Semen analysis test - Sri Lanka male female infertility treatment ...

AYURVEDIC HOSPITAL - Nugegoda, Sri Lanka.FOR INFERTILITY TREATMENTSCall Dr. R. A. R. P. Susantha on +94 112 812814 for Free Consultancywww.ayurvedic-hospital.comdr_susantha@yahoo.comSEMEN ANALYSISThe male factor infertility is most commonly defined as abnormalities in thenumber of sperm present, proportion of the motile and morphologically normalsperm. WHO has defined normal values for human ejaculate.Semen analysis testSemen analysis is not a test for fertility.Fertility determination is a couple-related phenomenon that requires the initiationof a pregnancy. The patient cannot be considered fertile based only on normalsemen analysis. It was shown that 30% of all patients with normal semenanalysis have abnormal sperm function.Typically two to three semen analyses are obtained over a 3 month period priorto making any final conclusion regarding baseline sperm quality or quantity.However, if the first semen analysis is normal, the repeat test is not required.Recent febrile illness or exposure to gonadotoxic agents may affectspermatogenesis for up to 3 months, therefore semen analysis has to bepostponed.Semen analysis includes the examinations of :1. Spermatozoa2. Other cells present in semen3. Seminal fluid

Altogether these data give indications on the testicular function and of theintegrity of the male genital tract.SpermatozoaSpermatozoa (sperm) are the male sex cells that carry a man's genetic material.Pregnancy occurs when a sperm penetrates a woman's egg (ovum).Sperm quality and quantity (sperm analysis test)A semen analysis measures the quantity and quality of both the liquid portion,called semen, and the microscopic, moving cells, called sperm.Semen is the turbid, whitish substance that is released from the penis duringejaculation. A sperm contains one copy of each chromosome (all of the male’sgenes) and fuses with the female’s egg, resulting in fertilization.A typical semen analysis measures:• The volume of semen,

• The semen consistency (thickness),• Sperm concentration,• Total number of sperm,• Sperm motility (percent able to move as well as how vigorously andstraight the sperm move),• The number of normal and not normal (defective) sperm,• Coagulation and liquefaction,• Fructose (a sugar in semen),• PH (acidity),• The number of immature sperm, and• The number of white blood cells (cells that indicate infection).Additional tests may be performed if semen is abnormal, such as a test for spermantibodies. If assisted reproductive technology is contemplated (for example, invitro fertilization), sperm function tests may also be performed. Sometimes a testcalled cryosurvival is done to see how well semen will survive for long-termstorage, if a couple would like to store sperm for future pregnancies.How is the sample collected for testing?Most labs require samples to be collected on-site as the semen needs to beexamined within one hour after ejaculation. Semen is collected in a private area,usually a bathroom. The man masturbates and collects the semen in a jar. Somemen, for religious or other reasons, might want to collect semen during the act ofintercourse, using a condom. If this is the case, the doctor should provide thecondom or sheath because lubricated condoms can affect test results.Semen specimen are obtained by masturbation into a sterile wide-mouthcontainer after 3 days of abstinence. Therefore, the patients should be stronglyrecommended to collect samples within clinic area.How to prepare for the test• There should be no sexual activity that causes ejaculation for 3 daysbefore the test.How the test will feel

• If the man is uncomfortable about how the sample is to be taken, thisshould be discussed with the health care provider.Why the test is performed• The test is performed if the patient's fertility is in question. It is helpful indetermining if there is a problem in sperm production or quality of thesperm as a cause of infertility.Some Commonly used normal semen parameters ( Normal values of semenvariables)VOLUME>2.0 MLpH 7.2-7.8CONCENTRATION>20x10 6 /MLMOTILITY >50%MORPHOLOGYWBC>30% WITH NORMAL MORPHOLOGY< 1x10 6 /MLNormal values of semen parameters have been issued by WHO in 1992 that aregenerally used as reference. Ideally each laboratory should set its own normalvalues reflecting the specific population analyzed, but this is practically limited bythe availability of semen from proven fertile men who have recently achieved apregnancy.It should be emphasized that semen is an exception amongst biological fluidssince its parameters display very wide intra and inter-individual variations.Therefore semen analysis should be repeated to take intra-individual variationsover time into account and confirm abnormal parameters.Standard testsNormal valuesVolume2.0 ml or morepH 7.2-7.8

Sperm concentrationTotal sperm countMotilityMorphologyVitalityWhite blood cells20x10 6 spermatozoa/ml or more40x10 6 spermatozoa or more50% or more with forward progression or 25% ormore with rapid progression within 60 min aftercollection30% or more with normal morphology b75% or more liveFewer than 1x10 6 /mlImmunological testsImmunobead testMAR testFewer than 20% spermatozoa with adherentparticlesFewer than 10% spermatozoa with adherentparticlesSeminal plasma biochemical analysisEpididymal markersa-glucosidase (neutral)Carnitine20 mU or more per ejaculate0.8-2.9 mmole per ejaculateProstate markersZinc (total)Citric acid (total)Acid phosphatase (total)2.4 mmole or more per ejaculate52 mmole or more per ejaculate200 U or more per ejaculateSeminal vesicle markerFructose (total)13 mmole or more per ejaculateSemen analysisSemen volumeNormal:1.0–6.5 milliliters (mL) per ejaculationAbnormal: An abnormally low or high semen volume ispresent, which may sometimes cause fertilityproblems.Liquefaction Normal: Less than 60 minutes

timeSperm countAbnormal: An abnormally long liquefaction time is present,which may indicate an infection.Normal:20–150 million sperm per milliliter (mL)0 sperm per milliliter if the man has had avasectomyAbnormal: A very low sperm count is present, which mayindicate infertility. However, a low sperm countdoes not always mean that a man cannot father achild. Men with sperm counts below 1 million havefathered children.Sperm shape(morphology)Normal:At least 70% of the sperm have normal shape andstructure.Abnormal: Sperm can be abnormal in several ways, such ashaving two heads or two tails, a short tail, a tinyhead (pinhead), or a round (rather than oval)head. Abnormal sperm may be unable to movenormally or to penetrate an egg. Some abnormalsperm are usually found in every normal semensample. However, a high percentage of abnormalsperm may make it more difficult for a man tofather a child.Spermmovement(motility)Normal:At least 60% of the sperm show normal forwardmovement.At least 8 million sperm per milliliter (mL) shownormal forward movement.Abnormal: Sperm must be able to move forward (or "swim")through cervical mucus to reach an egg. A highpercentage of sperm that cannot swim properlymay impair a man's ability to father a child.Semen pHNormal: Semen pH of 7.1–8.0White bloodcellsAbnormal: An abnormally high or low semen pH can killsperm or affect their ability to move or to penetratean egg.Normal:No white blood cells or bacteria are detected.Abnormal: Bacteria or a large number of white blood cells arepresent, which may indicate an infection.Fructose level Normal: 300 milligrams (mg) of fructose per 100 milliliters(mL) of ejaculate

Abnormal: The absence of fructose in the semen mayindicate that the man was born without seminalvesicles or has blockage of the seminal vesicles.Certain conditions may be associated with a low or absent sperm count. Theseconditions include• Orcuitis,• Varicocele,• Klinefelter syndrome,• Radiation treatment to the testicles,• Diseases that can cause shrinking (atrophy) of the testicles (such asmumps).If a low sperm count or a high percentage of sperm abnormalities are found,further testing may be done. Other tests may include• Measuring hormones-, such as testosterone, luteinizing hormone (LH),follicle-stimulating hormone (FSH), or prolactin.• A small sample (biopsy) of the testicles may be needed for furtherevaluation if the sperm count or motility is extremely low.• Several semen analyses at intervals of two or more weeks are necessaryin a man with an abnormality in the first test because of the variability ofresults.Even with complete collection of samples there is variability caused by countingerror, other technical errors and differences in the ejaculate from day to day.These large variations need to be remembered when interpreting results ofsemen analysis.

Variability of semen analysis results in a fertile sperm donor.C, sperm concentration; V, semen volume; M, total motility;MI, motility index-product of grade and percentage of spermwith progressive motility graded 0 to 3. (Mallidis C et al:Variation of semen quality in normal men. Int J Androl 14:99-107, 1991. Used by permission Blackwell ScientificPublications.)What abnormal results meanIf the sperm count is very low or very high there is a likelihood of being lessfertile. The percent of normal sperm has an effect on infertility. The acidity of thesemen and the presence of white blood cells (suggesting infection) may influencefertility. The use of many recreational and prescription drugs, alcohol, andtobacco use may affect fertility.Additional conditions under which the test may be performed:Klinefelter's syndrome

What’s the risks are sperm analysis test• There are no risks.Special considerationsApproximately half of couples unable to have children have a male infertilityproblem. One of the first tests done to evaluate a man's fertility is the semenanalysis. There are many unknowns in male infertility. The results from the testmay fail to explain the cause. If a low sperm count or abnormal semen is found,further testing may be required.Normal ejaculate volumeVolume is between 2 and 6 ml.• Seminal vesicles-----------------65%• Prostate----------------------------30-35%• Vasa---------------------------------05%Low volume is associated with absence or decrease of seminal vesiclecomponent of ejaculate( absence of SV, complete or partial obstruction ofejaculatory ducts) or retrograde ejaculationSource Volume CharacteristicsUrethral andbulbourethral glandsTestes,epididymides,vasadeferentia0.1-0.2cc0.1-0.2ccViscous, clearSperm presentProstate 0.5-1.0cc Acidic,waterySeminal vesicles 1.0-3.0cc Gelatinous, fructosepositive

Complete ejaculate 2.0-5.0cc Liquefies in 20-25minNormal semen pH• PH is 7.2-8.0. (Normal semen PH)• Prostatic secretion is acidic while• Seminal vesicle fluid is alkaline (seminal fructose is derived from seminalvesicles).• Acidic ejaculate (pH8.0_Azoospermia with low ejaculate volume,• Fructose negative and• Acidic may imply obstruction of the ejaculatory ducts.• PH over 8.0 may indicate infection.The semen is initially in liquefied state but quickly coagulate by the action ofprotein kinase secreted by the seminal vesicles. Proteolytic enzymes from theprostate liquefy coagulum in 20-25 minutes. Abnormal liquefaction may be casedby prostatic abnormalities, e.g. prostatitis. Increased viscosity may affect spermmotilityConcentration:• Evaluated in Mackler or Cell-VU chambers.• Azoospermic specimen contains no sperm,• Oligospermic specimen reveals concentration of less than 20x10 6 andNormospermic specimen contains more than 20x10 6 .Motility and forward progression:• Normally >50% of sperm in the specimen are motile.• Forward progression describes how fast the motile sperm are moving(normal 2+ in the scale from 0 to 4)

0 No movement1 Movement, none forward1+ Occasional movement of a few sperm2 Slow, undirected2+ Slow , directly forward movement3- Fast, but undirected movement3 Fast, directed forward movement3+ Very fast forward movement4 Extremely fast forward movementMorphology =• Shape of spermatozoa: Several techniques have been described toevaluate sperm morphology.• Sperm are classified into normal-oval shaped, tapered, amorphous,duplicated and immature.• Normal spermatozoid must have an oval form with smooth contour,acrosomal cap encompassing 40-70% of head, no abnormalities ofmidpiece, or tail and no cytoplasmic vacuoles of more than half of thesperm head.• Head size is 5-6 M x 2.5-3.5 M. Any borderline sperm are counted asAbnormal (amorphous, tapered, duplicated, immature, coiled tail, bluntedtail, midpiece abnormalities).• The predictive value of sperm morphology in determining pregnancy ratesis low.A. WHO criteria: >30% normal forms (100 cells evaluated)B.Strict criteria (higher predictive value in determining rates of pregnancy inIVF program) are based on the morphology of postcoital spermatozoa found at

the level of the internal cervical os. 100 cells evaluated for only normal sperm(>14% normal forms). Men with fewer than 4% normal forms usually failed tofertilize without micromanipulation. Strict criteria for normal sperm morphologyinclude:Sperm head:Smooth oval configuration.• Length-5-6 microns.• Width: 2.5-3.5 microns.• Acrosome comprises 40-70% of the anterior sperm headMidpiece:• Axially attached,• 1.5 times the head length,• 1micro/ m in widthTail:• Straight,• Uniform,• Slightly thinner than the midpiece,• Uncoiled,• 45Micro/ m longWhite blood cells (WBC):All semen samples have WBC in them. If greater than 1 million WBC per 1 ml arepresent, there is concern of infection.Generally leukocytospermia (WBC in the semen) affects 5-10% of the patientpopulation, but can rise to 20% in certain patients groups.

Semen has to be cultured for aerobic and anaerobic infection as well asChlamydia and Mycoplasma. Additionally, leukocytes have to be differentiatedfrom immature germ cells using immunohistochemical methods.WBC cells are deleterious because of their ability to stimulate the release ofreactive oxygen species (ROS), thereby inhibiting sperm motility and spermfunction. Reactive oxygen species (ROS) are produced by polymorphonuclearcells .The three main ROS are superoxide anion, hydrogen peroxide, and thehydroxyl radical.On the other hand, seminal plasma contains a number of antioxidants thatprotect sperm from oxidative damage from exposure to ROS.Men who have higher concentrations of such antioxidants may be able to tolerategreater concentrations of seminal leukocytes.Despite an apparently abnormal threshold level for leukocytes within the semen,a wide range of conflicting evidence exists as to the significance of seminalleukocytes and infertility.The impact of this condition and its treatment on semen quality are extremelycontroversialViability.Viability tests are used in cases of low motility to determine the presence of livesperm vs. necrozoospermia.The eosin test is based on the fact that eosin is excluded by live cells which arenot stained.The tail of only live spermatozoa is swelling in the hypoosmotic solution(Hypoosmotic swelling test)Fructose (13 mili/mol or more per ejaculate):Fructose is androgen-dependent and is produced in the seminal vesicles.Fructose levels should be determined in any patient with azoospermia andespecially in those whose ejaculate volume is less than 1 ml, suggesting seminalvesicle obstruction or atresia.

Absence of fructose, low semen volume, and failure of the semen to coagulateindicate either congenital absence of the vas deferens and seminal vesicles orobstruction of the ejaculatory duct.Semen analysis has comparatively limited predictive value for the ability of theindividual to achieve pregnancy.Additionally, 10-20% of infertile couple will not have any abnormalities. In order toenhance the diagnostic power of semen analysis, new tests have beendeveloped to identify functional defects and fertilizing potential of the sperm.The clinical data to support their use are not conclusive.1. Antisperm antibodies test.Sperm agglutination, reduced sperm motility, abnormal postcoital test aresuspicious for the presence of antisperm antibodies. Several tests are presentlyavailable including• Sperm Immobilization test,• Sperm Agglutination tests,• Indirect immunofluorescence test,• Enzyme-Linked Immunosorbent Assay,• Radiolabelled Antiglobulin Assay.• Immunobead RosetteTest is one of the most informative and specific and can identified differentantibody classes involved (IgG, IgA, IgM) and location on the sperm cell (head,body or tail)2.CASA- Computer Assisted Semen Analysis.Mostly for assessment of sperm concentration and specific patterns of spermmotility (velocity, linearity etc).The available clinical data show that the measurement obtained by CASA arecorrelated with conception in vivo and fertilization in vitro, but comprehensivequality control and quality assurance programs are necessary to ensureaccuracy.The equipment is highly expensive.3.Acrosome reaction.

Absence of acrosome reaction implies poor prognosis for fertilization.The test for acrosome reaction is very expensive, labor intensive, subjective andnot cost-effective since only 5% of infertile patients do not demonstrate anacrosome reaction.4.Hamster egg penetration testTo check sperm fusion ability. The diagnostic value is controversial because ofdifficulty in optimizing protocol. However, a zero test score may indicate a majorimpairment of sperm fusion capacity.5.In Hemizona test (to evaluate sperm zona-binding capacity)The two halves of human zona pellucida is incubated with patient's capacitatedsperm.6.PCR-based detectionOf the pathogens in the semen in patients with asymptomatic genital infection.7. Biochemical markers e.g. Creatine Kinase, Reactive Oxygen Species8. Post ejaculate urine analysis. Retrograde ejaculation is commonly seen inpatients with diabetes,• After transurethral surgery,• Retroperitoneal lymph nodes dissection,• Spinal cord injuries.• When bladder neck coaptation is not complete,1. The seminal fluid may travel retrograde into the bladder duringperiurethral muscular contraction.2. Patient may present with low semen volume,3. Low motility and sperm concentration.Urinalysis performed immediately after ejaculation.Unspun specimen examined for sperm under the microscope.If sperm are present, specimen is processed further to evaluate concentration,motility and morphology.

In certain situations sperm retrieved from the urine may be used for assistedreproduction.Type of assays1. Descriptive assay: Spermogram (Table 3)2. Functional assays: penetration of cervical mucus (postcoital test, in vitropenetration assay)binding of spz to the zona pellucidafusion of spz with zona-free hamster oocytehypoosmotic swelling of the flagella3. Immunologicalassay:mixed agglutination testimmunobead testsperm immobilization in cervical mucusSpermogramTable 3. Common or Characteristic Patterns of Semen AbnormalityVolume(mL) 1-6*Concentration(106/mL) >20*Motility(%) >50NormalMorphology(%) >15CommentCause0.4 0 - - Fructose 1nmol/L(low)pH 6.5(low)Congenitalabsence of vasaEjaculatory ductobstructionPartial retrogradeejaculationTesticular failurewith androgendeficiency(Spill or incompletecollection)4.0 0 - - Fructose 15nmol/LGenital tractobstructionPrimaryseminiferous tubalfailureSecondaryseminiferoustubule failure withandrogentreatment

3.0 100 0 35 Live 70% (Contamination orcondom collection)Immotile cilia3.0 100 5 35 Live 20% (Contamination ordelayedexamination)NecrospermiaSpermautoimmunity3.0 100 65 0 Small roundheads3.0 100 25 10 LiquefactiondelayedSpermaggregation2+Live 40%Polymorphs 1x 106/mL3.0 4 30 3 Mixedabnormalmorphology3.0

VARIATIONS IN SEMEN VOLUME AND APPEARANCE.• Low semen volume suggests incomplete collection,• Short duration of abstinence from ejaculation before the test,• Absence or obstruction of the seminal vesicles or androgen deficiency.• High semen volume (>8 ml) may be seen in association with oligospermiabut is of little practical significance.• Hemospermia is usually is the result of minor bleeding from the urethrabut serious conditions, such as genital tract tumors must be excluded.• Other discoloration of the semen may indicate inflammation of accessorysex organs.• The semen may be yellow with jaundice or salazopyrine administration.• Defects of liquefaction and viscosity are relatively common andpresumably result from malfunction of the accessory sex organs.• Although these may cause problems with semen analysis and preparationof sperm for ART, they are probably of little relevance to fertility.• Sperm agglutination is common with sperm autoimmunity but can alsooccur for other reasons.AZOOSPERMIA.• The total absence of sperm from the semen needs to be confirmed inrepeated tests with vigorous centrifugation of the semen and carefulexamination of the pellet.• Rarely an illness or difficulty with collection will cause transientazoospermia. With severe spermatogenic disorders and someobstructions sperm may be present in the semen intermittently. If any livesperm can be found,• These can be treated from auyrveda.

OLIGOSPERMIA.• Sperm concentrations of less than 20 million/ml are classified asoligospermic.• This figure probably derives mainly from the work of MacLeod, who foundthat only 5 per cent of fertile men had sperm concentrations less than 20million/ml.• There is a correlation between sperm concentration and other aspects ofsemen quality, such as percentage motility and normal morphology.• Both motility and morphology are usually poor with oligospermia.ASTHENOSPERMIA.• Asthenospermia is defined as a sperm motility of less than 50 per centtotal or less than 25 per cent with rapid progressive motility.• Spurious asthenospermia because of exposure of sperm to rubber(particularly condoms), spermicides, extremes of temperature, or longdelays between collection and examination should be excluded beforeaccepting that sperm motility is poor.• Low sperm motility is a frequent accompaniment of oligospermia, andthere usually is a mixed picture of morphological defects. It presumablyarises because of defective spermiogenesis.• Severe asthenospermia requires evaluation by electron microscopy.• Specific ultrastructural defects of the axoneme are associated with zerosperm motility or extreme asthenospermia (less than 5 per cent motilesperm) and sterility.• Absent dynein arms, other less common axonemal defects, mitochondrialabnormalities, disorganized fibrous sheath or outer dense fibres withstumpy tails, or normal ultrastructure may be found.• The gene defects in some of these disorders are being discovered.• Standard semen analyses of these patients usually show normalsperm concentrations and normal sperm morphology, although

some have tail abnormalities at the light microscopic level-short,straight, or thick tails or mid-piece defects.• Viability tests help to distinguish this group of patients from those withnecrospermia.• Patients with structural defects in the sperm that cause completeimmotility are untreatable and sterile except for Ayurvedic treatments fewmonths.• Asthenospermia may also be associated with sperm autoimmunity.• The causes of other motility defects of moderate degree are unidentified.• Some suggested abnormalities of protein carboxylmethylase, lactatedehydrogenase C4, and energy generation are unconfirmed andcontentious.NECROSPERMIA.• Necrospermia is important to distinguish from other types of severeasthenospermia because some patients produce pregnancies despite thelow sperm motility.• Necrospermia is characterised by usually less than 20-30 per cent totalmotility, less than 5 per cent progressive motility and a viability test lessthan 30-40 per cent indicating a high proportion of dead sperm.• The condition may fluctuate in severity, particularly with changes in coitalfrequency.• Necrospermia may be caused by defective storage of sperm in the tails ofthe epididymides or stasis in the genital tract.• There are ultrastructural features of degeneration in the ejaculated spermbut normal structure of late spermatids in testicular biopsies.• Characteristic of necrospermia is an improvement of sperm motility withincreased frequency of ejaculation.• Treatment with Ayurvedic drugs may have a beneficial effect, this isproved by me.

• The couple should have intercourse once or twice every day for three tofour days up to the time of ovulation.TERATOSPERMIA.• If there is a reduced percentage of sperm with normal morphologyassessed by light microscopy, the classification of teratospermia is used.• The microscopic assessment of sperm morphology is highly subjectiveand difficult to standardize between laboratories.• Various approaches to morphology are used. The simplest is to assigndefects with a priority in order head, mid-piece, and tail, and to record asnormal only those that conform to ideal shape with no defects in anyregion.• In the Strict morphology approach, marginally abnormal sperm areassigned abnormal.• Differential counts give the proportions of abnormal sperm with large,small, tapered, pyriform, or amorphous heads; normal heads but midpiecedefects; or normal heads and mid-pieces but abnormal tails.• Indices based on the average number of defects per spermatozoon arealso used (teratospermia index).• Automated methods involving image analysis by computer are beingdeveloped which promise to overcome the between-laboratory variabilityand greatly improve the predictive value of semen analysis for naturalconception.• The percentage of sperm with normal morphology assessed by strictcriteria after treating by Ayurvedic drugs the sperm and adjusting theconcentration to 80 million/ml provides one of the most useful predictors ofnormal conceving rates.• There is a progressive reduction in fertilization rates from 60 per cent to 20per cent as abnormal morphology increases from less than 70 per cent tomore than 95 per cent.

• Patients with high proportions of sperm with abnormal morphology shouldhave Ayurvedic treatmentsI because of the risk of failure of fertilization.It is important to distinguish mixed abnormalities of sperm morphologyfrom those in which all or the majority of sperm show a single uniformdefect, such as spherical heads with absence of the acrosomes and pinhead sperm, which are produced by defective spermiogenesis.• Pin-head sperm result when the centrioles from which the sperm tailsdevelop are not correctly aligned opposite the developing acrosome sothat the sperm heads are lost and absorbed during epididymal transit.• Both these conditions are extremely rare.Definitions of semen classificationsNormozoospermiaWhen all the spermatozoal parameters arenormal together with normal seminal plasma,WBCs and there is no agglutination.OligozoospermiaAsthenozoospermiaTeratozoospermiaOligoasthenoteratozoospermiaAzoospermiaAspermiaWhen sperm concentration is < 20 million/ml.Fewer than 50% spermatozoa with forwardprogression(categories (a) and (b) or fewer than25% spermatozoa with category (a) movement.Fewer than 50% spermatozoa with normalmorphology.Signifies disturbance of all the three variables(combination of only two prefixes may also beused).No spermatozoa in the ejaculate.No ejaculate.Scheme of spermogram

Spermatozoa parameters:The spermatozoa parameters assessed in the spermogram are• the number of sperm cells,• The viability,• The motility• The morphology of the sperm population.Immunological analysis:• Immunobead or mixed-agglutination (MAR) tests allow to detect antispermatozoa-antibodiesSeminal fluid parameters:• Biochemical assays of markers for prostate, seminal vesicles andepididymis assess the function of these accessory glands.Microscopic analysis on live sperm Search for:1. Aggregated spermatozoa2. Epithelial cells3. White blood cells4. BacteriaSperm concentration, total number and viability• The major part of the ejaculate volume is contributed by secretionsfrom the accessory glands (seminal vesicles and prostate), so theejaculate volume is not directly related to spermatogenesis andhence the sperm cell concentration (sperm cells/ml) variesaccording to the ejaculate volume.• The total number of spermatozoa per ejaculate reflects thespermatogenesis and is related to the time of sexual abstinencebefore collection.

• In normal situation spermatogenesis is considered to be a constantprocess over time and therefore the total number of sperm perejaculate should increase with abstinence time.• The ejaculate volume is related to the secretory function of theseminal vesicles and prostate.• Decreased ejaculate volume and increased sperm concentrationreflects impaired accessory glands function.• Vital staining of the spermatozoa allows to quantitate the fraction ofliving cells independently from their motility.Spermatozoa motility• The fraction of motile sperm in semen is measured either by manualcounting or using a computer assisted semen analysis (CASA) system.• Motility is assessed at the time of semen liquefaction and after 1 and 3hours to detect asthenozoospermia.Manual counting classifies sperm cells into 4 categories1. Immotile2. Locally motile3. Non linear4. Linear motileusing qualitative subjective criteria of selection.Many infertility centers now use CASA systems for objective measurments ofsperm motion and positive correlations have been found between motionparameters such as the amplitude of lateral head displacement, curvilinearvelocity, linearity and straight-line velocity and fertilization rates, but the thresholdlevels for these motion characteristics have not yet been established to meet ageneral consensus.

Bacteriological analysis• Direct measurement of infectious contamination is obtained frombacteriological cultures of both aerobic and anaerobic germs.• In normal conditions semen is not sterile but rather colonized at low levelsby a variety of germs.• Recent studies have shown that bacterial colonization did not havenegative impact on sperm-cervical mucus interaction.Biochemical analysis• Biochemical analysis of secretion components from prostate, seminalvesicles and epididymis in semen give informations about the functionalstate of these organs.• These markers include fructose as seminal vesicles marker, zinc or acidphosphatase as prostate marker and carnitine as epididymis marker.• Zinc can be measured by colorimetric assay while fructose and carnitineare measured using enzymatic assays.Anti-sperm antibodies• The presence of anti-sperm antibodies in semen can alter the spermfertilizing ability.• Spermatogenesis starts at puberty, after the "education" of the immunesystem to recognize self antigens is finished and are thereforeimmunogenic.• Under normal circumstances they are protected from the man's immunesystem by the hemato-testicular barrier that separates the inner part ofseminiferous tubules from the blood.• When this barrier is ruptured sperm cells induce the synthesis of antispermantibodies.• Antibodies adsorbed on the sperm surface can be detected byimmunological assays using secondary, Ig class-directed antibodies thatare coupled to beads. The percentage of sperm adhering to the beads

eflects in a semi-quantitative manner the presence of anti-spermantibodies.• We currently use Mar-test kits to detect anti sperm-IgG in semen.• Positive and dubious samples are subsequently tested for anti-sperm IgA.IgA antibodies are more significant clinically, but very rarely occur withoutassociated IgG.• Therefore the test of anti-sperm IgG antibodies in semen is sufficient forthe first screening procedure.• Anti-sperm IgG can be tested in serum but this is of little benefit as serumanti-sperm IgG do not correlate with anti-sperm Ig in semen and do notinfluence fertility prognosis.Morphology analysis• The evaluation of sperm morphology is performed after Papanicolaou orsimilar staining and consists in detailed examination of 100 sperm cells aswell as other cells present in the ejaculate, including leucocytes andimmature sperm cells.• Sperm cells represent a unique population where over 50% of the cellscan have morphological defects in normal fertile individuals.These defects affect the1. Head2. Midpiece3. Flagella of the sperm cell.• The percentage of sperm with normal morphology will be recordedas well as the individual abnormalities, in order to detectpredominant abnormalities, which suggest genetic defects affectingspermatogenesis.• The rare cases of monomorphic teratozoospermia as well assevere asthenozoospermia can be subjected to EM analysis todetect specific defects at the ultrastructural level, particularly in theflagella where abnormal microtubule assembly can be found as inthe immotile cilia syndrome.

• In recent years, Menkveld, Kruger and collegues have describedstrict criteria for spermatozoa morphology assessment with whichthey obtained good predictive value.• They report normal cases whith > 14% normal sperm morphology.The need to follow Kruger’s criteria of selection of normal spermmorphology is still debated. Nevertheless, numerous studies havenow clearly established that sperm morphology is an importantparameters in the evaluation of sperm fertility.• The evaluation of sperm morphology also includes the identificationof other cell types present in semen such as immature sperm cellsand leukocytes.• The presence of immature germ cells in semen indicatesspermatogenesis dysfunction at the testicular level whereasleukocytes in concentrations exeeding 1x103/ml indicateinflammatory conditions possibly related to infection.• Distinction between leucocytes and spermatogenesis cells• The distinction between spermatogenesis cells and leucocytes isnot always obvious, and it is important particularly in cases ofazoospermia.• Indeed, in azoospermia, the presence of spermatogenesis cellsindicates a testicular malfunction, while the presence ofleucocytes in the absence of any spermatogenesis cellssuggests that the azoospemia might be due to an obstuctionproblem.• Spermatogenesis cells present in semen are usually degenerating,and can sometimes be confused with leucocytes. In particular,multinucleated spermatids can be confused withpolymorphonuclear leucocytes (PMN).• Staining of endogenous peroxidases present in PMN can help todistinguish between these two cell types.

Sperm – cervical mucus interactions• Cervical mucus is the major physical barrier that sperm cells have to crossto access to the female upper genital tract.• Less than 1% of the sperm deposited in the vagina successfully penetratethe cervical mucus.• Evaluation of sperm-cervical mucus interactions includes the post-coitaltest, the sperm cervical mucus contact test and the in vitro sperm-cervicalmucus penetration assay.Post-coital test• The post-coital test is the analysis of cervical mucus a few hours afterintercourse. It reflects the physiologic situation in vivo and assesses boththe quality of cervical mucus and the penetration ability of sperm.• The quality of cervical mucus varies during the cycle and is favourablyinfluenced by estrogens, becoming more abundant and fluid at the time ofovulation.• Therefore post-coital tests are scheduled just before ovulation asdetermined by basal body temperature, or more accurately by follicularsizing by ultrasonography.• The number of motile sperm per high power microscopic field is recordedand the test is considered positive when 10 or more motile sperm arefound per field according to WHO guidelines.• Cervical mucus evaluation (including volume, consistensy, ferning,spinnbarkeit, cellularity and pH) is of utmost importance for theinterpretation of post-coital test results with respect to sperm function.• A decreased number of sperm in cervical mucus when the cervical mucusscore is low reflects inadequate mucus rather than impaired spermfunction.

• Cervical mucus is colonized by sperm that are stored for several hours incervical crypts. Sperm cells then gradually migrate trough the cervix.Therefore sperm are present in cervical mucus constantly for at least 12hfollowing intercourse and the timing of post-coital test (6-12h afterintercourse) allows testing the viability of sperm in this environment.• Abnormal penetration of cervical mucus by sperm has been associatedwith the presence of immobilizing anti-sperm antibodies, and repeatedlyabnormal post-coital test with normal score cervical mucus associatedwith normal sperm concentration and motiliy should be investigated byadditional tests such as anti-sperm antibodies detection in semen, spermcervicalmucus contact test and in vitro cervical mucus penetration assay.• The post-coital test has been used for several decades but is reported tobe inaccurate and to lack consensus normal values and methodology.Part of the problem may be due to the non-homogeneous nature of thecervical mucus that prevents quantitative determination of spermconcentration.• The post-coital test however remains an inexpensive and noninvasiveprocedure that gives information about the occurrence of ejaculation andthe ability of the sperm cells to function within the cervical environment.Sperm-cervical mucus contact test• Sperm-cervical mucus contact test consists in mixing semen and cervicalmucus in vitro and measuring the appearance of immobilized "shaking"motile sperm.• This is interpreted as the adherence of antibody-coated sperm cells tocervical mucus and was shown to correlate with semen anti-spermantibodies and pregnancy rate.• This test can be performed in parallel with donor semen or donor cervicalmucus, and therefore allows discriminating between a male versus femaleorigin of the sperm immobilizing factor.

In vitro cervical mucus penetration test• The third test of sperm-cervical mucus interactions involves thepenetration of cervical mucus by sperm in vitro.• The mucus is placed in a capillar tube, one end of the tube is dipped insemen and penetration and motility of sperm in the mucus column ismeasured. It can be performed with homologous or donor cervical mucus.• Using cervical mucus standardized by estrogen treatment, this test wasshown to have good predictive value of fertility. Alternatively to humancervical mucus this test can be performed with commercial midcyclebovine cervical mucus or hyaluronic acid gels.• The use of alternative material to human cervical mucus has practicaladvantages (availability, reproducibitity) but may be less informative thanhuman material, due to differences in the nature of the hydrogels.• Therefore whenever possible human cervical mucus should be used andfor specific male factor detection oestrogen standardized donor mucusshould be preferred.• Sperm functional assays• Sperm functional assays have been developed in an attempt to find agood predictive test of male fertility. We will discuss the hemizona assay,the hamster egg penetration assay and the sperm hypo-osmotic swellingassay.Hemizona assay• The hemizona assay (HZA) measures the binding of capacitated sperm toisolated human zona pellucida.• Human oocytes are bisected by micromanipulation, thus allowing for aninternally controlled comparison of sperm binding (from patient versus afertile control) to matching hemizonae surfaces.

• The two matched hemizona of the human oocytes have the advantage ofproviding functionally equal surfaces allowing a controlled comparison ofsperm binding and therefore limiting the amounts of oocytes used.• Ethically this assay is acceptable since the microsurgical bisection of theoocyte prevents any inadvertent fertilization.• The HZA has been found to be predictive of IVF outcome with positive andnegative predictive values of 83% and 95% respectively.• The major problem with this assay is the limited availability of humanoocytes. Eventually it could be replaced by a standardized kit in whichrecombinant human zona sperm receptors mimic the natural functionalhemizonae used now.Sperm penetration into zona-free hamster oocytes• The zona-free hamster oocyte sperm penetration assay is a heterologousbioassay has originally been developed to test capacitation, acrosomereaction, fusion and sperm chromatin decondensation.• Cross-species fertilization is made possible by removing the zonapellucida or hamster oocytes.• Using the original test conditions the limiting step is the low incidence ofspontaneous acrosome reaction in human sperm populations incubated invitro, and therefore it has been described as measuring the ability ofsperm to undergo acrosome reaction rather than the overall fertilizationprocess.• Optimized procedures including acrosome reaction induction by Ayurvedicdrugs are giving good predictive value.Hypo-osmotic swelling of sperm flagella• The hypo-osmotic swelling test (HOS) measures sperm membraneintegrity as ability to swell when exposed to hypo-osmotic media.

• The biologic significance of this test is unclear and its validity to predictIVF fertilization rate is controversial, and it is equivalent to viabilitystaining.• The HOS test and correlates with semen analysis data but not with thehamster oocyte penetration test.Immunobead testTests for sperm antibodies should be done routinely on all men being evaluatedfor infertility because no semen analysis pattern is characteristic of spermautoimmunity.• The immunobead test (IBT) with beads binding to more than 50 per centof motile sperm is regarded as positive, but there usually is more than 70to 80 per cent IgA binding with clinically significant sperm autoimmunity.Tail-tip only IBT binding is not significant• The indirect IBT in which normal donor sperm are exposed to test serumor seminal plasma can be used to test men with too few motile sperm forthe direct IBT.• An alternative screening method for men with sperm in the semen wouldbe to perform a sperm-mucus penetration test.• The mixed antiglobulin reaction test is an alternative to the IBT• Sperm-mucus penetration tests can be performed by postcoitalexamination of sperm in cervical mucus collected at midcycle or afterestrogen treatment (ethinyl estradiol, 50 mg twice daily for four days) toproduce mucus of equivalent quality.• In vitro capillary mucus penetration (Kremer) tests are particularlyimportant for evaluating the significance of sperm autoantibodies; failure ofsperm to penetrate more than 2 cm in one hour indicates severe spermautoimmunity with a poor prognosis if untreated.

Sperm function testsA number of tests of sperm function are available to examine the humanfertilization process.• These are only performed in specialist laboratories. If simpler approachesor active preparations of zona pellucida (ZP) or sperm receptor proteinsbecome available they will be widely used to improve the assessment ofhuman sperm.• IVF has permitted many conventional and new tests of sperm function tobe examined.• Groups of sperm variables that are independently significantly related tothe proportion of oocytes that fertilize in vitro can be determined byregression analysis.• This approach has confirmed the importance of sperm morphology andthe ability of sperm to interact with the coverings of the oocyteHuman Sperm-Oocyte InteractionStages of human fertilization. Spermatozoa swimthrough the surrounding medium and cumulus mass(not shown) and bind to the surface of the zonapellucida. The acrosome reaction is stimulated by zonaproteins and the acrosome reacted sperm penetratesthe zona, enters the perivitelline space and binds to theoolemma via the equatorial segment. Oocyte processessurround the sperm head and it enters the ooplasm anddecondenses. Infertility could result from defects of anyof these processes. For example, abnormal spermparticularly with defective head morphology bind poorly

to the zona.HUMAN SPERM-ZP BINDING RATIO TEST• The number of sperm bound to the ZP is strongly related to the fertilizationrate, human sperm-ZP interaction tests have been developed usingoocytes that failed to fertilize in vitro.• These oocytes can be used either fresh or after storage in concentratedsalt solutions.• Because the ZP binding capacity is variable, control (fertile donor) and testsperm are labeled with different fluorochromes (fluorescein andrhodamine).• After incubation with equal numbers of control and test sperm, the oocytesare aspirated through a wide bore pipet to dislodge loosely adherentsperm and the numbers of sperm tightly bound to the ZP are counted witha fluorescence microscope.• Results are expressed as a ratio of binding to the ZP of test and controlsperm for four oocytes.• An alternative method is to cut the zonae in half and expose each to testand control sperm (Hemizona assay).HUMAN SPERM-ZP PENETRATION TEST.• It is difficult to determine the number of sperm penetrating into the ZPwhen many sperm are bound to the surface.• The sperm bound to the surface of the ZP can be sheared off byrepeatedly aspirating the oocyte with a pipet with internal diameter lessthan the diameter of the oocyte (120? m).

• The sperm penetrating into the ZP or perivitelline space can be thencounted easily and the results of this test are the most predictive offertilization rates with standard IVF.ZP-INDUCED ACROSOME REACTION TEST.• Sperm dislodged from the ZP can be stained with a fluorescein labeledlectin such as pisum sativum agglutinin or an antibody specific for theacrosomal contents to determine the proportion that are acrosomereacted.• This test is useful for diagnosing disordered ZP induced acrosomereaction.HUMAN SPERM-OOLEMMA BINDING RATIO TEST.• Sperm-oolemma binding has been studied in the same way as the sperm-ZP binding test but using oocytes that have had the ZP removed.ZONA FREE HAMSTER OOCYTE PENETRATION TEST.• In a number of countries human sperm-zona free hamster oocytepenetration tests are performed to assess the ability of sperm tocapacitate, acrosome react, fuse with the oolemma, and undergo nucleardecondensation in the ooplasm.• This test does not evaluate sperm interaction with the ZP.

Figure 1. Clinical examination of thescrotum: note testicular atrophy (15mL)absence of pubic hair and bronzecoloured skin in a Caucasian man withhemochromatosis.ORCHIOMETRY.The volume of the testis is determined by comparison with an orchiometer(Normal 15 to 35 ml).• In the absence of varicoceles the right and left testes are about equal insize.

• Testicular volume is related to body size and number of sperm perejaculate.• As seminiferous tubules occupy more than 90 per cent of the volume ofthe testes, impairment of spermatogenesis is reflected by reducedtesticular size.• Testicular atrophy suggests severe impairment of spermatogenesis.TESTICULAR ABNORMALITIES.• Pain on palpation or excessive tenderness suggests inflammation.• Loss of normal testicular sensation may occur with chronic inflammations,neuropathy, or neoplasia.• Reduced consistency or softness of the testes is a feature of reducedspermatogenesis.• Abnormalities of shape and hard lumps suggest tumors or scars.EPIDIDYMAL ABNORMALITIES.Palpable abnormalities include:• Congenital absence of the vas or other failures of development;• Enlargements of the heads or nodules in the tails of the epididymides withobstruction,• Spermatoceles and other cysts and tumors.• In men with very small testes (

VASAL ABNORMALITIES.• Abnormalities of the vas include• Absence,• Nodules• Gaps with vasectomy,• Thickening• Beading of the vas• With severe postinflammatory• Scarring as from tuberculosis.MISCELLANEOUS ABNORMALITIES.Incidental scrotal findings include:• Scars from surgery,• Scrotal dermatitis• Pubic fat pads around the genitals in extreme obesity.• Inguinal hernias and lipomas and encysted hydroceles of the cord arepalpated above and behind the epididymis.• Cysts "hydatids" of the appendix testis or epididymis are typically anteriorto the head of the epididymis.• Spermatoceles and cysts of the paradidymis are in the head or body of theepididymis.Retroversion of the testes is common:• The vas and epididymis are anterior rather than posterior to the testes.• Hydroceles of mild degree are common.• A tense hydrocele may hide a testicular tumor.• Unilateral absence of the vas may be associated with ipsilateral agenesisof the kidney and ureter on the same side.• Many of these anomalies have little relationship ithinfertility.CHECKING FOR VARICOCELE.• With the man standing up, the scrotum can be inspected for swelling ofthe pampiniform plexus and a cough or Valsalva impulse seen or palpated

y holding the spermatic cords between the thumb and index finger ofeach hand and elevating the testes toward the external inguinal ring.• This manoeuvre reduces the risk of confusing contractions of thecremaster muscles with venous impulses.Varicocele size is graded:(grade 1),(grade 2),Cough impulse without palpable enlargement of the spermatic cordPalpable enlargement(grade 3). Visible enlargement• Although predominantly a left-sided condition, varicoceles occasionallymay be on the right side.• The accuracy and reproducibility of clinical examination even forstructures as accessible as those in the scrotum may not be high.• Varicoceles may vary in size from day to day.• Even absence of the vasa may be overlooked. With practice, orchiometrycan be repeated to within one to two orchiometer sizes.• The most common identifiable cause of infertility in men is varicocele. Thisis a condition of enlarged veins in the scrotum that causes abnormalities inthe temperature regulation of the testis.• Enzymes that are responsible for both sperm and hormone (testosterone)production have an optimal temperature at which they operate mosteffectively.• If this temperature is elevated by even one degree, sperm andtestosterone production are adversely affected.• The evidence for the negative effect of varicocele on testicular function inmale fertility is now overwhelming.• What is less certain, however, is the effect of repairing the varicocele onrestoring testicular function..

• Dozens of reports have been published demonstrating the benefit ofvaricocele surgery. However in most of these reports, controlled studieswere lacking.• Microscopes were not used in older surgical procedures, which made itextremely difficult to locate the tiny artery that provides the major source ofnourishment for the testis.• Subsequently this artery was often tied off which clearly was unlikely toimprove testicular function. Tiny lymph ducts were also inadvertently tiedoff, often causing a condition called "hydrocele," which is a bag of fluidthat develops around the testicle.• This enabled positive identification and preservation of the main artery andthe lymph ducts eliminating potential damage to the testicle as well aseliminating the complication of hydrocele.• Using these techniques in several thousands of patients, the averagehealthy sperm count after treating of the large varicoceles has beenshown to increase 128%.• The results showed the pregnancy rate in couples where men withvaricoceles underwent treatment, was three times higher than when menundergo surgery.Azoospermia• The second major cause of infertility in men is blockages or obstructionsof the male reproductive tract. This is particularly true for men with zerosperm count, a condition called "azoospermia." Men with zero spermcount can be divided into two broad groups:1. Men who have an obstruction problem or blockage, meaning they aremaking sperm, but the sperm can't get out,2. Men who have a production problem, meaning they are not makingsperm, a condition called "non-obstructive” azoospermia."• We can easily determine which group an infertile male is in by doing atesticular biopsy, also using a microscope to minimize discomfort andcomplications.

• Blockage can also be caused by a urinary tract infection or by the sexuallytransmitted diseases chlamydia and gonorrhea.• Bacteria can infect the tiny duct called the "epididymis," which isessentially a swimming school for sperm before they are able to swim tofertilize an egg.• Infection of the epididymis can cause scarring and blockage, inhibiting thesperm from leaving the duct to fertilize an egg. With the use ofayuevedictreatment, blockage repair success rates are extremely high.• One of the most common causes of blockage is vasectomy.• Approximately 500,000 to a million men undergo vasectomy each year inAmerica for permanent birth control.• With an increase in divorce rates coast-to-coast, the demand for reversalof vasectomy is also growing.• Currently, using a new technique developed, Called microdot technique.• Achieved return of sperm in 99% of men undergoing vasectomy reversalin whom we find sperm in at least one of their vas ducts.• Approximately 1% of all infertile men are born with the congenital absenceof the vas deferens, the "equivalent" of a vasectomy. Unfortunately, thereare no artificial tubes strong enough to replace the vas deferens.• However, we are now able to help such men conceive using an Ayurvedicdrugs for the epididymis.• The most exciting new development in the field of male infertility is theability to treat men with severe sperm production problems called nonobstructiveazoospermia.• Even though these men may have no sperm in their semen, we can nowfind sperm between the cells of the testicles in almost half of these cases,Using an Ayurvedic drugs.• Genetic testing of these men with non-obstructive azoospermia hasrevealed that 10% to 15% are missing a tiny piece of their Y chromosome.This condition is called micro Y deletion.

• Human beings have 46 chromosomes, males have one X chromosomeand one Y chromosome and females have two X chromosomes. The Ychromosome carries the genes that are responsible for producing sperm.• Men who have low to no sperm count might be missing a small piece ofthat Y chromosome.• Fortunately helping men with Ayurvedic drugs have children almostguarantees their male children wil not have the same infertility problem.Our ability to combat male infertility has never been stronger. Itis entirely possibly that, now we are able to increase spermcount and quality by using Ayurvedic drugs to normally fertilizean egg. The steps in such a process are very complex and notunderstood at present. Once the process is mastered, however,in near futur male infertility will become a thing of the past.Hormone Assessment• It is not necessary to perform hormone measurements routinely.• Follicle-stimulating hormone (FSH) levels in patients with azoospermia,normal testicular volume and normal virilization may help distinguishgenital tract obstruction from a spermatogenic disorder.• But some men with primary seminiferous tubal failure have normal FSHlevels. Normal FSH is common with germ cell arrest at the primary

spermatocyte stage. Rarely, high FSH levels are seen with normalspermatogenesis.• Measurement of FSH, luteinizing hormone (LH), and testosterone is usefulin men with reduced testicular volume and signs of androgen deficiency todistinguish primary from secondary hypogonadism.• Inhibin B measurement may provide additional information about the stateof spermatogenesis.• Isolated FSH deficiency due to mutations in the FSH b gene has beendescribed.• Prolactin should be measured in men with galactorrhea or androgendeficiency and loss of libido.• Other hormone investigations occasionally are required, for example,1. Thyroid function tests with hyperprolactinemia,2. 17-hydroxyprogesterone measurements with congenital adrenalhyperplasia,3. Estradiol with liver disease or tumors,4. Human chorionic gonadotropin (hCG) with tumors and estrogenexcess,5. Pituitary function tests for panhypopituitarism.Genetic Studies• Karyotypes are performed in men with clinical evidence of primarytesticular failure and small testes to confirm a clinical diagnosis ofKlinefelter syndrome, in which the karyotype usually is 47, XXY, but theremay be higher numbers of X chromosomes or a sex-reversal 46, XXkaryotype.• While most men with Klinefelter syndrome produce no sperm some areoligospermic and very rarely, fertile.• Also sperm for ICSI may be obtained by testicular biopsy.• Defective spermatogenesis may occur with 47, XYY but the clinical pictureis much less uniform than it is for Klinefelter syndrome.

• It appears that the extra sex chromosome is deleted early ingametogenesis as the embryos and children generally have normalkaryotypes.• However an increased rate of sex chromosomal aneuploidy has beennoted in some studies of sperm from XXY and XYY men.• Because of the increased frequency of autosomal abnormalities:• Reciprocal and Robertsonian translocations and inversions,• In patients with defective spermatogenesis and the risk of transmittingthese in the unbalanced form in offspring, karyotypes should be performedbefore treatment in all men with moderate to severe oligospermia (forexample average sperm concentrations less than 5 million per milliliter) ofprimary testicular origin.• Cystic fibrosis gene studies are important for evaluation of patients withcongenital absence of the vas and their partners.• If the woman has a CF gene mutation, preimplantation genetic diagnosisof their embryos can be offered.• Microdeletions in the long arm of the Y chromosome have been found in5-15% of men with severe primary spermatogenic disorders.• Sons of men with these microdeletions have been found to have the samemicrodeletions.• Androgen receptor defects have also been found in some men withunexplained primary spermatogenic disorders.• Mutations in the gene impairing androgen receptor activity produceandrogen insensitivity which has a variable phenotypic expression fromtesticular feminization to otherwise normal males with gynecomastia orhypospermatogenesis and oligospermia.• Increases in the number of CAG repeats in exon 1 over about 40 areassociated with Kennedy disease - progressive spinobulbar atrophy andmen with this condition may be infertile.• It is considered that the number of CAG repeats has an inverse effect onthe activity of the androgen receptor.

• Several studies of the CAG repeat numbers in men with primaryspermatogenic defects have indicated significant increases in repeatnumber compared with those in controls although the numbers of repeatsare within the normal range. However this has not been confirmed in allstudies.• A relationship between lower sperm concentration and longer CAG tractsin normal men has also been reported.• Whether increasing intratesticular testosterone levels would increasespermatogenesis in men with underactive androgen receptors remains tobe determined.Other specific genetic tests and family studies may be indicated on clinicalgrounds for example: Kallmann syndrome, FSH and FSH receptormutations, myotonic dystrophy, mitochondrial gene mutations and defectsof sperm ultrastructure.• At present it is not clear what genetic disorders should be screened for ininfertile men.• Patients should be counseled about the possibility of transmitting knownand unknown genetic defects.Testicular Biopsy• Testicular biopsies are necessary to assess spermatogenesis in men withpresumed genital tract obstruction.• A significant proportion of men with azoospermia, normal testicular size,and normal FSH, are found to have severe spermatogenic disorders.• Some severe spermatogenic defects may be incomplete and as ICSI canbe performed with a few sperm obtained from the testes, diagnostictesticular biopsies can be offered to men with severe primary testicularfailure with persistent azoospermia.• If any tubules containing elongated spermatids can be found it should bepossible to perform ICSI.

• However, if no areas of spermatogenesis to this stage are seen in thediagnostic biopsies there is less chance that more extensive removal oftesticular tissue for sperm retrieval from the testes will be successful.• Open biopsies may be performed under local or general anesthesia.• It is most important that the tissue be removed from the testes withminimal damage and placed in a suitable fixative, such as Bouin orSteive's solution. Standard formalin fixatives destroy the cytoachitecture.• Needle biopsy procedures have become popular, and although manyobtain only isolated cells, these cells may be sufficient for diagnosis on thebasis of cytology or for flow cytometry assessment.• A technique for obtaining small amounts of tissue by needle aspirationbiopsy under local anesthesia has been developed (Fig. 4) that usuallyprovides sufficient material for a histological diagnosis of the state of theseminiferous epithelium.• The aspiration biopsy techniques do produce some deformation artefactsin the tissue.• Provided there is not a severe spermatogenic defect, needle aspirationbiopsy is useful for obtaining testicular sperm for ICSI.• Complications of this procedure include failure to obtain tissue particularlywith very small or fibrosed testes (

Figure 4. (A) Fine-needle tissue aspiration biopsy of thetestis. Local anesthetic is injected around the vas to blocktesticular sensation. (B) Fine-needle tissue aspiration biopsyof the testis A 21 gauge butterfly needle is inserted into thetestis. An assistant applies suction to the needle tubing via a10mL syringe and the operator makes thrusting movementsof the needle into the substance of the testis. (C) Fineneedletissue aspiration biopsy of the testis While

maintaining the suction the needle is removed carefully andany seminiferous tubules protruding from the needle aregrasped with fine forceps to avoid them falling back into thepuncture hole. With this technique seminiferous tubulesections are sucked into the needle and these are expelledinto some culture medium. Portions can be sent for histologyand the remainder used for extraction of sperm in the IVFlaboratory by stripping the seminiferous tissue out of theconnective tissue membrane of the seminiferous tubule.For clinical purposes, testicular histology is classified asfollows:Normal or hypospermatogenesis (all the cellular elements of spermatogenesisare present but in reduced numbers),Germ cell arrest (the initial cellular elements of spermatogenesis are present butat a certain stage the process stops, most often at the primary spermatocytes),Sertoli cell-only syndrome or germ cell aplasia (the tubules contain Sertoli cellsbut no germ cells),Hyalinization (the cellular elements have disappeared leaving only thickenedseminiferous tubules walls as in Klinefelter syndrome),Immature testis (no gonadotropin stimulation, prepubertal appearance).Examples are shown in Fig. 5.

Figure 5A. Testicular histology from fine-needleaspiration samples. Normal.Figure 5B. Testicular histology from fine-needleaspiration samples. Left mildhypospermatogenesis with elongated spermatidswith poor head morphology: right normal.

Figure 5C. Testicular histology from fine-needleaspiration samples. Mild-moderatehypospermatogenesis.Figure 5D. Testicular histology from fine-needleaspiration samples. Moderate-severehypospermatogenesis.

Figure 5E. Testicular histology from fine-needleaspiration samples. Germ cell arrest at theprimary spermatocyte stage.

Figure 5F. Testicular histology from fine-needleaspiration samples. Sertoli cell-only syndrome:low and high power.

Figure 5G. Testicular histology from fine-needleaspiration samples. Germ cell arrest at thespermatogonial stage from gonadotropindeficiency. Upper panel atrophic Leydig cellstained with antitestosterone antibody.

Figure 5H. Testicular histology from fine-needleaspiration samples. Carcinoma in situ, onlytransformed spermatogonia and Sertoli cellspresent.Other InvestigationsUltrasonography is useful for checking for tumors in the testes, particularly whenthe testes are difficult to palpate because of a tense hydrocele.It can also be used to measure testicular size and confirm the presence andnature of cysts or other abnormalities in the scrotum.Doppler blood flow assessment is valuable in assessing a painful swollen testisfor torsion or inflammation and for evaluating varicoceles.Other tests of a varicocele, including thermography, technetium scans, andvenography, may be performed but as pointed out below the value of treatingvaricoceles to improve fertility is uncertain.Rectal ultrasound may demonstrate cysts in the prostate, enlarged seminalvesicles or dilated ejaculatory ducts associated with distal genital tractobstructions.Clinical suspicion of the presence of a pituitary tumor should be followed up byappropriate radiology.

Abdominal imaging is necessary to check the position of an impalpable testis.