 % of patients, its absence (congenital bilateral aplasia of the vas deferens; CBAVD) is an expression of a mutation affecting the CFTR gene corresponding to a minimal form of cystic fibrosis [10].

The prostate is palpated by digital rectal examination. The physician should inspect its size, shape, boundaries, tenderness, consistency, and search for any nodules, hardening, or fluctuations. Enlargement of the normally firm, chestnut-shaped organ is often correlated with benign prostate hyperplasia. Pain during the examination is a sign of prostatitis; solid, nodular changes are a sign of cancer. Usually palpation does not detect a carcinoma until there is advanced-stage disease. Due to its low sensitivity, it is not well suited for early detection [11]. In addition, the manual inspection should be done after blood is taken for the prostate–specific antigen (PSA) test. Mechanical irritation (e.g., due to riding a bicycle, sex, etc.) may lead to temporarily elevated PSA serum levels and hence false-positive results in tumor marker blood tests.

The seminal vesicles, which are located above the prostate, may also be palpated.


An ultrasound completes the physical examination. Considering that it often yields far more precise results (e.g., in the examination of the testes), it may also replace individual steps in the clinical examination. If the testis and epididymis are healthy, there will be a homogeneous echo pattern. A scrotal ultrasound may be used to measure testicular volume relatively precisely (Figure 2). The vas deferens, visible by its thick muscle wall and narrow lumen, is seen at ultrasound as the continuation of the epididymis.

Figure 2.

Normal scrotal ultrasound showing a homogenous pattern of the testicle. Determination of testicular volume is computerized by measuring the elliptical area.

An inhomogeneous internal pattern of the testes may be due to an organized hematoma following trauma, (granulomatous) orchitis, or a carcinoma in situ.

For the latter, testicular microcalcifications (“starry sky” appearance) are also significant. Although their exact etiology remains uncertain, there is an association with impaired Sertoli cell function and anomalies affecting the LKB1 gene, as well as testicular malignancies (especially seminomas) [12]. Regular palpation and ultrasound studies (2 ×/year), along with assessing testicular tumor markers and instructing the patient in self-examination, are important measures for such patients. If the patient has additional risk factors (maldescensus testis, infertility, testicular atrophy, contralateral testicular tumor), a testicular biopsy is indicated to rule out cancer [13].

Testicular tumors manifest on ultrasound as an echo-poor / hypoechoic or “mixed echo” lesion in the parenchyma with increased circulation (Figure 3). The patient should be referred to an urologist for comprehensive staging, surgery, and chemotherapy.

Figure 3.

Hypodense area within the testis, suspicious for malignancy. Histology revealed a seminoma.

An inhomogeneous internal pattern in the epididymis with echo-rich structures and irregular boundaries is usually correlated with chronic epididymitis.

Hydrocele is an accumulation of serous fluid around the testis (hydrocele testis) (Figure 4) in a portion of the vaginal process that is not obliterated; there may or may not be a connection to the abdominal cavity (hydrocele funiculi spermatici or hydrocele communicans). In patients with abdominoscrotal hydrocele, the inguinal portion of a large hydrocele is pressed into the abdomen; obliteration occurs in the area of the internal inguinal ring. This disorder may be congenital or acquired (trauma, infection, testicular tumor, testicular or hydatid torsion, abnormal lymph drainage after a varicocele or inguinal hernia surgery). Treatment consists of either surgery or sclerosis and is mainly done for cosmetic reasons. There is lacking evidence of a positive effect on fertility [14].

Figure 4.

Ultrasound of a hydrocele testis showing accumulation of clear fluid in the tunica vaginalis.

A spermatocele may be seen on ultrasound as a smoothly bordered round structure on the epididymis with no reflectivity. These are sperm-filled retention cysts on the epididymis or, occasionally, on the spermatic cord. Spermatocele is a very common, harmless finding; treatment is only warranted if it causes clinical symptoms or grows.

Varicocele is a visible and palpable enlargement of the veins of the pampiniform venous plexus. It mainly occurs on the left side and may be a primary or secondary disorder. It leads to reflux into the testicular veins. Four degrees of severity may be distinguished (Table 3) [15].

Table 3. Classification of varicocele acc. WHO [51]
Varicocele – degrees of severity
Subclinical varicoceleOnly visible on Doppler ultrasound or thermography
Grade I varicoceleOnly palpable with Valsalva maneuver, Vein diameter < 1 cm
Grade II varicocelePalpable, but not visible, Vein diameter 1–2 cm
Grad III varicoceleVisible through the scrotal skin (and palpable), Vein diameter > 2 cm

In some patients, there is impaired sperm quality. In patients with fertility disorders, the reported frequency of varicocele (grades I–III) is 14–40 %, compared to 8–10 % in unselected men. Treatment of varicocele significantly improves the concentration and motility of spermatozoa. The effect of treatment on any improvement in spontaneous pregnancy in infertile couples is uncertain, although recent meta-analyses support a relationship, at least for grade II or worse [16].

A transrectal ultrasound of the prostate can help identify changes in prostatitis or benign prostate hyperplasia (BPH), and it can aid in diagnosing prostate cancer. Intraprostatic cysts and dilation of the ejaculatory duct have also been shown to cause occlusive azoospermia.

The seminal vesicles should be examined before obtaining a semen sample, because they are more difficult to visualize on ultrasound afterward. The detection of cystic dilations, in particular in occlusive azoospermia, is clinically valuable. Other pathological findings include aplasia and hypoplasia.


The basic spermiogram includes an examination of the color, consistency, pH, and ejaculate volume, as well as a microscopic analysis of sperm concentration, motility, morphology, vitality, and leukocyte count.

Evidence-based reference values for the basic spermiogram

In the 5th edition of the WHO laboratory manual from 2010 [17] (which appeared in 2011 in German), the reference values for all parameters of the basic spermiogram were adjusted. Previously, the classification was based on a consensus procedure; the new reference values for the standard spermiogram are evidence-based, using results from prospective studies with about 1,800 men worldwide who were able to father a child within one year of unprotected intercourse [18]. The current normal values for a spermiogram are listed in Table 4.

Table 4. Evidence based reference values given in the latest WHO manual [17]
ParameterReference values (2010)
Ejaculate volume≥ 1.5 ml
Sperm count / ejaculate≥ 39 mil.
Sperm concentration≥ 15 mil./ml
Total motility≥ 40 % sperm
Progressive motility≥ 32 % sperm
Vitality≥ 58 % living sperm
Normal morphology≥ 4 % normomorphic sperm
Additional values (consensus recommendation)
Peroxidase-positive leukocytes< 1 mil./ml

The findings of the main spermiogram parameters are classified as normozoospermia (normal sperm), oligozoospermia (low sperm concentration), asthenozoospermia (reduced motility), teratozoospermia (reduced percentage of morphologically normal sperm), oligoasthenoteratozoospermia (density, motility, and morphology are pathological), azoospermia (no sperm in ejaculate) or aspermia (no semen). These in no way represent clinical diagnoses. Intra-individual discrepancies are the norm rather than the exception; if the results of the spermiogram are poor, a second one should be performed after 7 to 21 days [17].

A couple's fertility depends on the reproductive health of both partners. Thus, the point at which poor spermiogram results are clinically effective varies.

Depending on the partner (i.e., her age), fertility may even be possible despite very poor values on the spermiogram; conversely, a couple may not be able to conceive despite normal spermiogram results [18]

Macroscopic evaluation

The inspection of the ejaculate begins with a gross inspection about 30 minutes after the sample is obtained, which corresponds to the normal liquidation time. During the liquidation time, semen should be stored at 37 °C. If, after one hour, it forms a thread more than 2 cm long on the pipette, this is referred to as viscopathy.

To enable further tests, a pinch of α-chymotrypsin or 10 IU/ml bromelain is added in a phosphate buffer (ratio of 1:2). For the latter, the dilution should be included when determining the concentration [19].

If the analysis cannot begin within three hours, a new sample should be obtained to avoid artificial damage due to the non-physiological environment [17].

After liquidation, the color (gray/opalescent) and odor (chestnut-blossom-like) are examined. Volume was formerly measured by pipetting the semen into calibrated containers. The resulting loss of material (e.g., in the pipette tips) and inaccuracies [20] led to the WHO recommendation for indirect volume measurement by measuring the weight of the sample in a collecting tray (ejaculate density of about 1 g/ml) [17]. The pH may be determined (color change of a pH indicator) within an hour after obtaining the sample. Table 5 gives an overview of all macroscopic spermiogram parameters.

Table 5. Macroscopic evaluation of the ejaculate
ParameterNormal ejaculateDeviations

Watery/transparent appearance with low sperm concentration

Brown/red color with blood mixed in (hematospermia)

Volume≥ 1.5 ml

Lower with:

▸ Mishandling of specimen or shortened waiting period

▸ Partial or complete retrograde ejaculation

▸ Occlusion of seminal ducts (e.g., utriculus cysts)

▸ Abnormal function of seminal vesicle and prostate (e.g., in hypogonadism)

pH value≥ 7.2

Lower with:

▸ Presence of prostate secretions only due to obstruction or malformation of draining seminal ducts (concomitant azoospermia, reduced volume and fructose concentration)

Increased (pH > 8.0) with:

▸ Use of lubricants

▸ Semen sample collected > 1 hour before analysis

▸ Inflammation

Plain specimen

At the beginning of the microscopic analysis of the ejaculate, an initial plain specimen is prepared for estimating spermatozoa density and motility. In addition, one may identify agglomerations (unspecific clumping of non-motile sperm, debris) and agglutinations (motile sperm sticking together as a sign of sperm antibodies), as well as spermatogenesis cells, inflammatory cells (leukocytes, macrophages), erythrocytes, epithelial cells, and bacteria.

Sperm concentration

Sperm concentrations may be determined using immobilized sperm. Prior dilution of the sample is based on the number of sperm in the field of view (400×) in the plain specimen. A hemocytometer is especially well suited for obtaining a sperm count (recommended counting chamber: Neubauer improved, Figure 5). Two independent dilutions of the immobilization solutions should be prepared and analyzed in two counting chambers [21]. The current reference value for sperm concentration is ≥ 15 × 106 sperm/ml [17]. The total number of sperm per ejaculate is calculated by volume (in ml) and sperm concentration (in mil/ml). The current reference value is ≥ 39 million.

Figure 5.

Counting chamber (Neubauer improved) to measure sperm concentration. The cover slip should be moved with slight pressure on the surface of the chamber until the so called Newton rings appear.

If there are no sperm in the plain specimen, the semen should be centrifuged (3000 × g for 15 minutes). If there is still no sperm in the pellet, azoospermia is present. Yet, even under these conditions, sperm have occasionally been detected in the supernatant [22].

Sperm motility

An analysis of sperm motility is done with a plain specimen at 400× magnification with phase-contrast microscopy using two independent assessments of separate samples, counting 200 consecutive sperm from each sample and then determining the mean. The use of a counting device facilitates the procedure.

The 5th edition of the WHO laboratory manual has simplified the evaluation of sperm motility in plain specimens (Table 6) [17, 23]. It is now easier to learn how to perform a sperm motility analysis, and there is also better comparability of laboratory values due to lower error rates. A disadvantage is a certain loss of information, such as the percentage of sperm with progressive motility. For in-vitro fertilization (IVF), in particular, the percentage of fast progressive sperm is a good predictor of success [24]. Changes in motility assessment are thus the controversially discussed [25].

Table 6. Differences in motility evaluation given in the older and the newest WHO manual [17, 23]
CategoryPrior categorizationCurrent categories
WHO aRapid linear progressive motility (≥ 25 μm/s)Progressive motility (= WHO a+b)Total motility (= WHO a + b + c)
WHO bSlow progressive motility (5–25 μm/s)
WHO cLocally motile sperm (< 5 μm/s)Non-progressive motility
WHO dImmotile spermImmotile sperm 

Computer-assisted systems (CASA) are also available for detailed assessments of sperm motility, including various indicators of speed [26].

Diminished sperm motility is a main indicator of reduced fertility. Various forms of sperm damage are evident as reduced motility or immotility. The causes of asthenozoospermia may be a disorder of spermatogenesis or maturation in the epididymis. Other possible sources include post-testicular disorders such as genital tract inflammation (reactive oxygen species, pro-inflammatory cytokines), sperm antibodies, or viscopathy.

Artificial sources of loss of motility, which can occur during the examination, include a long time between obtaining the ejaculate and performing the analysis, exposure to non-physiological temperatures (optimal body temperature), and toxic effects of buffers, etc.

Sperm morphology

The analysis of sperm morphology is an important component of andrological laboratory tests. Yet, it involves significant amounts of material and time; also, proper evaluation requires a great deal of practice.

Aliquots of the fresh semen samples are placed on a coated slide after complete liquefaction, allowed to air dry, and then stained (modified Papanicolaou stain or Shorr stain or the Diff-Quick method). A light microscope is used to evaluate 200 sperm each (duplicate testing) at 1,000× enlargement with oil immersion. The current WHO laboratory manual recommends using “strict criteria” [27, 28] for determining the percentage of normal-shaped sperm. This has resulted in a significant decrease in the now evidence-based reference value from ≥ 15 % (1999) to ≥ 4 % normomorphic spermatozoa [17, 23].

Pathological forms may also be divided into the categories: head, neck and mid-piece defects; disorders of the flagellum; and cytoplasmic droplets. Determining the absolute number of normomorphic sperm may also be useful for making decisions related to assisted reproduction [29].

The percentage of vital sperm is determined, based on the WHO stipulations, using eosin and negrosin staining. Only dead cells with a permeable cell membrane stain with eosin; after one to two minutes they appear pink under bright field or phase contrast microscopy at 400x magnification. The current, evidence-based threshold for sperm vitality is ≥ 58 % [17]. The test is therefore unnecessary if total sperm motility is ≥ 60 %.

Leukocyte concentration in ejaculate

Another part of the basic examination of the ejaculate is determining the leukocyte concentration using simple peroxidase staining of a plain semen sample [17]. The WHO threshold, which was arrived at by consensus, is ≤ 1 million peroxidase-positive leukocytes/ml [17]. If there a genital tract inflammation, further microbiological examinations of the ejaculate (obtained under sterile conditions) should be performed to assess the need for treatment. The most important pathogens are chlamydia, mycoplasma, and ureaplasma urealyticum, as well as enterobacteria. Asymptomatic, chronic inflammation of the seminal ducts is also often present which impair sperm quality. Further tests include determining the inflammatory markers granulocyte elastase or interleukin 6 or 8 in the ejaculate [30].

Direct damage of sperm related to inflammatory processes is caused by the release of reactive oxygen species from the leukocytes. As a result of lipid peroxidation, there is damage of the sperm membrane and disruption of important functions; in particular, the motility or DNA integrity may be negatively affected [31].

Further spermiogram parameters

Depending on the necessity of doing so, and the options available, the spermiogram may also be broadened to include the following standardized parameters: detection of sperm antibodies using a mixed antiglobulin reaction (MAR) test or immunobead test and spectrophotometric evaluation of zinc, fructose, and α-glucosidase levels in seminal plasma. In accordance with the current WHO laboratory manual, the normal values are based on expert consensus recommendations (Table 7).

Table 7. Sperm antibody test, markers of epididymal, seminal vesicle and prostate function [17]
Sperm antibodies< 50 % motile sperm with bound particles (MAR test) or beads (immunobead test)Sign of immune-mediated infertility, anti-sperm IgA and IgG antibodies cause agglutinations
α-glucosidase in seminal plasma> 20 mU/ejaculate

Parameter of epididymal function, use for differential diagnosis of azoospermia:

Lower values with occlusive azoospermia, normal values in non-obstructive azoospermia

Fructose in seminal plasma> 13 mol/ejaculate

Parameter of seminal vesicle function, differential diagnosis for azoospermia:

Lower values in occlusion near urethra (occlusion after opening of the seminal vesicle) or aplasia of the spermatic duct (CBVAD), normal values in patients with disorders of semen transport or occlusion of the seminal ducts before the seminal vesicle as well as in non-obstructive azoospermia

Zinc in seminal plasma> 2.4 mol/ejaculateParameter of secretory capacity of prostate

In addition, a wide array of sperm function tests which simulate the individual steps in the fertilization process may be used to assess the fertility of the sperm cells. These include computer-assisted semen analysis (CASA), the sperm-mucus interaction test, the zona pellucida binding test, an analysis of the percentage of spontaneous acrosomal reacted sperm, and the hamster oocyte penetration test [17].

DNA fragmentation rate

An assessment of the DNA fragmentation rate is widely used in routine clinical practice as an independent marker of male infertility. A high rate of DNA fragmentation in sperm impairs the occurrence of pregnancy in otherwise healthy couples in IUI cycles (and to a lesser extent in IVF cycles), but not in intracytoplasmic sperm injection (ICSI) cycles. In addition, the DNA fragmentation rate has a high predictive value for abortion rates in IVF/ICSI cycles [32].

The current threshold for the DNA fragmentation index is 25 %. Values above this indicate a statistically increased probability of (a) longer time until natural pregnancy or failure to achieve pregnancy, (b) failure of intrauterine insemination, and (c) abortions and miscarriages [33]. For such couples, ICSI is advised. Use of sperm separation processes, such as density gradient centrifugation and annexin V-MACS, may be used to select sperm with a reduced fragmentation index [34, 35].

Sources of a sperm with a DNA fragmentation rate include disorders affecting nuclear remodeling in spermatogenesis and other post-testicular factors. Lifestyle factors, such as smoking (even passive smoking), are also a cause [36], as are obesity and diabetes mellitus [37, 38], as well as urogenital infections (leukocytospermia). These lead to direct damage to the integrity of the sperm DNA due to increased oxidative stress. The formation of 7-hydro-8-oxo-2´-deoxyguanosine (8-oxo-dG) is a key element in oxidative damage affecting DNA [39].

The activation of an apoptotic signaling cascade with subsequent DNA fragmentation has also been discussed [40], especially related to heat exposure, e.g., from sauna use or sitting on heated seats [41, 42]. There are also often extremely high DNA fragmentation rates after radiation treatment or chemotherapy [43, 44]. This means that DNA fragmentation rate may be improved by changing one's lifestyle, as well as changing medication use [33].

Despite its special significance for infertility diagnosis, the determination of the sperm DNA fragmentation rate presents certain problems. Assays which have been evaluated on somatic cells cannot be used without testing on sperm due to the special condensation of sperm DNA in the sperm head. In actual practice, both direct (TUNEL) and indirect detection methods are used. Examples of the latter include the Sperm Chromatin Structure Assay (SCSA®), acridine orange staining, the sperm chromatin dispersion test (SCD), and the comet assay. These tests measure the susceptibility for DNA fragmentation.

There are excellent data available on the significance of the DNA fragmentation rate as a parameter in male fertility; the results are based primarily on SCSA and TUNEL [45]. Both methods are fluorescence-based and relatively complex; they are thus performed either by specialized institutions or by sending the sample to an appropriate center. This should change with the Halosperm® kit, which recently became commercially available. The kit is a variant of the sperm chromatin dispersion test; simple Diff-Quick staining leads to halo formation of DNA-intact sperm. The analysis is done using light microscopy. A problem related to the test is the comparably small amount of data available on the clinical value of a sperm chromatin dispersion test. The data also show that the sperm chromatin dispersion test has no predictive power in terms of the success of assisted reproduction techniques [46, 47]. Thus the use of the Halosperm® kit should still be viewed critically.

German Medical Association Guidelines on Quality Assurance

According the guidelines of the German Medical Association on Quality Assurance of Laboratory Medical Tests (Rili-BÄK), the requirements for quality assurance in semen tests (part B4) were modified as of 1 January 2011 (with a transition period ending 3 December 2012) [21].

An important point for clinical practice is the mandated duplicate analysis for the spermiogram parameters concentration, motility, morphology (at least 200 sperm each). In addition, andrological laboratories must participate in routine internal and external quality controls. In Germany, the only accredited reference institution for the bi-annual inter-laboratory comparison is a quality assurance program called “QuaDeGA” (Qualitätskontrolle der Deutschen Gesellschaft für Andrologie).

Hormone diagnosis

The male reproductive tract is governed by a well-regulated system of hypothalamic (GnRH), hypophysial (LH, FSH), and testicular hormones (androgens, inhibin B) (Figure 6). Any disruption of the hormonal cycle may lead to testicular dysfunction and impairment of spermatogenesis.

Figure 6.

Regulation of hormones by hypothalamus, hypophysis and testis.

Basic andrological hormonal diagnosis thus includes an assessment of FSH, LH, testosterone, and inhibin B as well as serum prolactin. Taken together with clinical appearances, hormonal disorders may be identified and differentiated. The spectrum ranges from age-related hypogonadism (primary testosterone deficiency) to forms of hypogonadotropic and hypergonadotropic hypogonadism (e.g., in Kallmann or Klinefelter-syndrome), or prolactinoma.

Along with the spermiogram results, hormonal analysis yields information about the patient's fertility reserve (Table 8). An evaluation of inhibin B is also part of the fertility diagnosis. The hormone and inhibin B/FSH ratio are more sensitive markers for spermatogenesis quality than FSH alone [48].

Table 8. Estimation of fertile capacity by means of FSH and spermiogram values
NormalNormal rangeNormal finding
LimitedIn lower normal rangeStimulation of spermatogenesis possible with medication
LimitedElevatedDrug stimulation of spermatogenesis is impossible, feedback loop exhausted

Additional studies

Testicular biopsy

A testicular biopsy may be helpful for definitively distinguishing between obstructive and testicular azoospermia. The diagnostic and treatment biopsy are the same. In addition, testicular tissue is cryconserved. If testicular sperm are isolated, these may be used for subsequent intracytoplasmic sperm injection. The terms TESE or cryo-TESE refer to testicular sperm extraction. The probability of detecting sperm in the testis may be estimated in advance using hormonal diagnosis. The higher the serum FSH concentration, or the lower the serum inhibin B concentration, the more unlikely it is that any sperm will be found. Inhibin B is somewhat more specific than FSH; studies have shown that at serum concentrations of 20 ng/l there is no detection of sperm in the testes [48]. If the germinal epithelium is absent (Sertoli-cell-only syndrome), inhibin B will not be detected [49].

An isolated diagnostic testicular biopsy should only be performed if there is suspicion of a malignant tumor.

Genetic diagnosis

Genetic testing is advisable in patients with very poor spermiogram results (sperm concentration < 5 mil/ml) and before artificial insemination using intracytoplasmic sperm injection [50]. Table 9 provides an overview.

Table 9. Genetic counseling: overview of indications
Genetic diagnosisIndication
KaryotypingRepeated abortions, suspected Klinefelter syndrome, before planned intracytoplasmic sperm injection (ICSI)
Detection of Y-chromosomal microdeletionSevere oligozoospermia or azoospermia, before planned ICSI
Mutation analysis of CFTR gene (cystic fibrosis)Occlusive azoospermia, especially in congenital bilateral aplasia of seminal der spermatic duct, in known CFTR mutation in the partner

Fragen zur Zertifizierung durch die DDA

  1. Welche Erbkrankheit kann Ursache einer Verschlussazoospermie sein?
    1. adrenogenitales Syndrom
    2. Trisomie 21
    3. Mukoviszidose
    4. Phenylketonurie
    5. Neurofibromatose Typ 1 (Morbus Recklinghausen)
  2. Welches der im Folgenden aufgeführten Medikamente verursacht häufig Erektionsstörungen?
    1. Desloratadin
    2. Doxycyclin
    3. Metoprolol
    4. Vardenafil
    5. Methotrexat
  3. Welche Aussage zu Hydrozelen trifft nicht zu?
    1. In der Regel beeinträchtigen sie nicht die Spermienqualität.
    2. Es handelt sich um Retentionszysten.
    3. Die Therapie erfolgt meist aus kosmetischen Grunden.
    4. Sie können auch am Samenstrang auftreten.
    5. Sie können nach einer Herniotomie auftreten.
  4. Welche Aussage zur Spermien-Motilität nach WHO 2010 trifft nicht zu?
    1. Mindestens 40 % der Spermien sollten motil sein.
    2. Die Gesamtmotilität berechnet sich als Summe aus progressiv und am Ort motilen Spermien.
    3. Die Progressivmotilität schließt sowohl schnell als auch langsam vorwarts schwimmende Spermien ein.
    4. Mindestens 32 % der Spermien sollten progressiv motil sein.
    5. Die Gesamtmotilität berechnet sich als Summe aus schnell und langsam progressiv motilen Spermien.
  5. Welche Aussage ist falsch?
    1. Verflüssigungszeiten des Ejakulates über 1 Stunde sprechen für eine Viskosipathie.
    2. Eine rotbraune Tingierung des Ejakulates spricht für eine Hämatospermie.
    3. Der Zinkspiegel im Ejakulat ist ein Marker der Prostatafunktion.
    4. Der α-Glucosidase-Spiegel im Ejakulat ist ein Marker der Samenbläschenfunktion.
    5. Der Nachweis erhöhter Leukozytenkonzentrationen im Ejakulat kann die Fertilität beeintrachtigen.
  6. Der Referenzwert für den prozentualen Anteil normomorpher Spermien im Ejakulat liegt bei…
    1. ≥ 4 %
    2. ≥ 15 %
    3. ≥ 25 %
    4. ≥ 50 %
    5. ≥ 60 %
  7. Welche Bedeutung hat die Bestimmung der Fruktose im Seminalplasma?
    1. Erfassung der metabolischen Versorgung der Spermien
    2. Erfassung des Zuckerstoffwechsels im Seminalplasma
    3. Ursachenanalyse von Spermien-Motilitätsstörungen
    4. Analyse einer Spermaallergie
    5. Nachweis und Differenzierung einer Verschlussazoospermie
  8. Welcher der folgend genannten Tests weist Fragmentationen der Spermien-DNA direkt nach?
    1. Comet-Assay
    2. TUNEL-Assay
    3. Spermienchromatindispersionstest (SCD)
    4. Acridinorangefärbung
    5. Sperm Chromatin Structure Assay (SCSA)
  9. Welche Parameter lassen Rückschlüsse auf die Spermatogenese im Hoden zu?
    1. Östrogen und Progesteron
    2. LH und Testosteron
    3. GnRH und SHBG
    4. FSH und Inhibin B
    5. Prolaktin und Oxytocin
  10. Welche der folgenden Aussagen zur Richtlinie der Bundesärztekammer zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen (Rili-BÄK) ist nicht korrekt?
    1. Doppelbestimmungen der Spermienparameter Konzentration, Motilität, und Morphologie sind zwingend vorgeschrieben.
    2. Die Durchführung interner und externer Qualitätssicherungsmaßnahmen ist zwingend vorgeschrieben.
    3. Die Spermiogrammparameter müssen durch Auszählung von 100 Spermien bestimmt werden.
    4. Die Übergangsfrist zur Umsetzung der Anforderungen an die Ejakulatdiagnostik endete zum 31.12.2012.
    5. Das Qualitätssicherungsprogramm QuaDeGA der Deutschen Gesellschaft für Andrologie ist zur Durchführung der Ringversuche in Deutschland akkreditiert.

Liebe Leserinnen und Leser,

der Einsendeschluss an die DDA für diese Ausgabe ist der 18. Oktober 2013. Die richtige Lösung zum Thema ,,Opportunistische Hefe-Mykosen: Candidose, Kryptokokkose, Trichosporonose und Geotrichoseg” in Heft 5 (Mai 2013) ist: 1a, 2c, 3e, 4d, 5d, 6b, 7d, 8c, 9e, 10c.

Bitte verwenden Sie für Ihre Einsendung das aktuelle Formblatt auf der folgenden Seite oder aber geben Sie Ihre Lösung online unter http://jddg.akademie-dda. de ein.