Male Infertility Testing

Male Infertility Testing.


The commonest and simplest male fertility testing is a routine semen analysis. The male is normally requested to abstain from ejaculation for a minimum of 2 and a maximum of 5 days before he is advised to dispose of a specimen for assessment. The sample is normally collected in a sterile semen container at the clinic and delivered immediately to the andrology laboratory where the evaluation test is undertaken.

While the most important parameters are the count, motility and morphology of spermatozoa in the ejaculate other factors inclusive of progression, volume, consistency (viscosity) and agglutination reactions are of significance. The most recent World Health Organisation (WHO) criteria for a normal semen specimen are 1.5 ml (and not more than 5 ml) volume, 15 million of spermatozoa per ml ejaculate, 32% progressive motility and 4% of morphologically normal spermatozoa.

Macroscopic Examination

The specimen should liquefy within a maximum of 15 min following disposal (although the WHO criteria allow for 60 min). Specific enzymes (secreted by the prostate gland) are activated immediately following ejaculation aiming at liquefying the semen. Circumstantially semen may not completely liquefy or appears in the form of mucous streaks containing jelly-like grains. This may be indicative of prostate functional impairment or infection such as prostatitis (possibly chronic).

The homogenous opalescent appearance may also be disturbed by the presence of a brownish colour suggestive of red blood accumulation in the ejaculate and which may be of clinical significance. The pH of the specimen which determines its acidity (when lower than 7) or alkalinity (above 7) should be in the range of 7.2- 8.0. If the pH exceeds 8.0 (more alkaline) infection should be suspected based on a diminished prostate secretion of acidic by-products while if lower than 6.8 (severely acidic) agenesis of the seminal vesicles (glands which secrete a significant proportion of the fluid which ultimately becomes semen) should be considered.

Microscopic Examination

This involves the estimation of motility, progression, concentration and morphological assessment of spermatozoa while it aims also at determining the population presence of other cells, agglutination reactions between spermatozoa and debris (waste-disintegration products).

Conventional assessment of progressive motility and morphology entails the inspection of a small volume (8-12μl) of raw semen under light microscopy at magnifications of x200-x600 and at room temperature (23°C). At least 100 spermatozoa are evaluated within one or more microscopic fields and the motility is graded from 1 to 4 based on the following criteria.

Rapid linear progressive motility (covering a distance of at least 20 μm or half the length of a spermatozoon per second). Slow or sluggish progressive motility Non-progressive motility.


The mature spermatozoon is divided into three distinct parts; the head, the neck and the tail. Morphologically abnormal are considered those spermatozoa which curry a defect in their oval-shaped head and/or their compact spiral shaped neck and/or their flagellum type tail. A head piece defect can be due to an abnormality in the size or shape of the sperm head i.e. large, small, amorphous, elongated, duplicated, round, pin-like, tapering etc. A neck or middle piece defect may be associated with absent tail, non inserted or bent tail, irregular, larger or smaller sized structured necks. Tail piece defects may include coiled tails, pin sized, broken, multiple or with irregular widths.

The concentration of spermatozoa into the seminal fluid is a strong indicator of male fertility and while a 30 million total sperm count is recognised as normal the duration of abstinence and volume of the ejaculate should be considered for correct evaluation. Analysis of sperm density is conventionally undertaken using a simple cell counter namely a haemocytometer.

Good Quality Sperm Poor Quality System

Evidence of immunological factor infertility can be obtained from the microscopic observation of sperm to sperm agglutination reactions. Spermatozoa appear to stick to each other most usually in a head to head, head to tail or tail to tail pattern. Such presentations seriously compromise progressive sperm motility which is evident with time.

The simplest test to determine the presence of this autoimmune condition is the mixed antiglobulin reaction test (MAR).

The principle of this technique is to mix raw semen with latex particles quoted with a human antibody (IgG) and adding in the suspension an antihuman (IgG) antibody in view of observing any reaction with subsequent formation of mixed agglutinates between the particles and motile spermatozoa. A prevalence of 50% or more spermatozoa with adherent particles is indicative of immunological infertility.

Sperm antibodies which may account for up to 10% of male factor infertility impair sperm fertilisation capacity mainly by rendering progressive motility sub-optimal and by their direct interference with the events preceding egg and sperm encounter such as capacitation and the acrosomal reaction. Penetration into the egg can also be impaired due to the obliteration of important enzyme complexes mediated by antibody adherence.


This is the condition where no spermatozoa could be identified using high-powered microscopy following rigorous concentration of the seminal plasma. It affects approximately 1% of the human male population and accounts for up to 15% of male factor infertility. It is distinct from aspermia which is the complete absence of seminal fluid at ejaculation. There are three separate categories of azoospermia:

(i) pre-testicular which associate with endocrine abnormalities.

(ii) testicular where the dysfunction is inherent to the testicles themselves (primary testicular failure)

(iii) post-testicular where ejaculatory and ductal dysfunctions prevent sperm from reaching the urethra. While the first and third condition can be reversed, testicular causes of azoospermia cannot be rectified with the exception of varicocele.


A suboptimal or an azoospermic result of a routine semen analysis is normally followed by several evaluation tests which fall within 3 main categories:

(1) medical history such as childhood illnesses or disorders, exposure to toxins such as radiation or chemotherapy, genital trauma or surgery, infectious diseases, previous and current prescribed medication, family history and prior fertility.

(2) physical examination aiming at assessing the size and consistency of the testes, the presence of trauma or scar at the inguinal and/or scrotal areas, secondary sex characteristics such as gynaecomastia, hair distribution and body physique, the presence and consistency of spermatic cord ducts and epididymes and the presence of varicoceles (dilated scrotal veins).

(3) endocrine assessment incorporating a hormonal profile test for the reproductive hormones FSH and LH and total testosterone concentration.

DNA Sperm Fragmentation

DNA sperm fragmentation (DSF) is a condition which when present at abnormal rates compromises the genetic integrity of spermatozoa. While fertilisation of eggs is possible the normal developmental potential of early cleaving embryos is seriously affected and this is consistent with pre-implantation embryo arrest or high implantation failures or early miscarriage. Men with poor semen parameters (who are most usually also subfertile) are more likely to be affected although DSF can be seen in individuals with optimal semen quality.

The higher the percentage rates of affected sperm the lower the chances for a healthy pregnancy to result. The adverse effects of the condition may be mitigated when younger eggs are fertilised since these have a higher capacity to raise repair mechanisms than older oocytes. Consequently, the younger the age of the female spouse the better the prognosis.

While the commonest cause for DSF is oxidative stress a collection of other factors normally contingent to deranged DNA replication mechanisms (topoisomerase activity) and regulation of programmed cell death (apoptosis) can provoke the condition. Most importantly exposure to environmental and occupational pollutants, lifestyle (i.e. diet, cigarette smoking and narcotics), infection, increased testicular temperature (i.e. due to varicocele) and advanced age (normally over 45) are possible causes.

DNA Sperm Fragmentation Assessment

Antisperm Antibodies

Effective treatment of DSF is difficult if possible at all and while changes in lifestyle and administration of antioxidant agents which include amongst others selenium, cod liver oil and vitamin C may have ameliorating effects against oxidative stress, initiatives against DSF due to the rest of the aetiologies described above may prove ineffective. It has been reported that antibiotic treatment due to infection mediated DSF or varicocele repair may have some positive feedback. Evidence exists that the damage to the DNA integrity occurs at the post-testicular level. Retrieving testicular sperm surgically for ICSI has been shown to be associated with improved outcomes compared with spermatozoa from the ejaculate.

The conventional approach to screening DSF is the halosperm methodology which is based on the Sperm Chromatin Despersion test (SCD) technology. Processing of the specimen enables two different morphologies of spermatozoa to evolve. These depend on the presence of absence of haloes of chromatin dispersion consistent with the level of DNA fragmentation.

Briefly, intact spermatozoa obtained from raw semen are immersed in an inert agarose micro-gel on a pre-treated microscopic slide. An initial acid treatment denatures DNA only in those sperm cells with fragmented DNA. Following this a lysing solution is used to remove almost all of the nuclear proteins and in the absence of massive DNA breakage produces nucleoids (nucleus like structures) with large halos of spreading DNA loops emerging from a central core. However the nucleoids from spermatozoa with fragmented DNA either do not show a dispersion halo or the halo is minimal. The determination can be done with the use of either bright field or fluorescence microscopy while the technique is relatively quick to complete.

Sperm Aneuploidy

Aneuploidy is a condition in which the number of chromosomes in the nucleus of a cell is not an exact multiple of the monoploid number of a particular species. An extra or missing chromosome is common cause of genetic disorders including human birth defects. All cells in our organism known as somatic cells have a 46 complement of chromosomes other than the germ cells (the egg and the sperm) which curry 23. While chromosomal abnormalities in somatic cells can easily be screened by blood Karyotype analysis exploring the chromosomal content in spermatozoa necessitates the use of specialised techniques which can examine chromosomes directly.

Approximately 10% of all sperm in fertile men are known to curry a genetic abnormality while this rate is further increased in men with poor semen quality subjecting them also subfertile. Morphologically normal spermatozoa may present aneuploidy too although a higher prevalence is observed in spermatozoa with abnormal morphology. Fertilisation of eggs with aneuploid sperm will result in early embryonic arrest or a miscarriage or more rarely in the birth of defective offspring.

Unfortunately, there is no effective treatment against aneuploidy. It is believed that a healthier lifestyle may be beneficial while administration of folic acid has also been shown to have improving effects. Aneuploidy in sperm other than advancing age can be a consequence of exposure to toxins inclusive of radiation, chemotherapy, environmental and occupational pollutants, excessive cigarette smoking and alcohol and caffeine consumption. Individuals with very poor morphology and count sperm, abnormal reproductive hormone profiles, repeated IVF failures and recurrent miscarriages are advised to undertake the aneuploidy screening test. The technique namely FISH (Fluorescence In Situ Hybridization) makes use of fluorescent probes to label individual chromosomes in several hundreds of spermatozoa. A limitation of the technique is that only a few chromosomes can conventionally be screened (13, 18, 21, X and Y). A normal spermatozoon is described by the appearance of a single fluorescent signal in its nucleus for each of the labeled chromosomes.

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