Advanced techniques for the diagnosis of male infertility

Specialist diagnosis of male infertility.

Medical interview and physical examination

The basis for the diagnosis of male infertility is the medical interview and physical examination of the reporting patient. It is important to create an intimate atmosphere during the examination, which will help to secure the patient’s full confidence in the doctor.

During the interview, attention should be paid to the exact duration of trying for a child and the frequency of sexual intercourses as it often happens that the patients talk about many years of trying but forget to add that they see each other at several weeks’ intervals.

Further questions should concern the patient’s physical and intellectual development as well as the development of reproductive organs (whether testicles have always been in the scrotum). The patient should be asked about past surgeries, infectious diseases – especially mumps during and after adolescence. Definitely, the patient should be asked about the proper function of olfaction (a symptom which suggests Kallman’s syndrome) and potential erectile dysfunction (it is best to ask whether the patient experiences spontaneous morning erections). We can’t omit the questions concerning the presence of chronic inflammation of the respiratory system. If the symptom mentioned above is accompanied by the presence of immobile sperm, we can suspect the Young’s syndrome (damaged cells with cilia and damaged sperm)(2).

Information about the medications taken by the patient is also important as they may affect spermatogenesis. We mustn’t forget about the patient’s job – exposure to toxic substances, excessive warmth, etc.

We pay attention to the patient’s body structure. We look for eunuchoid characteristics, examine the thyroid gland, look for gynecomastia and galactorrhea. Physical examination should include the assessment of the condition of epididymides, vasa deferentia, size of testicles (normal size: approximately 20mL). Testicular atrophy is observed in primary testicular failure, Klinefelter’s syndrome, endocrinopathies, after mumps infection and in case of hepatic dysfunction.

The examination should rule out the presence of higher grade varicoceles which are considered to be one of the probably more common causes of male infertility (24).

During penis assessment, we should pay attention to the presence of hypospadias or epispadias which may cause abnormal depositing of semen during intercourse and lead to infertility. In the per rectum examination, we can assess the prostate gland, its size, signs of infection and the presence of cysts.

General semen analysis

General semen analysis is the basis of diagnosis in male infertility. It should be carried out, if not in the first place, in parallel with the start of diagnosis in a woman with the aim to look for the causes of couple infertility.

The analysis is carried out between the 2nd and the 7th day of sexual abstinence. It should be repeated after 3-4 weeks as the semen parameters change over time (11). Normal semen parameters obtained twice significantly exclude the male infertility factor in a high percentage of patients. Naturally, abnormal semen parameters do not exclude conception. There are well-known studies where 7.7% of couples achieved pregnancy despite severe oligozoospermia in the male partner.(4)

WHO recommends that semen should be collected by masturbation to a clean, dry container (1). However, “naprotechnologists” recommend that semen should be obtained during sexual intercourse with the use of a special condom. This conduct isn’t only related to the teachings of the Catholic Church but it is based on studies conducted by Zavos which report better semen parameters in ejaculates obtained during sexual intercourse than by masturbation. This concerns in particular patients with oligozoospermia(5).

Assessment of semen parameters

Semen volume – 1.5–5 mL.
Decreased semen volume or its absence is observed in case of obstruction or absence of deferent ducts and seminal vesicles, retrograde ejaculation, hypogonadism, incomplete semen collection. Increased semen volume may indicate the inflammation of the prostate gland or sample contamination with urine. When the semen volume is below 1 mL, urinalysis has to be performed in order to rule out retrograde ejaculation.

• pH>7.2
  Decreased pH is observed in the case of seminal vesicle dysfunction or ejaculatory duct obstruction.
• Liquefaction: normal liquefaction occurs within 60 minutes.
  Incomplete liquefaction may indicate abnormal prostate function, liquefaction that is too watery suggests the dysfunction or absence of seminal vesicles.

• Semen concentration/density:
  According to WHO 2010 norms, normal sperm concentration is 15 million sperm cells in 1 mL of ejaculate, but not less than 39 million in the whole volume.

Using these criteria, we can diagnose oligozoospermia when the sperm number is below 39 million/ ejaculate. We talk about severe oligozoospermia when the sperm number is below 5 million/1 mL of ejaculate. Cryptozoospermia is the condition where there are no sperms in the direct preparation but they are present in the pellet remaining after sample centrifugation. Azoospermia means the absence of sperm in direct preparation and after centrifugation of the preparation. In the case of azoospermia and when the ejaculate volume is low, urine passed after ejaculation has to be assessed.

• Sperm motility >32% of the total sperm count.
  Sperm motility defines the percentage of the total number of sperm cells making movements of any type. WHO in its actual guidelines divides sperm only into motile and immotile sperm cells.. It has been reported that the probability of conception increases when sperm motility increases up to approximately 60% (6).
• Vitality >58%
  In the vitality assessment, we most often use the eosin test. Live sperms do not stain when the dye is added to the ejaculate.

Another vitality test is hypo-osmotic swelling. Under the influence of hypo-osmotic solution, sperms coil their tails depending on the cell membrane permeability. As compared with other vitality tests, this one is reversible. Decreased sperm vitality correlates with the increased rate of early recurrent miscarriage of unexplained etiology.

• Sperm morphology: minimum 4%.
  It includes sperm structure examination. Sperm cells are observed under a microscope with 100x objective, after the semen smear has been stained using the Schorr’s technique. Abnormalities are classified by their location in the sperm’s structures. In the most recent WHO criteria the percentage of normal sperm was set at only 4%. Increased percentage of abnormal sperm (teratozoospermia) is associated with varicoceles and secondary testicular failure.(1) We can also assess the semen levels of fructose, zinc, citric acid and acid phosphatase. These tests are used to assess the function of seminal vesicles.
• Round cells <5 million/mL Round cells are all cells which are found in semen and which are not sperm. If the number of round cells exceeds the acceptable norm, a precise number of leukocytes should be determined. If this number exceeds 1 million /mL, an infection of reproductive organs can be suspected. The next diagnostic step is semen smear with antibiogram (3).
• Agglutination
  Sperm aggregation may be caused by an inflammatory factor and indicate the presence of antisperm antibodies.
• Antisperm antibodies
  Sperm cells have specific antigens which are not recognized by the immune system as their own. When the blood-testicle barrier is damaged, the body can produce
  antisperm antibodies. The significance of the presence of antisperm antibodies is not clear. We can find publications which report lower pregnancy rates in the partners of men with over 50% of sperm connected to antibodies(9). There are also studies suggesting the presence of two groups of antibodies – causing and not causing infertility(10).
• Computer-aided semen analysis- CASA
  Due to the advances in computerization, automated systems of semen analysis were developed. With these specialist devices, diagnostician bias can be ruled out, thus making the analysis more objective. However, such devices have to be subject to precise system calibrations and rigorous quality controls as the obtained results strictly depend on the manner of semen sample preparation.

Sperm function tests

• Sperm penetration test allows to assess the sperm’s ability to undergo acrosomal reaction, fusion with oolemma and to track the sperm nucleus decondensation in cytoplasma.
  Technically, it consists in assessing the proportions of oocytes (oocytes devoid of zona pellucida obtained from female golden hamsters) penetrated by the sperm of the diagnosed patient, and comparing the results with those of a man with proven fertility (1). The test is not carried out routinely as it is expensive and time-consuming.
• Post-coital test (PCT)
  It allows to assess the mucus penetration by sperm and sperm survival in cervical mucus. It is carried out shortly before the expected ovulation through evaluation of the female partner’s mucus for sperm presence and motility. The test is carried out after 8 to 10 hours from sexual intercourse. Approximately 10% of infertile couples have abnormal PCT result. The test result is considered normal if at least 10 motile sperm cells are visualized per high power field. The abnormal result can suggest the presence of antisperm antibodies, infections of reproductive system in female and/or male partner, hostile mucus, poor semen quality and sexual disorders in a man.
• Assessment of the free radical level
  Normal oxygen metabolism generates free oxygen radicals which can be toxic in excessive amounts. Free radicals are produced both by sperm and leukocytes. Their increased levels are observed in infertile men in connection with sperm dysfunction (14)
• Motile sperm organellar morphology examination – MSOME
  MSOME is among the most modern tests assessing sperm morphology. The assessment of motile sperm allows the detailed analysis of sperm cell structures. The examination is performed at a magnification of minimum 600x. MSOME does not rule out the risk of aneuploidies in sperm. The number of cells with abnormal sperm morphology is proportional to the patient’s age. Such cells contain a large number of vacuoles. MSOME applied in IVF techniques increases the chances of proper pregnancy development.(6)

Hormonal evaluation

Although hormone disorders are detected in only about 3% of infertile men, every patient with abnormal results of semen analysis, erectile disorders, decreased libido or features of endocrinopathy in physical examination should undergo the hormone test panel. The hormonal assessment includes the determination of levels of FSH, LH, prolactin, testosterone, SHBG, estradiol and TSH. Depending on the results of medical interview and physical examination, an appropriate range of tests is recommended.

In patients with hypogonadotropic hypogonadism, we will observe low levels of FSH, LH and testosterone. In hypogonadotropic hypogonadism, high levels of FSH and LH are observed with normal or low testosterone levels. Hyperprolactinemia may cause both disorders of spermatogenesis and sexual disorders. In some patients with oligozoospermia, decreased testosterone to estradiol ratio can be observed, which normalizes to a large extent with the improvement in semen parameters after aromatase inhibitor administration (15).

Imaging tests

In patients with abnormal semen parameters, the risk of testicular cancer is 20 times higher. Therefore, one should always consider the extended diagnosis to rule out a cancer disease (16).

 Ultrasound exams
In patients with azoospermia and severe oligozoospermia, transrectal, scrotal and possibly abdominal ultrasound examination is performed. Transrectal ultrasound is recommended for patients with low ejaculate volume and normal testicular volume, acidic semen pH and decreased fructose level in the case of suspected obstruction of the ejaculatory duct (17). Scrotal ultrasound is carried out to rule out the testicular tumor, verify the absence of a testicle in the inguinal canal and the absence of varicoceles. Veins of pampiniform plexus dilated by more than 3 mm and noticeable reverse flow in color Doppler imaging during the Valsalva maneuver indicate the presence of varicocele (18, 19). Abdominal ultrasound is recommended in men with agenesis of the vas deferens, as there is the increased risk of unilateral renal agenesis (20).

 X-ray exam

 Vasography
Vasography is performed to examine the vas deferens when its obstruction is suspected. The examination is performed with the use of a contrast medium for the purpose of determining the site and size of the obstruction and stenosis of the vas deferens, i.e. in case of azoospermia (3).
Testicular and epididymal aspiration or biopsy (TESA, TESE, MESA). Testicular biopsy is performed in patients with azoospermia. It is possible to perform fine-needle aspirational biopsy with the aim to obtain sperm for IVF-ICSI procedure.

Surgical testicular biopsy

During diagnostic (surgical) biopsy, two sections are collected – one for histopathological examination and one for potential sperm extraction. Histological examination of tissue sections collected from testicles is of significance when we assess the grade of the lack of development of seminiferous epithelium in incomplete forms of hypogonadotropic hypogonadism for the purposes of choosing the therapeutic procedure (21).

Genetic tests

Genetic tests most often performed in the diagnosis of male infertility include karyotype assessment, test for mutations in the CFTR gene, assessment of the Y chromosome microdeletions and sperm DNA fragmentation.

 Assessment of chromosomal disorders
Karyotyping is recommended in men with azoospermia and severe oligozoospermia. Chromosomal disorders occur in approximately 72-9% of infertile men. In 66% of these cases, the abnormal karyotype test result is associated with the diagnosis of the Klinefelter’s syndrome. Karyotyping is recommended in all men with oligozoospermia who start preparations to in vitro fertilization procedure (22).

 Test for CFTR mutation
CFTR mutations are the cause of cystic fibrosis. It has been determined that most men with cystic fibrosis do not have both vasa deferentia (congenital bilateral absence of the vas deferens, CBAVD). Some men who are heterozygous for CFTR mutation are also CBAVD-affected. It should be remembered that a couple with CFTR mutation can use preimplantation diagnosis (23).

 Microdeletions in the Y chromosome
A region in the long arm of the human Y chromosome where genes important for spermatogenesis are located is called the AZF region (azoospermic factor). It is considered that approximately 10% of azoospermic men may have microdeletions in the AZF region (3). Within this region, we differentiate 4 subregions: AZFa, AZFb, AFc, AZFd. We observe the lowest chances of semen collection in men with the absence of sperm when microdeletions cover the AZFa and AZFb subregions. (24)

 Sperm DNA fragmentation
Every sperm has very tightly packed DNA in its head. Various external factors as well as genetic factors can have impact on the degree of sperm genetic material damage. An increased rate of sperm DNA fragmentation is associated, among others, with infections, fever, elevated temperature of the testicular area, cigarette smoking, inappropriate diet, substance abuse, exposure to environmental contamination, as well as the advanced age. Treatment, namely reducing the fragmentation rate, depends on a causative factor. If chromatin damage has been caused by oxidative stress, a change in the lifestyle and a well-balanced diet may improve the DNA damage rate. In case of infections, the use of antibiotic therapy may also improve the fragmentation rate. The efficacy of activities undertaken has to be checked and the genetic material has to be assessed again, 2-3 months after the first analysis.

It has been proved that infertile men have higher level of fragmentation than men with confirmed fertility. Sperm DNA fragmentation is recommended before IVF-ICSI procedure with regard to the health of future children, in connection with the absence of natural barriers in IVF-ICSI procedures (25). In cases of increased fragmentation, sperm separation is recommended, in order to use in ICSI only non-fragmented sperm.

Currently the best technique for the assessment of DNA fragmentation is the microscopic assessment of the sperm DNA damage level (Terminal deoxynucleotidyl transferase dUTP nick end labeling – TUNEL).

It is assumed that the normal values are <15% sperm with DNA damage, decreased fertility – 15%-30%, problems with fertility – >30%.

Many various methods are used in the diagnosis of male infertility, from general semen analysis, hormone tests to genetic testing.

Despite such an extensive arsenal of tests, we can still diagnose to a significant degree the idiopathic infertility in a patient. Nevertheless, thanks to the assisted reproductive technologies (ART), many infertile men from couples who accept ART as the way of treatment may become biological parents.

Authors of the article: Mariusz Łukaszuk, MD, professor Krzysztof Łukaszuk, PhD, MD, Chief of INVICTA Fertility Clinics.

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