The sperm formation involves two steps : in the first step spermtogenic cells form rounded cells called spermatids which in the second step differentiate into specialized cells known as sperms. These processes are labeled respectively asSpermatocytogenesisSpermiogenesis
The primitive sex cells appear earliest in 4th week of intra uterine life in the wall of yolk sac as primordial germ cellsThey migrate to the developing testes and lie dormant among the cells lining the seminiferous tubules
At puberty the germ cells awaken and start the actual process of spermato genesisThese cells increase in number by simple mitosis to form cells known as spermatogonia ; type- A and type- B.Type-B spermatogonia, replicate DNA to have 46 double structured chromosomes to begin meiosis-1 and are called primary spermatocytes.
In prophase1 pairing and crossing over of chromosomal segments takes place and genetic recombination occursIn metaphase1 homologous pairs arrange on equatorIn anaphase 1 homologous pairs separate to go to opposite polesAfter telophase 1 meiosis 1 ends and 2 secondary spermatocytes form each with 23 double structured chromosomes with X or Y sex chromosome complementIt takes about 22 days to complete meiosis-1
Meiosis 2 follows immediately without DNA replication. Only 23 double structured chromosomes are involved2 secondary spermatocytes quickly undergo meiosis-2 (Centromeres split in metaphase 2) and end with the formation of 4 sperrmatids each with 23 single structured chromosomes and1N DNATwo spermatids bear X chromosome complement and other two bear Y chromosome complement
As steps of spermatogenesis continue the spermatocytes progressively move from basement membrane to the luminal side of seminiferous tubuleThe cells of Sertoli provide nutrition and pockets of support to developing spermatocytes
The spermatocytes in different stages of development remain attached by cytoplasmic bridgesAll the spermatocytes are not in the same stage of development in the seminiferous tubules
Spermatids are rounded cells.They modify to assume specific shape of the sperm.This process is called Spermiogenesis. In it they elongate and reorganize internal structure to acquire the particular shape.
The changes include ; Golgi apparatus forms acrosomal cap-proteolitic enzymes Nucleus is condensed Centriols: make collar around neck Microtubules, forrm flagellum, Mitochondria arrange as spiral around neck Excess cytoplasm cast off as residual body Cyto plasmic bridges break and sperms release from Sertoli cells to lie free in lumen of seminiferous tubules.About 64 days are required to go from a spermatogonium to a sperm
A mature sperm has head, neck and tailFrom lumen of seminiferous tubules sperms enter duct of epididymisThey take 20 days to travel this 4-6 meter long tortuous ductIf ejaculation does not occur they die and degenerate
Semen is body fluid that is ejaculated at the time of orgasm, contain sperm & secretion of seminal vesicle, prostate, cowper’s gland & urethral gland.
Semen is viscous, neutral or slightly alkaline & whitish opaque.60 % semen volume is derived from seminal vesicle which is also a major source of high FRUCTOSE content of semen.Seminal vesicle secretion also provide the substrate for the coagulation of the semen following ejaculation.
About 20 % of the volume of semen is contributed by the prostate gland.It is milky in appearance & also rich in proteolytic enzymes are responsible for the liquefaction of semen.
About 10 -15 % of semen volume is also contributed by EPIDIDYMIS, VASDEFERENS, COWPER’S GLAND & URETHERAL GLAND.Less than 5 % of semen volume is contributed by Spermatozoa.
The process of ejaculation result in the mixing of these distinct fraction of semen.These enter the urethra individually in the rapid succession.
The function of first clear fluid fraction may be to cleanse & lubricate the urethra in preparation for the bulk of following ejaculate - It originate from urethral & cowper’s gland.
The second fraction consist of small amount of secretion from epididymis & vasdeferns & large proportion of prostatic secretion which contain spermatozoa.
The third & final fraction consist of mucoid secretion resulting from emptying of seminal vesicle.The semen specimen collected for routine examination should contain all above mention fraction.
Sample should be collected in wide mouth clean & dry bottle.1. Semen collected following 3 days of abstinence.2.The specimen should be collected by MASTURBATION in the clinical pathology laboratory.This allow a complete examination of semen particularly liquefication time.
3. Alternate method for collection is in pateint’s house by coitous interuruptus or masturbation.The specimen should be deliverd within 30 min to the laboratory.( This specimen may be not satisfactory).
1. Specimen should not be collected in ordinary condoms since the powder or lubricant applied to the condom may be spermicidal.The container in which the semen sample is collected should be free from detergent.
The semen specimen should be examined immediately after collection.It should be kept at room temperature.
To determine the fertility of the man.After a male has undergone vasectomy to check the completeness of the procedure.In medicolegal situations such as disputes about the paternity of a child.After reversal of vasectomy to confirm the success of the procedure.
HISTORY TO BE NOTED:•Name•Date and time of collection•Length of abstinence•Interval between collection and analysis•History of fever•Drug intake•Alcohol abuse
NOTE:Prolonged abstinence will lead toincreased volume, but reducedmotility.The patient should evacuate hisbladder before specimen collection. Ifretrograde ejaculation is suspected, apost-ejaculate urine sample iscollected and examined for presenceof sperms.
ASSESSMENT OF SEMEN:(according to WHO)Standard tests:1. Volume2. pH3. Sperm concentration4. Total sperm count5. Motility6. Morphology7. Vitality8. White blood cells9. Immunobead test10. MAR test
Optional tests:• Alpha galactosidase(neutral): 20mU or more• Zinc (total): 2.4micromol or more• Citric acid(total):52micromol or more• Acid phosphatase:200U or more• Fructose:13micromol or more
VOLUME:Measured by aspirating into a pipette or by using asyringe(non-toxic 1,2 or 5ml)Normal-1.5ml or more.Low volume<1.5ml•B/l ejaculatory duct obstruction•B/l congenital vasal aplasia•Inadequate erection & improper mood at collection•Incomplete collectionHigh volume>10ml: Dilutional oligozoospermiaAspermia Absence of ejaculate•Retrograde ejaculation•Anejaculation•B/l ejaculatory duct obstruction
COLOUR:Homogenous grey opalescent appearance.After prolonged abstinence, slightly yellow.Deep yellow- Pyospermia.Rust colour- Small bleedings in seminal vesicles.Red or brown indicates presence of blood. •Trauma to the genital tract. •Inflammation. •Tumour of the genital tract.Increased turbidity indicates inflammatory process in some partof the reproductive tract.
VISCOSITY: Freshly ejaculated semen is highly viscous due to substrate produced by seminal vesicles. The coagulum liquefies spontaneously to form a translucent, viscous fluid in a three stage process. Action of a prostatic clotting enzyme. Liquefaction is initiated by enzymes of prostatic origin. Protein fragments are further degraded into free amino acids and ammonia. Failure to liquefy indicates inadequate prostaticsecretion. To liquefy, add bromeline, plasmin orchymotrypsin.
Normal liquefaction time: 20-60minViscosity of liquefied semen can be estimatedby: Gentle aspiration into a 5ml pipette, then allowing thesemen to drop by gravity. Observe the length of the thread.Normal semen leaves as small discrete drops.Increased viscosity is associated with poorinvasion of cervical mucus in post-coital studies as well asdecreased ability to fertilize ovum.Absence of viscosity points to reduced cell content.The semen from males with b/l congenital absence of vasdeferentia & seminal vesicles fails to coagulate due to absenceof substrate.
pH:Measured by using a pH meter or pH paper.Normal:7.2-8Semen is the strongest buffer in the body.Seminal vesicle & vas deference secretions alkalineProstatic secretion acidic(due to citric acid, proteolyticenzymes, acid phosphatase)Motility is reduced in acidic medium.pH<7 is associated with largely prostatic secretions due tocongenital aplasia of vas & seminal vesicles and whencontaminated with urine.pH>8 is associated with acute infection of prostate, seminalvesicles or epididymis.
SPERM CONCENTRATION:WBC Micropipette Semen-0.5mark Diluting Fluid- 11 markCharge in counting chamber.Count the number in four corner squares.Sperm/ml = Nx10x20x1000 4 =Nx50,000If it is very viscid, add mucolytic agent in 1:1 dilution andmultiply by 2.If count is high(>100m), then use higher dilution.
Composition of diluting fluid:1. Sodium bicarbonate- 5g. Counteracts mucus and allows even dilution of this viscous fluid.2. Phenol-1ml. Kills sperms and stops their movement. Also acts as preservative.3. Distilled water-100ml.A man should not be termed oligozoospermic until atleast 3 samples are evaluated at an interval of 3wks and 3 months.Azoospermia is a condition where the semen sample has no spermatozoa in a fresh sample or in a centrifuged resuspended sample.
MOTILITY:Routinely used technique:•Place a drop of liquefied semen on a glass slide.•Cover with coverslip and rim its edges with vaseline.•Examine under 40X with reduced illumination.•Count the number actively motile sperms out of 200.•Calculate the percentage.For accuracy,Coverslip- 22mm x 22mmSemen- 10ul , depth of 20um.Phase contrast microscopy – 400x/600x at 37deg C.
VIABILITY:If motility is < 40% a viability should be performed.Supravital staining by eosin Y with nigrosin. Dead cells take up the stain. 100 sperms are counted. Live/Dead sperm ratio calculated.Hypo osmotic saline test (HOS): Sperms are added to hypo osmotic saline and incubated at 37deg C. Swelling of the sperm tail is examined under phase contrast microscope. Sperms which have active membrane swell after 30mins. Many immotile live sperms direct towards immotile cilia syndrome. Do electron microscopy.
Normal Motility: After liquefaction orIf less than this, asthenozoospermia. within 60mins after ejaculation.Causes:ColdRadiationSpermicides, PesticidesProlonged heat exposuresProlonged abstinenceAutoimmunityProgressive loss of motility by 5%/hr after 3hrs.
SPERM MORPHOLOGY:•Thin smears similar to blood smears are made featheringtechnique.•Before staining, mucoid material is removed by gentle washingwith semen dilution fluid. Then wash gently with buffereddistilled water.•Several staining techniques are used 1)Pap is the best. 2)Haematoxylene technique. 3)Giemsa.4)Leishman/basic fuchsin(0.25% acqeous).5)Crystal violet. 6)Diff-Quick stain•On basic fuchsin, Sperm head caps: light blue. Nuclear post: dark blue. Body &tails: red/pink.
Sperm morphology: Ideal spermatozoon Menkveld et al. 1991, WHO 1999, ESHRE 2002 Head oval shaped regular contour Length: 4-5.5 micron Width 2.5-3.5 micron Darker posterior region Base of head should be broad Single tail symmetrically attached BORDERLINE FORMS = ABNORMAL
COMPUTER AIDED SPERM ANALYSIS:Automation seeks to establish standard methods ofanalysis and set acceptable levels of accuracy inmeasuring various parameters to promoteinterlaboratory comparisons.Advantages:Subjective errors avoidedMotility can be assessed quantitativelyCapable of handling many samples without undergoingfatigueMorphological defects can be made out
AGGLUTINATION OFSPERMS:Motile sperms stick to eachother in various orientations like-head to head, midpiece tomidpiece, tail to tail or incombinations depending on thespecificity of antispermantibodies. Agglutination pointsto immunological cause ofinfertility.
ANTISPERM ANTIBODIES:Can occur in the:a)Serum of male or female b)Seminal plasma c)SpermatozoaEffects:a)Lowered progressive motility. b)Decreased ability to penetrate cervicalmucus. c)Decreased ability to penetrate egg.Antibodies are found to react with: a)The front part of acrosome. b)Post-nuclear cap c)Tail piece d)Equatorial part of acrosome
Antisperm antibodies are found in followingconditions:•Testicular disease•Autoimmune azoospermatogenesis•Following vasectomy•Repeated infections•Obstruction of ducts•Cryptorchidism•Varicocele•Testicular biopsy•Trauma•Torsion•Genetic predisposition
OTHER CELLS:Round cells –1) germinal cells (single or double highly condensed nucleus with abundant cytoplasm). 2)leucocytes < 1 million/ml or 1- 2/hpf. Increased no. in infection of reproductive tract.RBCs – normally absent.Present in 1. TB of seminal vesicles 2. rupture of blood vessels 3. Infection of prostate 4.Vit. C deficiencyEpithelial cells from urogenital tract.
Determination of fructose:Procedure:Pipette 5 ml of resorcinol reagent in a test tube. [Resorcinolreagent: resorcinol-50mg, Conc.HCl-33ml, distilled water-67ml]Add 0.5 ml of semen.Mix and place in a boiling waterbath for 5min or heat.Observations: Red coloured ppt. in 30 secs.In quantitative assays, this is compared with a known fructosestandard at 490nm.Normal level of fructose: 150-300mg/dl.Reduced levels: -Seminal vesicle dysfunction -High sperm count
MICROBIOLOGICAL ASSAYS:Indications: 1) Accessory gland infection. 2) High number of leucocytes in semen(>1million/ml)Precautions should be taken to avoid contamination.Culture should be done to detect both aerobic & anerobicorganisms.If >1000CFUs/ml, then do antibiotic sensitivity tests.E. Coli can cause sperm agglutination and immobilization. Thisis mediated by mannose and mannose binding cell surfacestructures present on both cell types.
SPERM FUNCTION TESTS:Factors responsible for defective sperm function:• Peroxidase damage by excessive generation of reactive oxygen species.• High activity levels of creatine phosphokinase and a low ratio of muscle( CK-M) to the combined activities of muscle and brain isoforms( CK-M+B) abnormal activities of mid piece.The tests include:a. Sperm penetration assay (SPA)b. Hemizona assayc. Acrosin assayd. Hypo osmotic saline teste. Cervical mucous penetration test
a. Sperm penetration assay:Uses zona denuded golden hamstereggs as hosts for the penetration ofhuman sperms. This measuresSperm capacitationSperm oocytes fusionSperm incorporation in oocytesDecondensation of sperm chromatinb. Hemizona assay:Utilizes unfertilized oocytes
c. Acrosin assay:Measures acrosin, a trypsin like serineprotienase specific to spermacrosome. It is responsible for spermpenetration of the zona pellucida, afterits release is triggered by the bindingof sperm to zona.
HYPO-OSMOTIC SWELLING TEST:-For successful union test of the spermatozoa with female gametes, integrity of sperm membrane is very essential.Sperm capacitation, acrosomal reaction & penetration of egg is also dependent upon membrane integrity of spermatozoa.Therefore assesment of membrane function is a useful indicator of fertilizing ability of spermatozoa.
f. Cervical mucous penetration test/ Postcoital test/ Sims-Huhner test:Aims : -To study the quality of cervical mucous. -To know the ability of spermatozoa to penetrate the cervical mucous and maintain activity.After 8-10 hrs of coitus during the ovulatory phase, theendocervical mucous is collected in a Luer syringe. Thevolume, colour and viscosity (Spinbarkeit ) of themucous noted. A drop of mucous is placed on a slideand examined for the presence of sperms. Atleast 10motile sperms should be present per hpf. The materialshould be examined for leucocytes, erythrocytes andtrichomanads.
EXAMINATION FOR THE PRESENCE OF SPERMS IN MEDICOLEGAL CASES:Obtaining the sample:1. From vagina: direct aspiration or saline lavage.2. From clothing or other fabrics: preliminary scan with UV light green fluorescence 1sq cm of stained fabric soak in 1-2ml of physiological saline for 1hr. Then fluid is subjected to tests.Tests:1)Examination for sperms:• Direct smears from vagina• Smears from aspirate• Washings from fabric after centrifugationStain with H & E.
2) Determination of acid phosphatase:More sensitive 2500 king armstrong units3) Blood group substances4) Florence test: screening method Depends on presence of cholines5) Precipitin test:Using specific antiserum by capillary tubereaction.6) Determination of sperm specific LDH isoenzyme.