Diagnostic Methods in Male Infertility

What is Male Infertility?

Despite significant advances in reproductive techniques over the past 10-15 years, there have been no major breakthroughs in correcting impaired spermatogenesis. This is largely due to a lack of understanding about the causes of infertility. Various genetic anomalies and environmental gonadotoxins, among many other factors, can contribute to male infertility. Therefore, the more accurately the etiology is determined, the more successful the treatment will be.

The chance of conception for couples is approximately 20-25% per menstrual cycle (1-3% in infertile couples),75% within 6 months, and 90% within 1 year. About 15% of couples experience infertility issues. The highest fertility chance for both men and women is at age 24. In couples presenting with infertility, the problem is solely male-related in 20% of cases, and in 30-40% of cases, there is pathology in both partners.

In conclusion, about half of the couples who do not have children have a male-related problem. Even without any treatment, 25-35% of these infertile couples will conceive with just conscious sexual activity, most of which happens within the first 2-3 years. While keeping this in mind, both the duration of infertility and the age of the couple at the time of consultation are very important.

As with other medical problems, there are three fundamental components in diagnosing male infertility: medical history, physical examination, and laboratory tests. Based on these, differential diagnosis is made, and if necessary, more specific tests are conducted.

How is Male Infertility Diagnosed?

Taking a thorough sexual and medical history is the most important tool in reaching a diagnosis and will guide the physical examination. It is crucial to learn about the duration of infertility, previous miscarriages, abortions, stillbirths, contraceptive methods used, frequency and timing of sexual intercourse.

The couple’s knowledge about fertilization and conception should be reviewed. It should be reminded that ovulation occurs exactly mid-cycle and that intercourse outside this period usually does not contribute to pregnancy. Sexual intercourse timing does not always have to coincide exactly with ovulation because sperm can survive in cervical mucus for up to 48 hours.

Since the lifespan of the ovum is short, intercourse after ovulation reduces the chance of pregnancy. Although there are some debates, having intercourse every other day during the ovulation week is generally accepted. Very frequent intercourse may reduce the number of sperm in the vagina, while too infrequent intercourse may result in missing ovulation.

Lubricants used during intercourse (such as K-Y jelly, Astroglide, saliva) often impair sperm motility, so they should be avoided or used minimally if possible. Erectile and ejaculatory function must always be questioned.

Developmental history should include inquiries about undescended testicles, puberty age, gynecomastia, and congenital abnormalities of the urinary and central nervous systems.

Surgical history is particularly important. Pelvic and retroperitoneal surgeries (such as RPLND) may impair erectile and ejaculatory function. Surgeries involving the bladder neck can cause retrograde ejaculation. During hernia repair, the vas deferens may be injured or its blood supply compromised. Similarly, surgeries such as hydrocelectomy can injure the vas deferens or epididymis. Testicular trauma or torsion can cause testicular atrophy.

Medical history must include past urinary infections, sexually transmitted diseases, kidney disease, chemotherapy, radiotherapy, epididymitis, diabetes, tuberculosis, other chronic diseases, and anosmia. Mumps usually affects the testes after puberty (in 10-30% of cases) and bilateral involvement has been reported in 20-60%. Testicular tumors and lymphomas are associated with oligospermia in about 60% of cases at diagnosis.

Recently experienced febrile illnesses can impair spermatogenesis for up to 3 months. Therefore, to assess basal fertility status, a sperm analysis should be performed at least 3 months after such infections. Chronic upper respiratory infections can be associated with infertility. Kartagener syndrome (immotile cilia syndrome) should be suspected if sperm motility is absent and there is a history of frequent respiratory infections. Nearly all males with cystic fibrosis have congenital absence of the vas deferens.

In congenital bilateral absence of the vas deferens, most men have mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. The combination of frequent respiratory infections and azoospermia suggests Young syndrome, where secretions cause epididymal obstruction. Pituitary tumors may present with severe headaches, galactorrhea, and visual field defects. Kallmann syndrome features congenital hypogonadotropic hypogonadism along with anosmia and midline defects.

Many drugs and substances (nitrofurantoin, cimetidine, sulfasalazine, cocaine, nicotine, marijuana) can impair spermatogenesis. The androgenic components of anabolic steroids can cause hypogonadotropic hypogonadism. Exposure to pesticides and environmental toxins may have gonadotoxic effects. Individuals with suboptimal sperm parameters should avoid saunas and hot tubs.

The effect of smoking on spermatogenesis is not fully clear. A meta-analysis including 21 studies showed smoking reduces sperm concentration by 13-17%, whereas 14 other studies did not show this effect.

Evaluation of the Female Partner

Abnormalities are detected in approximately three-quarters of infertile couples in the female partner. Ovulation disorders are seen in 30%, fallopian tube abnormalities in 25%, endometriosis in 4-5%, cervical mucus abnormalities in 4%, and hyperprolactinemia in 4%. The following methods are used to assess ovulation and the luteal phase:

Menstrual History: A regular menstrual cycle occurring every 21-35 days indicates the presence of ovulation. A cycle shorter than 21 days or longer than 35 days suggests ovulation problems.
Basal Body Temperature Monitoring: There is a temperature rise of at least 0.4°F approximately 12-15 days after ovulation. Just before this rise, a slight temperature drop occurs, which indicates ovulation. However, it is not always possible to detect this reliably.
Mid-luteal Phase Serum Progesterone Level: A level above 3 ng/mL indicates ovulation, and a level above 10 ng/mL suggests a normal luteal phase.
LH Levels in Blood and Urine: LH levels increase just before ovulation.
Endometrial Biopsy: Performed a few days before the expected menstruation to detect luteal phase defects.
Transvaginal Ultrasound: Used to evaluate follicular development before ovulation.
Day 3 FSH Level: An FSH level above 10 mIU/mL on the 3rd day of the menstrual cycle indicates diminished ovarian reserve.

Evaluation of the Fallopian Tubes

Pelvic inflammatory disease (PID),endometriosis, and previous abdominal surgeries may cause tubal disease.

Hysterosalpingography (HSG): Evaluates the patency of fallopian tubes and uterine cavity. It is performed in the early follicular phase of the menstrual cycle to avoid radiation exposure to a fertilized ovum. Cornual spasm can often mimic proximal tubal obstruction; therefore, this test should be repeated before making a diagnosis. It is later confirmed with laparoscopy.
Laparoscopy: Combined with the trans-cervical injection of diluted dye to assess tubal disease.
Sonohysterography: Involves the injection of contrast material through the cervix combined with ultrasound imaging.

Evaluation of the Uterus

The uterus is evaluated using HSG, ultrasound (USG),and hysteroscopy.

Evaluation of the Peritoneal Cavity

If the history and physical examination suggest endometriosis, tubal, or ovarian adhesions, the most sensitive and specific diagnostic test is laparoscopy.

The evaluation of women takes more time compared to men; therefore, the woman’s age must always be taken into account before recommending treatment. Conception rates decline rapidly in the 35-39 age group.

Physical Examination of Male Infertility

The examination begins as the patient enters the doctor’s office. Abnormalities in secondary sexual characteristics may indicate an endocrine pathology. Gynecomastia can be due to elevated prolactin (PRL) or an imbalance between estrogen and androgen. Situs inversus should raise suspicion for Kartagener syndrome.

Genital Examination

The penis should be examined for hypospadias and significant chordee.
The testes should be evaluated for the presence of masses and size. About 80% of testicular volume is composed of seminiferous tubules and germinal elements. A reduction in these leads to decreased testicular volume. Normal testicular volume (measured by orchidometer or ultrasound) is approximately 4x3 cm or greater than 20 ml.
When there is obstruction in the epididymis, induration or cystic dilation may be detected on examination. Spermatoceles and epididymal cysts can be palpated.
Palpation of the vas deferens is very important to confirm their presence.
The spermatic cords should be evaluated for varicocele. Grade I varicoceles are palpable only during the Valsalva maneuver; Grade II varicoceles can be palpated when standing; Grade III varicoceles are visible even through the scrotal skin. For diagnosing subclinical varicoceles, venography, Doppler stethoscope, and duplex ultrasound are used. On ultrasound, the presence of multiple veins with at least one having a diameter greater than 3 mm confirms the diagnosis of subclinical varicocele. Since there are no controlled studies showing increased pregnancy rates after diagnosis and treatment of subclinical varicoceles, extensive evaluation of these cases is not generally recommended.
Although some recommend a digital rectal exam for prostate and seminal vesicle evaluation, it should be remembered that many pathologies detected by transrectal ultrasound (TRUS) are missed during the physical exam. Rectal examination is more appropriate if there are clues in the patient’s history.

Basic Laboratory Evaluation of Male Infertility

At least 2 or 3 semen analyses should be performed for all patients. The first and most fundamental test to evaluate an infertile man is the semen analysis. However, except for azoospermia, semen analysis results do not definitively classify a man as fertile or sterile. Abnormalities in semen parameters only indicate a statistically reduced probability of fertility.

Semen Analysis

Typically, a semen sample is provided after 3–5 days of sexual abstinence. During the first 4 days, sperm count increases by about 25% per day, while sperm motility and morphology remain unchanged. If abstinence exceeds one week, a decrease in motility has been reported. If the sperm sample is brought from outside the laboratory, it must be kept at body temperature and delivered within 1–2 hours.

The coagulum of the ejaculate is formed by seminal vesicle secretions and liquefies within 5–25 minutes due to prostate-derived proteases such as PSA and plasminogen activators. If the seminal vesicles are absent or hypoplastic, the semen usually does not coagulate, appears acidic, and has a reduced volume. Hyperviscous semen and non-liquefied semen are not the same. Non-liquefied semen maintains its coagulum properties, while hyperviscous semen loses coagulum characteristics over time but remains relatively thick and does not drip like liquefied semen. Previously, genital tract infections were blamed for hyperviscosity, but this is currently unclear. If semen liquefaction is absent or hyperviscous, a postcoital test is recommended. If adequate numbers of motile sperm are present in the cervical mucus during this test, semen viscosity is not considered problematic. However, if cervical mucus is good but sperm count is low, artificial insemination is often recommended. Adding alpha-amylase or semen extender can improve liquefaction, but there is no evidence that this improves fertility.

Low Ejaculate Volume: According to WHO, the normal volume is 2 ml or more. The complete absence of seminal fluid is called aspermia. This can result from either retrograde ejaculation or absence of emission. The most common cause of absent emission is spinal cord injury, frequently seen in diabetes and multiple sclerosis. Retroperitoneal surgery can damage sympathetic ganglia controlling ejaculation, but nerve-sparing techniques (e.g., RPLND) have reduced this complication. Psychological disorders such as inability to orgasm can also cause aspermia. The most common cause of low ejaculate volume is improper specimen collection; repeating the test is important to avoid this error. Partial retrograde ejaculation, due to neurological disorders, medications, bladder neck surgery, or idiopathic causes, is another common cause. Seminal vesicle secretions make up most of the seminal fluid. Low ejaculate volume can also indicate partial or complete ejaculatory duct obstruction. In vasal agenesis, seminal vesicles are often absent, resulting in low ejaculate volume. Since seminal vesicles and prostate are androgen-dependent, androgen deficiency also reduces volume.

Some men may produce more sperm during intercourse and may bring the specimen using a condom. Abstinence duration should also be considered. In cases of low ejaculate volume, the first step is centrifuging the post-ejaculatory urine specimen at 300 g for 10 minutes. Finding 10–15 sperm/HPF in the pellet confirms retrograde ejaculation. If negative, obstruction should be evaluated by transrectal ultrasound (TRUS).

The average sperm count reported in fertile populations is 70–100 million per milliliter. Fertility depends on multiple semen parameters and factors involving both partners, so sperm counts may overlap between fertile and infertile groups. According to WHO reference values, the minimum normal sperm count is 20 million/ml. Counts below this are termed oligospermia. Isolated oligospermia is rare, usually idiopathic, and sometimes linked to androgen deficiency. If count is below 10 million/ml, testosterone and FSH levels should be measured. Elevated FSH alone indicates impaired spermatogenesis and a full endocrine evaluation is usually not needed. The most common identifiable cause of oligospermia is varicocele, which usually causes multiple seminal parameter defects.

Azoospermia refers to complete absence of spermatozoa in the ejaculate. It occurs for three reasons: insufficient hormonal stimulation (hypogonadotropic hypogonadism),spermatogenesis failure, and obstruction. The initial step is centrifuging the semen and examining the pellet for sperm presence. If sperm are found, ductal obstruction is excluded. Physical examination findings such as testicular size and consistency, epididymal fullness, and vas deferens presence guide diagnosis. Obstructed testes are usually larger than normal with prominent epididymes. Although not a strict rule, testes with testicular failure tend to be smaller. Hypogonadotropic hypogonadism patients show decreased virilization and secondary sexual characteristics with very small testes. Serum LH, PRL, and testosterone should be measured in these cases. Patients with very small testes and 2-3 times elevated FSH have severe germ cell failure; if spermatozoa are found on biopsy, intracytoplasmic sperm injection (ICSI) is used to achieve fertility. Genetic tests may reveal chromosomal anomalies and Y chromosome microdeletions in these cases. About 6% of infertile men show chromosomal anomalies on karyotype analysis. The frequency of anomalies increases as sperm count decreases. Karyotype abnormalities are found in 10-15% of azoospermic patients, 4-5% of oligospermic patients, and 1% of normospermic men. The most common chromosomal anomaly is Klinefelter syndrome, with spermatozoa found in about 50% of TESE procedures. Y chromosome long-arm microdeletions occur in about 13% of azoospermic and 3–7% of oligospermic patients. The AZF (azoospermia factor) region on the Y chromosome long arm is subdivided into AZFa, AZFb, and AZFc. Larger microdeletions in the AZF region correlate with greater loss of spermatogenic activity. Defects in AZFa and AZFb predict poorer prognosis for sperm retrieval. Specific gene mutation analyses may be necessary in some cases. Mutations in the CFTR gene cause cystic fibrosis and congenital bilateral absence of the vas deferens (CBAVD). All azoospermic patients with CBAVD lack clinical cystic fibrosis but carry mild CFTR mutations. Due to the high carrier rate, genetic testing is strongly recommended for partners in some countries such as Northern Europe. Mutation panels commonly used detect frequent mutations; a negative test does not guarantee absence of mutations. Unknown gene mutations may also cause infertility.

Sperm Motility should be assessed within the first 1–2 hours. The quality of forward progression is evaluated on a scale of 0 to 5:

0: no movement
1: non-progressive movement
2: slow movement, often in place
3: moderate speed with straight-line movement
4: fast movement in a straight line

Another classification:

A: rapid progressive motility
B: slow progressive motility
C: non-progressive motility
D: immotile

According to WHO, the combined ‘a+b’ motility should be above 50%, and ‘a’ motility alone should be above 25%.

Asthenospermia refers to abnormal sperm motility and can be caused by ultrastructural defects in spermatozoa, prolonged abstinence, genital tract infections, antisperm antibodies, partial ductal obstruction, varicocele, and idiopathic factors. Toxic contaminants in collection containers or exposure of semen to extreme temperatures can also cause asthenospermia.

In patients with low ejaculate volume and asthenospermia, partial ductal obstruction should be considered and TRUS performed.

Rare sperm agglutination usually has no significance, but frequent agglutination warrants investigation for antisperm antibodies (ASA). The blood-testis barrier is formed by tight junctions between Sertoli cells in seminiferous tubules. Ductal obstructions disrupt this barrier, leading to ASA formation. ASA are present in about 60% of vasectomy patients and one-third of CBAVD patients. The role of testicular torsion, biopsy, varicocele, and cryptorchidism in ASA formation is debated. An abnormal postcoital test, especially with shaking immotile sperm, strongly suggests ASA presence. Direct tests identifying antibodies on sperm are clinically more important than indirect serum tests, as direct tests can be negative in some cases with positive indirect tests. Immunobead assay and mixed agglutination reaction (MAR) are used to detect ASA (IgA and IgG). Many labs consider a sample positive if more than 20–50% of sperm are bound by ASA. Direct tests are used for screening; indirect tests measure serum titers. Low concentrations of ASA have less impact on fertility. ASA affect sperm motility, cervical mucus penetration, capacitation, acrosome reaction, zona pellucida binding and penetration, and oolemma binding. ASA are detected in about 10% of infertile men versus 2% in fertile men.

If most sperm are immotile or motility is around 5–10%, a sperm viability test (e.g., Eosin Y, HOST test) should be performed. If sperm are non-viable, the condition is called necrospermia. If most sperm are viable but immotile, primary ciliary dyskinesia (immotile cilia syndrome) and Kartagener syndrome should be considered. These patients have defects in axonemal dynein arms of respiratory cilia and sperm flagella, diagnosed by electron microscopy. Another ultrastructural defect is fibrous sheath dysplasia.

Morphological Defects are called teratospermia. Accurate morphology assessment requires examination of stained semen samples. In older criteria, samples with more than 60% normal and less than 3% immature forms were considered normal, including borderline forms. Currently, stricter criteria are used, and borderline forms are included in the abnormal group. Kruger et al. reported fertilization rates of 37% with less than 14% normal sperm morphology and 81–91% with higher normal morphology in IVF patients with >20 million/ml sperm count and >30% motility (1988). Another study reported fertilization rates of 7.6% with less than 4% normal morphology and 63% between 4–14%. Pregnancy rates with intrauterine insemination (IUI) are significantly lower when normal morphology is below 4%. Morphology should be evaluated with other parameters. Spontaneous pregnancies may still occur with very low morphology. Some research suggests the acrosome index (percentage of sperm with normal acrosomes) is important, especially in those with less than 5% normal forms by strict criteria; an acrosome index of 5–15% corresponds to fertilization of over 50% of oocytes in IVF.

When sperm concentration, motility, and morphology are all impaired, the condition is called oligoasthenoteratospermia (OAT),most commonly caused by varicocele. This is the most frequent semen analysis abnormality.

According to WHO, leukocyte count should be less than 1 million/ml. Unstained wet mounts can confuse leukocytes with immature germ cells, collectively called round cells. Patients with poor semen quality have increased immature sperm counts. When 10–15 round cells per high power field (or >1 million/ml) are observed, leukocyte staining is required to differentiate leukocytes from immature sperm. Papanicolaou staining is traditional but requires expertise. Immunohistochemical techniques using monoclonal antibodies against leukocyte surface antigens are more complex. Peroxidase staining detects peroxidase inside leukocytes but not in monocytes. Only one-third of cases with increased round cells have true pyospermia (pus in semen). The significance of pyospermia is debated. Some patients with bacteriuria do not have pyospermia. Semen cultures are often contaminated with distal urethral organisms. DNA testing (PCR) for Chlamydia trachomatis in urine is used to assess pyospermia.

Semen pH is ≥7.2 due to acidic prostate and alkaline seminal vesicle secretions. Fructose (120–450 mg/day) in semen is produced by the seminal vesicles under androgen control. Absence or obstruction of seminal vesicles results in acidic, fructose-negative, non-coagulating, low-volume (<1 ml) azoospermic ejaculate.

Computer-assisted sperm analysis (CASA) does not provide a more reliable prognosis than manual methods and has limited clinical use.

Hormonal Evaluation

Less than 3% of infertile men have a primary hormonal cause. The most common hormonal abnormality is elevated FSH levels. Increased FSH indicates a significant problem in spermatogenesis, whereas normal FSH levels do not guarantee normal spermatogenesis. In hypothalamic and pituitary dysfunction, serum gonadotropin and testosterone levels are low, accompanied by impaired spermatogenesis. Since LH is released episodically, measurements ideally require pooling samples taken at 15-minute intervals; however, a single measurement often accurately reflects the patient’s clinical status. Because endocrine abnormalities are very rare in men with sperm concentrations above 10 million/ml, measuring serum FSH and morning testosterone levels is recommended only when the count is below this threshold. Some authors argue that PRL and LH should also be included in the hormonal panel. If isolated testosterone levels are normal or borderline low and LH is not elevated, bioavailable testosterone should be measured.

If prolactin (PRL) levels are elevated, pituitary MRI should be performed. Especially when visual field defects and headaches are present, central nervous system tumors should be considered. Not all pituitary tumors are functional; PRL levels may be normal. Prolactin-secreting tumors are usually macroadenomas (>1 cm). In these patients, PRL levels exceed 50 ng/mL, and gonadotropin and testosterone levels are suppressed. In infertile patients, lower PRL elevations (<50 ng/mL) may be seen, but their clinical significance is debated. Mild PRL increases can be caused by stress, renal failure, medications, chest wall irritation, and thyroid dysfunction.

GnRH stimulation tests and hCG stimulation tests have no routine role.

Infertility due to congenital adrenal hyperplasia (CAH) is very rare; many patients with CAH are fertile. It is proposed that excessive adrenal androgens inhibit pituitary axis and testicular stimulation, leading to infertility.

In morbidly obese men, aromatase enzyme in adipose tissue converts testosterone to estradiol, causing estrogen excess. Estradiol stimulates hepatic production of sex hormone-binding globulin (SHBG),which lowers bioavailable testosterone. Estrogen excess can cause bilateral gynecomastia, erectile dysfunction, and testicular atrophy. Estradiol levels in men are very low, making measurements unreliable. The use of testosterone/estradiol ratios has been suggested, but normal values are not clearly established.

Radiological Evaluation

The purpose of radiological evaluation is to detect partial or total ductal obstruction. However, distinguishing partial ductal obstruction from other causes of infertility, especially idiopathic oligospermia, is very difficult.

Vasography: Vasography is used to locate the site of obstruction in azoospermic cases where spermatogenesis has been confirmed by testicular biopsy. It should be performed together with reconstructive surgery because the procedure itself carries a risk of damaging the vas deferens. Typically, saline is injected to demonstrate distal patency at the vasal level. If saline does not flow freely, diluted non-ionic contrast medium is used to identify the obstruction site. If the contrast agent is seen in the vas deferens, seminal vesicles, ejaculatory ducts, and bladder, the vasography is considered normal. To check proximal patency, vasal fluid is examined microscopically, and the presence of sperm indicates patency. Another method for detecting ejaculatory duct obstruction (EDO) is seminal vesiculography, but if performed transrectally rather than transperineally, there is a higher risk of infecting the seminal vesicles.

Transrectal Ultrasonography (TRUS): Performed in azoospermic patients suspected of EDO to anatomically visualize the prostate, seminal vesicles (SV),and ampullary part of the vas deferens. On transverse imaging, the normal diameter of seminal vesicles behind the bladder is up to 1.5 cm. Absence or hypoplasia of seminal vesicles is easily identified, but in some patients with complete EDO, seminal vesicles may not be dilated. In these cases, seminal vesicle aspiration under TRUS guidance can be performed. Intraprostatic cysts and hyperechoic lesions in the prostate have been suggested to associate with EDO, but hyperechoic lesions are also seen in fertile populations.

Scrotal Ultrasound (USG): Used for detecting varicoceles but is unnecessary in patients with normal physical examination. If obesity prevents an adequate physical exam, color Doppler ultrasound may be used. A vein diameter greater than 3.5 mm is required for a clinical varicocele diagnosis. In some subfertile patients, testicular tumors can be detected by ultrasound. Leydig cell tumors are usually not palpable but can cause elevated serum testosterone and gynecomastia. Scrotal ultrasound is recommended in these cases.

Abdominal Ultrasound (USG): Renal agenesis occurs in approximately 80% of cases with unilateral absence of the vas deferens. Bilateral absence has a different mechanism and renal agenesis is less common. Renal anomalies should be evaluated by ultrasound in these cases.

Sperm Function Tests

Post-Coital Test (PCT): This test examines the interaction between sperm and cervical mucus. It should be performed when the cervical mucus is clear and thin, ideally just before ovulation. A drop of cervical mucus is examined under the microscope. A normal result shows 10-20 sperm per high-power field (HPF) at 400x magnification. A normal result indicates no problem with cervical mucus or semen accumulation. Abnormal results can be due to improper timing, antisperm antibodies (ASA),abnormal semen, or improperly timed intercourse. Due to issues with standardization and reproducibility, PCT is not routinely used. In cases with normal semen but abnormal PCT, concerns about cervical mucus quality arise. Some authors recommend an in vitro cervical mucus interaction test in this situation, while others do not, since it may not change treatment.

Acrosome Reaction: For sperm to fertilize the egg, capacitation and acrosome reaction are necessary. Transmission electron microscopy (the gold standard) allows observation of this process. Tests inducing acrosome reaction use agents such as ionophore A23187 or zona pellucida. Normally, spontaneous acrosome reaction occurs in less than 5% and induced reaction in 15-40% of sperm in fertile samples. In infertile samples, the opposite pattern is observed. This test is available only in very limited centers.

Sperm Penetration Assay: The zona pellucida surrounding the ovum is a glycoprotein barrier preventing fertilization by other species. If this barrier is removed, human sperm can fertilize hamster eggs. Scoring is based on the percentage of eggs penetrated by sperm or the number of sperm penetrating each egg. This assay evaluates sperm’s capacitation, acrosome reaction, fusion with the oolemma, and integration into the ooplasm.

Hemizona Test: The zona pellucida is microscopically bisected; one half is incubated with normal fertile semen, the other with the patient’s semen. The number of sperm bound to each half is counted. The ratio of sperm bound to the patient’s half relative to the control is calculated. If this ratio is below 0.6, the patient’s chance of fertilization is considered low. This test is not widely used.

Sperm Vitality Tests: Eosin Y and Trypan Blue stains are commonly used. Dead sperm take up the stain, while live sperm do not. These tests are performed when sperm motility is below 5-10%. The Hypo-Osmotic Swelling Test (HOST) relies on the principle that live sperm with intact membranes swell in a hypo-osmotic solution due to water influx; dead sperm do not swell. HOST is very useful in selecting sperm for ICSI; only swollen cells are used.

Reactive Oxygen Species (ROS) Test: ROS include superoxide radicals (O2-),hydrogen peroxide (H2O2),and hydroxyl radicals (OH-). Low levels of ROS are needed for sperm hyperactivation and capacitation. Excessive ROS causes damage to sperm cells. Both leukocytes and sperm produce ROS. Infertile men have higher ROS levels in seminal fluid than fertile men. Antioxidants normally neutralize ROS, but infertile patients often have low antioxidant levels. The balance between ROS production and total antioxidant capacity (TAC) is measured by the ROS-TAC score; a score below 30 is associated with infertility. There is no consensus on ROS test indications, so it is not routinely used.

Chromatin/DNA Integrity Tests: During spermatogenesis and epididymal transport, sperm chromatin undergoes specific changes, resulting in tightly organized, dense chromatin resistant to genetic damage. Although DNA damage can be repaired post-fertilization in the egg, chromatin organization defects can cause infertility. Factors such as smoking, pollution, industrial toxins, cancer, and fever cause abnormal DNA. Apoptosis is a normal process during spermatogenesis; defects in apoptosis lead to mature sperm with DNA breaks. ROS-induced oxidative stress also damages sperm DNA. DNA damage is measured by TUNEL and comet assays. The Sperm Chromatin Structure Assay (SCSA) does not directly measure DNA breaks but assesses chromatin susceptibility to denaturation in an acidic environment, yielding a DNA Fragmentation Index (DFI). Abnormal semen parameters usually correlate with abnormal DNA integrity, but even men with normal semen can have abnormal DNA tests. Studies show that DFI above 30% predicts low pregnancy chances with 15% sensitivity and 96% specificity. This means high DFI lowers pregnancy chances, but low DFI does not guarantee pregnancy. Results on the impact of DNA damage on ICSI outcomes are inconsistent. DNA/chromatin damage, occurring after sperm leave the testis, does not affect fertilization or early embryo development but impacts later embryonic stages and miscarriage rates. Using testicular sperm instead of ejaculated sperm with lower DFI can improve pregnancy rates in some cases. Testing is indicated in unexplained infertility, repeated IVF failures, and miscarriages, though universal standards are still needed.

Testicular Biopsy

Testicular biopsy has traditionally been performed for two reasons. The first is to differentiate between obstructive and non-obstructive azoospermia, and the second is to obtain sperm for IVF or ICSI. However, if the absence of the vas deferens is detected during clinical examination, performing a testicular biopsy becomes unnecessary. Bilaterally small testes and elevated FSH levels indicate testicular failure. Since sperm retrieval chances improve with extensive testicular dissection in all histological diagnoses (hypospermatogenesis, maturation arrest, germ cell aplasia),reliance on a single diagnostic biopsy has decreased.

Recommended References:

Sigman M, Jarow J. Male infertility. In Campbell-Walsh Urology. Eds: Wein A, Kavoussi L, Novick A, Partin A, Peters C. Saunders Co., 9th Edition, pp. 609-634, 2007.
Dohle GR, Jungwirth A, Colpi G et al. Guidelines on male infertility. In EAU Guidelines DVD, pp. 1-70, 2008.
World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, New York, Cambridge University Press, 1999.
Honig SC, Lipshultz LI, Jarow J. Significant medical pathology uncovered by a comprehensive male infertility evaluation. Fertil Steril 62: 1028-1034, 1994.