Male Infertility Workup

Updated: Jun 05, 2020
  • Author: Chirag N Dave, MD; Chief Editor: Edward David Kim, MD, FACS  more...
  • Print

Laboratory Studies

Semen analysis

The semen analysis is the cornerstone of the male infertility workup. A specimen is collected by masturbation into a clean, dry, sterile container or during coitus using special condoms (containing no spermicidal lubricants). The patient should be abstinent for 2-3 days prior to maximize sperm number and quality. Each day of abstinence is typically associated with an increase in semen volume of 0.4 mL and an increase in sperm density by 10-15 million sperm/mL, for up to 7 days.

The sample should be processed within 1 hour, and 2-3 samples (at a minimum of 2-3 days apart) should be evaluated because of daily variations in sperm number and quality. Various parameters are measured, such as ejaculate volume and sperm density, quality, motility, and morphology. Individual tests evaluate only one aspect of a quality necessary for fertility and do not imply the ability or inability to achieve conception (see the Table in the Procedures section).

The World Health Organization (WHO) published reference ranges for semen testing in 2010. [45] These include “lower reference limits” representing the 5th percentiles for semen characteristics. [46] Note that the lower reference limits do not serve as a cut-point between “fertile” and “infertile.”


Normal ejaculate volume is 1.5-5 mL, and the WHO lower reference limit (5th percentile) is 1.5 mL. A small ejaculate volume may be observed in patients with retrograde ejaculation, absence of the vas deferens or seminal vesicles, ductal obstruction, hypogonadotropism, or poor sympathetic response. An increased volume is rarely observed and is often caused by a contaminant, such as urine.

Semen quality

Semen is initially a coagulum that liquefies in 5-25 minutes due to prostatic enzymes. At this point, pouring the semen drop by drop should be possible. Semen that is not initially a coagulum is often an indication of an ejaculatory duct obstruction or the absence of seminal vesicles. Nonliquefication of the semen can be differentiated from benign hyperviscosity by a normal postcoital test finding. No excessive sperm agglutination should exist.

Sperm density

Normal sperm density is greater than 20 million sperm/mL. The WHO lower reference limit (5th percentile) is 15 million sperm per mL, or 39 million sperm per ejaculate. Oligospermia is defined as fewer than 20 million sperm/mL, severe oligospermia is less than 5 million/mL, and azoospermia is defined as no sperm present.

To verify azoospermia, the semen should be centrifuged and evaluated under a light microscope for the presence of sperm. Patients with azoospermia should have a postejaculatory urine sample analyzed for sperm, should be evaluated for ejaculatory duct obstruction, and should undergo a hormonal evaluation.

Sperm motility

Motility is described as the percent of sperm present with flagellar motion viewed on a bright-field or phase-contrast microscope. Normal motility is defined as more than 60% of sperm having normal movement, and the WHO 2010 lower reference limit (5th percentile) is 40%. Grading is as follows:

  • Grade 0 – No movement
  • Grade 1 – Sluggish movement
  • Grade 2 – Slow movement in a poorly defined direction
  • Grade 3 – Slow or curved forward movement
  • Grade 4 – Fast movement straight forward 

Patients with abnormal sperm motility should be evaluated for the following:

  • Pyospermia
  • Antisperm antibodies
  • Varicocele
  • Sperm ultrastructural abnormalities
  • Partial ductal obstruction

Sperm morphology

The head, acrosome, mid piece, and tail of individual spermatozoa are analyzed with phase-contrast microscopy after fixation with Papanicolaou stain. At least 200 sperm are analyzed. Normal sperm have a smooth oval head approximately 3-5 μm long and 2-3 μm wide. More than 60% of sperm should be normal, and less than 2-3% should be immature. These sperm show a high level of retained cytoplasmic droplets around the mid piece.

Teratospermia is defined as less than 30% normal morphology, and the WHO lower reference limit (5th percentile) is 4%. Abnormal head shapes are described as tapered, duplicated, small, large, amorphous, or pyriform. The acrosome should be 40-70% of the size of the head, and no mid piece or tail abnormalities should be present.

Patients with a high number of immature sperm should be evaluated for excessive exposure to heat or radiation or for infectious processes.

To help objectify sperm morphology and therefore enhance the consistency and reproducibility among laboratories, Kruger introduced a definition of "strict criteria" in 1986. Using these criteria, he reported a clinically significant threshold of 14% normal forms as an excellent predictor of IVF success. Patients with less than of 14% normal forms had a substantially reduced success rate.

Computer-aided semen analysis (CASA)

Introduced in the late 1980s, CASA uses a video camera and computer to visualize and analyze sperm concentration and movement. This semiautomated technique is thought to potentially standardize the evaluation of semen. CASA measures the following parameters:

  • Curvilinear velocity – The average distance per unit time between successive sperm positions)
  • Straight-line velocity – The speed of forward direction
  • Linearity – The straight-line velocity divided by the curvilinear velocity

In addition, the program measures the average path velocity, the amplitude of lateral head displacement, and the flagellar beat frequency, and it is used to evaluate for evidence of hyperactivation.

Although CASA produces good qualitative data, it is a labor-intensive procedure with a high initial cost and is plagued with inaccuracies when sperm concentrations are very high or very low. It has not been shown to improve patient outcomes but, rather, is helpful for research purposes.


An increased number of white blood cells in the semen may be observed in patients with infectious or inflammatory processes of the genital tract. Germ cells and white blood cells both appear as round cells on microscopic examination, so immunohistochemical stains are used to differentiate the 2 cell types. Immunohistochemical stains are performed if more than 5-10 round cells/HPF are present. 

Other tests

Semen may be analyzed for levels of zinc, citric acid, acid phosphatase, and alpha-glucosidase. These tests are used to determine gland failure or obstruction.

Antisperm antibody test

Sperm contain unique antigens that are not recognized as self by the body's immune system because of the blood-testis barrier.

Antisperm antibodies may form when the blood-testis barrier is breached because of infection, vasectomy, testicular torsion, cryptorchidism, or testicular trauma. Antibodies that are bound to sperm decrease the sperm’s ability to penetrate the cervical mucus and bind to the zona pellucida.

Although 60% of patients have evidence of antisperm antibodies after vasectomy, the clinical significance has not been completely elucidated. In addition, antibodies are present in 35% of patients with CBAVD. The presence of antibodies in serum or seminal plasma is less prognostic than antibodies bound to sperm.

Suspect antisperm antibodies when semen analysis reveals abnormal clumping, agglutination, unexplained decreased motility, or an abnormal postcoital test result.

Several methods are available to detect antisperm antibodies, such as radioimmunoassay and enzyme-linked immunosorbent assay, but the most specific test is the immunobead test. More than 15-20% bound is considered a positive test result.

Hormonal analysis

Around 3% of cases of male infertility are estimated to be due primarily to a hormonal cause. A routine part of the initial evaluation is testing of specific serum hormone levels, which usually includes FSH, LH, testosterone, and prolactin. Abnormalities may be a sign of a primary hypothalamic, pituitary, or testicular problem.


Imaging Studies

Transrectal ultrasonography

TRUS is indicated in patients with azoospermia or severe oligospermia to evaluate for complete or partial ejaculatory duct obstruction, particularly when the vasa are palpable and low ejaculate volume is noted. [25, 26]  TRUS is also useful to evaluate for the presence or absence of the seminal vesicles.

A 6.5- to 7.5-MHz probe is used with the bladder partially filled.

Obstruction is suggested by enlarged seminal vesicles (>1.5 cm width).

Scrotal ultrasonography

Scrotal ultrasonography is used to evaluate the anatomy of the testis, epididymis, and spermatic cord. It is a useful adjunct for evaluating testicular volume, testicular and paratesticular masses, and the presence or absence of varicoceles.

A large review reported a 38% rate of abnormalities on testicular ultrasonography in infertile men, including 30% with varicocele and 0.5% with testicular cancer. [47]

Routine testicular ultrasonography in infertile men is controversial, but some suggest it because of the increased risk of testicular cancer in infertile men (1 of 200 versus 1 of 20,000 in the general population). [48]

Color-flow ultrasonography is used to evaluate for varicocele using a 7- to 10-MHz probe.

A varicocele is diagnosed on a sonogram if a spermatic vein is greater than 3 mm or vein size increases with Valsalva. Repair of subclinical varicoceles (those diagnosed only with ultrasonography) has not been proven to improve fertility. [38]


Vasography is used to evaluate patency of the ductal system.

Indications for vasography include azoospermia with sufficient mature spermatids present on testicular biopsy and at least one palpable vas.

Relative indications include severe oligospermia with a normal finding on testis biopsy, antisperm antibodies, and decreased semen viscosity.

This test may be performed either as an open procedure at the same time as testicular biopsy or by a percutaneous puncture (see image below).

Male infertility. Technique of open vasography: Th Male infertility. Technique of open vasography: The vas distal to the site of incision is determined to be patent if saline is injected without resistance. Alternatively, radiographic contrast dye is injected through the vas deferens and radiography is performed, or blue dye may be injected and visualized in the urine to confirm patency. A vasovasostomy or vasoepididymostomy may then be performed at this level.

The patient may be placed in a 10-15° Trendelenburg position to bring the symphysis pubis out of the radiation field.

Unilateral patency rules out vasal or ejaculatory duct obstruction as the cause of azoospermia.


Other Tests

Postcoital test

An abnormal postcoital test result is observed in 10% of infertile couples. Indications for performing a postcoital test include semen hyperviscosity, increased or decreased semen volume with good sperm density, or unexplained infertility.

After coitus at mid cycle, the female's cervical mucus is examined for the presence or absence of sperm. Usually, 10-20 sperm/HPF are observed. Abnormal results may be due to antisperm antibodies, sperm ultrastructural abnormalities, an abnormal hormonal milieu, male or female genital tract infection, poor semen quality, inhospitable cervical mucus, or male sexual dysfunction. If no sperm are observed, the couple's coital technique should be analyzed.

If the test result is normal, consider a test of sperm function and ability to penetrate the egg.

Sperm function tests

When a primary sperm defect is suspected or when other tests do not reveal the cause of infertility, sperm function tests may determine if a significant sperm abnormality exists. These tests analyze specific sperm functions, such as the ability to undergo capacitation and the acrosome reaction and the ability to bind to and to penetrate the egg.

The capacitation assay is used to evaluate the ability of sperm to undergo capacitation. After capacitation, sperm have hyperactivated motility, which can be recognized under microscopy. Failure of sperm to undergo capacitation portends a poor response to IVF, and ICSI should be considered.

The acrosome reaction assay tests the ability of the sperm to undergo the acrosome reaction when exposed to inducing substances. The acrosome process, which covers the anterior two thirds of the sperm head, contains hyaluronidase and other enzymes used to digest the zona pellucida of the egg. After sperm binding and capacitation, the plasma membrane of the egg induces the acrosome to release its contents. This reaction occasionally occurs spontaneously (< 10% of the time), although a spontaneous reaction is more common in infertile men.

Under the microscope, acrosome-inducing substances are added to the sample after the sperm have undergone capacitation, which usually takes approximately 3 hours. Usually, 15-40% of the sperm undergo the acrosome reaction when stimulated, and fewer undergo the reaction in infertile men. The results of the test correlate with IVF success; patients with an abnormal test result may need to undergo ICSI.

Sperm penetration assay (SPA)

First described in 1976 by Yanagimachi et al, the SPA is used to check the ability of sperm to function in vitro by evaluating capacitation, the acrosome reaction, and the ability of the sperm to fuse with the oolemma. [49]  Because cross-species fertilization is usually prevented by the zona pellucida, the SPA uses hamster ova with the zona pellucida removed. These ova are incubated with the donor's sperm and the number of sperm penetrated per ovum is measured. A normal result is more than 5 sperm penetrations per ovum. Fewer penetrations probably indicate a problem. Patients with a poor SPA should proceed directly to ICSI.

Hypoosmotic swelling (HOS)

The HOS test is used to provide functional information to differentiate between viable but immotile sperm and dead sperm. Normal sperm are able to maintain an osmotic gradient when exposed to hypoosmotic conditions, whereas dead sperm cannot. After exposure to a dilute solution (150 mmol/L), sperm are observed under the microscope. Normal sperm swell, with bulging of the plasma membrane and curling of the tail. This test is commonly used clinically to select viable (but nonmotile) sperm for ICSI.

Inhibin B

Inhibin B is usually produced by sperm for the acrosome reaction. An increased level or an inability to clear acrosomal enzymes may lead to self-destruction and lipid peroxidation of the sperm membrane. Increased inhibin B levels may be caused by ductal obstruction or abnormalities within the seminiferous tubules.

Vitality stains

Vitality stains using substances such as eosin Y and trypan blue help determine whether sperm are alive and their membrane is intact or if the sperm are dead. Live sperm can exclude dye, while dead sperm cannot. These tests are of little use unless very low numbers of sperm exist or motility is absent and necrospermia must be ruled out. The subsequent process of slide fixation kills all of the sperm, thus preventing their clinical use.



Testicular biopsy

Testicular biopsy is performed in azoospermic men with a normal-sized testis and normal findings on hormonal studies, for the following indications:

  • Rule out partial obstruction in patients with severe oligospermia
  • Evaluate patients with hypogonadotropism to select those likely to respond to gonadotropin replacement
  • Retrieve spermatozoa in azoospermic patients undergoing IVF or ICSI.

The procedure may be performed under spinal, general, or even local anesthesia, and it may be performed as an open procedure or percutaneously. Open surgery allows better testicular control and generally results in a better test, allowing multiple areas to be sampled for the presence or absence of sperm. A touch preparation of the testicular tissue, obtained from either an open or needle-core biopsy, may aid in a prompt evaluation during the procedure and, if used on a sterile slide, may even be cryopreserved for later use.

An operating microscope is often helpful to assist in identification of healthy-appearing tubules, especially in patients with Sertoli-cell–only syndrome.

In addition, vasography may be performed at the same time to evaluate for obstruction.

Potential complications include pain, bleeding, and inadvertent epididymal biopsy that may give false results and can lead to secondary obstruction.

A small window should be used if a later reconstruction is anticipated, to decrease the risk of adhesions within the tunica vaginalis.

Hemostasis must be pristine to decrease the risk of a hematocele.

When performing diagnostic biopsies, consider obtaining biopsies from both testicles, due to a reported 40% discordance in pathology between the 2 sides.

Usually, it is recommended that testicular tissue be cryopreserved at the time of biopsy for potential future use in IVF.

Table. Abnormal Findings on Semen Analysis: Possible Follow-up Tests* (Open Table in a new window)



Conclusion/Further investigation

Ejaculate volume

Low (< 1.5 mL)

Postejaculation urine (retrograde ejaculation)

TRUS (absence of vas deferens)

Hormonal evaluation (hypogonadism)

High (>5 mL)

Likely contaminant

Semen quality

Does not coagulate

TRUS (ejaculatory duct obstruction)

Does not liquefy

Hormonal analysis

Sperm density

Oligospermia (< 20 million per mL)

Severe oligospermia (< 5 million per mL)

TRUS (partial ejaculatory duct obstruction)

Antisperm antibody evaluation

Hormonal analysis

Physical examination for varicocele


Sperm centrifuged to verify azoospermia

Postejaculation urine (retrograde ejaculation)

Hormonal evaluation

Testicular biopsy (testicular failure)

TRUS (ejaculatory duct obstruction)



Antisperm antibodies

Physical examination for varicocele

*All semen analyses with abnormal results should be repeated.


Histologic Findings

Biopsy samples in patients with infertility from pretesticular causes have atrophic cells due to a lack of gonadotropin stimuli. Prepubertal hypogonadotropism leads to small, immature seminiferous tubules with delicate tunica propria and a lack of elastic fibers. In contrast, patients with postpubertal hypogonadism show few or no germ cells, shrunken tubules, and a thickened, hyalinized tunica propria.

Primary testicular failure causes various defects. Normal-sized seminiferous tubules, normal Leydig cells and Sertoli cells, and a normal tunica propria characterize maturation arrest, but germ cells are arrested at any premature stage. Patients with hypospermatogenesis have a thin germinal epithelium and a decreased number of germinal elements. Germ cell aplasia (Sertoli-cell–only syndrome) is associated with vacuolated Sertoli cells and no germinal epithelium but otherwise normal seminiferous tubules. Klinefelter syndrome is characterized by a decreased number of spermatogonia, germ cell hypoplasia, Sertoli cell atrophy, tubular hyalinization, prominent Leydig cells (hyperplasia), and deformed tubules. Cryptorchid testes have small immature tubules, spermatogonia of variable size, and a hyalinized tunica propria.

Acute mumps orchitis is associated with interstitial edema, mononuclear infiltrate, and a degeneration of germinal epithelium, while recovery is characterized by a patchy loss of germ cells with tubular hyalinization and sclerosis.

Posttesticular obstruction leads to increased tubule diameter, increased thickness of the tunica propria, and a decreased number of Sertoli cells and spermatids. Sloughing of the germinal epithelium may be present.