Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Dr. Furquan Alam
PG Resident,
Department of Biochemistry,
SGRRIMHS & SMI Hospital, Dehradun.
SEMEN ANALYSIS
SPERMATOGENESIS
The sperm formation involves two steps :
 1) Spermatocytogenesis
In the first step spermtogenic cells fo...
Site Of Sperm Formation
Seminiferous tubules of Testis
3/13/2016 8:00:37
PM
DR. FURQUAN ALAM 3
3/13/2016 8:00:37
PM
DR. FURQUAN ALAM 4
3/13/2016 8:00:37
PM
DR. FURQUAN ALAM 5
Seminal Fluid
Semen is body fluid that is ejaculated at the time of
orgasm, contain sperm & secretion of seminal vesicle,
...
Source Volume Characteristics
Urethral and bulbourethral
glands
0.1-0.2cc Viscous, clear
Testes, epididymides, vas
deferen...
 Semen is viscous, neutral or slightly alkaline & whitish
opaque.
 60 % semen volume is derived from seminal vesicle whi...
 About 10 -15 % of semen volume is also contributed by
epididymis, vasdeferens, cowper’s gland & uretheral gland.
 Less ...
3/13/2016 8:00:38
PM
DR. FURQUAN ALAM 10
Normal Values From WHO Manuals, Editions 2- 4 And Lower
Reference Limits From New 5th WHO Manual (2010)
Semen parameter
WH...
Steps: Semen Analysis
In the first 5 minutes:
 Placing the specimen container on the bench or in an
incubator (37 °C) for...
Steps Of Semen Analysis
Between 30 and 60 minutes:
Assessing sperm vitality (if the percentage of motile cells is
low).
...
Steps
Between 30 and 60 minutes:
Assessing peroxidase-positive cells (if round cells are present).
 Preparing spermatozo...
Steps
After 4 hours:
 Fixing, staining and assessing smears for sperm
morphology.
Later on the same day (or on a subseque...
Sample Collection:
Preparation:
 The sample should be collected in a private room near
the laboratory, in order to limit ...
Sample Collection:
Preparation:
 The man should be given clear written and spoken instructions
concerning the collection ...
Sample Collection for Diagnostic/ Research purpose:
 The sample should be obtained by masturbation and ejaculated into
a ...
Sterile collection of semen for assisted reproduction:
 This is performed as for diagnostic collection but the
specimen c...
Sterile collection of semen for microbiological analysis:
 In this situation, microbiological contamination from non-seme...
Collection of semen at home:
 A sample may be collected at home in exceptional circumstances, such as a
demonstrated inab...
Collection of semen by condom:
 A sample may be collected in a condom during sexual intercourse only in
exceptional circu...
Safe handling of specimens:
 Semen samples may contain dangerous infectious agents
(e.g. human immunodeficiency virus (HI...
Precautions
 Specimen should not be collected in ordinary condoms
since the powder or lubricant applied to the condom
may...
Storage
 The semen specimen should be examined immediately
after collection.
 It should be kept at room temperature.
3/1...
Indications For Analysis:
 To determine the fertility of the man.
 After a male has undergone vasectomy to check the
com...
 History To Be Noted:
 Name
 Date and time of collection
 Length of abstinence
 Interval between collection and analy...
Sample Form For
Semen Analysis
3/13/2016 9:12:39
PM
DR. FURQUAN ALAM 28
3/13/2016 9:17:25 PM DR. FURQUAN ALAM 29
NOTE:
Prolonged abstinence will lead to increased volume, but
reduced motility.
The patient should evacuate his bladder ...
Assessment Of Semen:(according To WHO)
 Standard tests:
1. Volume
2. pH
3. Sperm concentration
4. Total sperm count
5. Mo...
 Optional tests:
 Alpha galactosidase (neutral): 20mU or more.
 Zinc (total): 2.4 micromol or more
 Citric acid(total)...
Volume:
Measured by aspirating into a pipette or by using a syringe(non-toxic 1,2 or
5ml): Normal: 1.5ml or more.
Low volu...
Colour:
Homogenous grey opalescent appearance (Normal).
After prolonged abstinence, slightly yellow.
Deep yellow- Pyosp...
Viscosity:
 Freshly ejaculated semen is highly viscous due to
substrate produced by seminal vesicles. The
coagulum liquef...
Normal liquefaction time: 20-60min
Viscosity of liquefied semen can be
estimated by: Gentle aspiration into a 5ml pipett...
pH:
Measured by using a pH meter or pH paper.
Normal: 7.2-8
Semen is the strongest buffer in the body.
Seminal vesicle & v...
Sperm Concentration:
WBC Micropipette Semen-0.5mark
Diluting Fluid- 11 mark
Charge in counting chamber.
Count the number ...
Composition of diluting fluid:
1. Sodium bicarbonate- 5g. Counteracts mucus and allows even
dilution of this viscous fluid...
Normal count : 15 to 150 million/ml.
Oligozoospermia : < 15million/ml.
Causes:
 Mumps: orchitis
 Prostatitis
 Hypopitui...
Motility:
Routinely used technique:
 Place a drop of liquefied semen on a glass slide.
 Cover with cover slip and rim it...
Grading:
Rapid progressive motility
Slow/Sluggish progressive motility
Non - progressive motility
Immotility
3/13/2016 8:0...
Viability:
 If motility is < 40% a viability should be performed.
 Supravital staining by eosin Y with nigrosin. Dead ce...
Normal Motility: > 32%
If less than this, asthenozoospermia.
Causes:
 Cold
 Radiation
 Spermicides, Pesticides
 Prolon...
Sperm Morphology:
 Thin smears similar to blood smears are made feathering
technique.
 Before staining, mucoid material...
Sperm morphology: Ideal spermatozoon
Menkveld et al. 1991, WHO 1999, ESHRE 2002
Head oval shaped regular contour
Length: 4...
Computer Aided Sperm Analysis:
Automation seeks to establish standard methods of
analysis and set acceptable levels of acc...
Agglutination Of Sperms:
Motile sperms stick to each other in various
orientations like- head to head, midpiece to midpiec...
3/13/2016 9:37:54 PM DR. FURQUAN ALAM 49
Antisperm Antibodies:
Can occur in the:
a)Serum of male or female
b)Seminal plasma
c)Spermatozoa
Effects:
a)Lowered progre...
Antisperm antibodies are found in following
conditions:
Testicular disease
Autoimmune azoospermatogenesis
Following vas...
Techniques of detecting antibodies:
Agglutination
Immobilization
Precipitation
Complement fixation
Passive hemaggluti...
Other Cells:
Round cells :
1) Germinal cells (single or double highly condensed nucleus with
abundant cytoplasm).
2) Leuco...
Biochemical Assays:
 Prostate gland function
 Zinc
 Citric acid
 Acid phosphatase
 Seminal vesicles
 Fructose
 Pros...
Determination of fructose:
Procedure:
Pipette 5 ml of resorcinol reagent in a test tube. [Resorcinol reagent:
resorcinol-...
Microbiological Assays:
 Indications:
1) Accessory gland infection.
2) High number of leucocytes in semen(>1million/ml)
...
Sperm Function Tests:
Factors responsible for defective sperm function:
 Peroxidase damage by excessive generation of rea...
a. Sperm penetration assay:
Uses zona denuded golden hamster eggs as hosts for
the penetration of human sperms. This measu...
c. Acrosin assay:
Measures acrosin, a trypsin like serine protienase
specific to sperm acrosome. It is responsible for spe...
D. Hypo-osmotic Swelling Test:
 For successful union test of the spermatozoa with female
gametes, integrity of sperm memb...
E. Cervical mucous penetration test/ Post coital test/ Sims-
Huhner test:
Aims :
To study the quality of cervical mucous....
Examination For The Presence Of Sperms In
Medicolegal Cases:
Obtaining the sample:
1. From vagina: direct aspiration or sa...
3/13/2016 9:16:24 PM DR. FURQUAN ALAM 63
2) Determination of acid phosphatase:
More sensitive 2500 king armstrong units
3) Blood group substances
4) Florence test...
TYPICAL HEAD DEFECTS
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 65
TYPICAL MID PIECE DEFECTS
Bent
Flat
implantation Too thin Irregular
Too
thick
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 66
TYPICAL TAIL DEFECTS
Hairpin
Cytoplasmic
drop Double Coiled Stumped
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 67
THANK YOU
3/13/2016 8:00:38
PM
DR. FURQUAN ALAM 68
Upcoming SlideShare
Loading in …5
×

Semen Analysis

12,594 views

Published on

As per WHO guideline 2010

Published in: Health & Medicine
  • Free Video Reveals 1 Weird Trick To Cure Vitiligo In 45 Days! Click Here: ☺☺☺ https://j.mp/3kTNHDZ
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • You can try to use this service ⇒ www.HelpWriting.net ⇐ I have used it several times in college and was absolutely satisfied with the result.
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Free Video Reveals 1 Weird Trick To Heal Vitiligo Forever! Click Here: ▶▶▶ http://tinyurl.com/y4d5dqxj
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • If u need a hand in making your writing assignments - visit ⇒ www.WritePaper.info ⇐ for more detailed information.
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Você pode obter ajuda de ⇒ www.boaaluna.club ⇐ Sucesso e cumprimentos!
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here

Semen Analysis

  1. 1. Dr. Furquan Alam PG Resident, Department of Biochemistry, SGRRIMHS & SMI Hospital, Dehradun. SEMEN ANALYSIS
  2. 2. SPERMATOGENESIS The sperm formation involves two steps :  1) Spermatocytogenesis In the first step spermtogenic cells form rounded cells called spermatids.  2) Spermiogenesis In the second step spermatids differentiate into specialized cells known as sperms. 3/13/2016 8:00:37 PM DR. FURQUAN ALAM 2
  3. 3. Site Of Sperm Formation Seminiferous tubules of Testis 3/13/2016 8:00:37 PM DR. FURQUAN ALAM 3
  4. 4. 3/13/2016 8:00:37 PM DR. FURQUAN ALAM 4
  5. 5. 3/13/2016 8:00:37 PM DR. FURQUAN ALAM 5
  6. 6. Seminal Fluid Semen is body fluid that is ejaculated at the time of orgasm, contain sperm & secretion of seminal vesicle, prostate, Cowper's gland & urethral gland. 3/13/2016 8:00:37 PM DR. FURQUAN ALAM 6
  7. 7. Source Volume Characteristics Urethral and bulbourethral glands 0.1-0.2cc Viscous, clear Testes, epididymides, vas deferentia 0.1-0.2cc Sperm present Prostate 0.5-1.0cc Acidic,watery Seminal vesicles 1.0-3.0cc Gelatinous, fructose positive Complete ejaculate 1.5-5.0cc Liquefies in 20-25min Semen 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 7
  8. 8.  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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 8
  9. 9.  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 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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 9
  10. 10. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 10
  11. 11. Normal Values From WHO Manuals, Editions 2- 4 And Lower Reference Limits From New 5th WHO Manual (2010) Semen parameter WHO edition and year 2nd - 1987 3rd - 1992 4th - 1999 5th - 2010 Volume (ml) 2.0 2.0 2.0 1.5 Sperm concentration (106/ml) 20 20 20 15 Total sperm count (106) 40 40 40 39 Motility (% progressive) 50 50 50 32 Vitality (% live) 50 75 75 58 Morphology (% normal) 50 30 (15) 4 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 11
  12. 12. Steps: Semen Analysis In the first 5 minutes:  Placing the specimen container on the bench or in an incubator (37 °C) for liquefaction. Between 30 and 60 minutes:  Assessing liquefaction and appearance of the semen.  Measuring semen volume.  Measuring semen pH (if required).  Preparing a wet preparation for assessing microscopic appearance, sperm motility and the dilution required for assessing sperm number. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 12
  13. 13. Steps Of Semen Analysis Between 30 and 60 minutes: Assessing sperm vitality (if the percentage of motile cells is low).  Making semen smears for assessing sperm morphology.  Making semen dilutions for assessing sperm concentration.  Assessing sperm number.  Performing the mixed antiglobulin reaction (MAR) test (if required). 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 13
  14. 14. Steps Between 30 and 60 minutes: Assessing peroxidase-positive cells (if round cells are present).  Preparing spermatozoa for the immunobead test (if required).  Centrifuging semen (if biochemical markers are to be assayed). Within 3 hours:  Sending samples to the microbiology laboratory (if required). 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 14
  15. 15. Steps After 4 hours:  Fixing, staining and assessing smears for sperm morphology. Later on the same day (or on a subsequent day if samples are frozen):  Assaying accessory gland markers (if required).  Performing the indirect immunobead test (if required). 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 15
  16. 16. Sample Collection: Preparation:  The sample should be collected in a private room near the laboratory, in order to limit the exposure of the semen to fluctuations in temperature and to control the time between collection and analysis.  The sample should be collected after a minimum of 2 days and a maximum of 7 days of sexual abstinence. If additional samples are required, the number of days of sexual abstinence should be as constant as possible at each visit. 3/13/2016 8:20:47 PM DR. FURQUAN ALAM 16
  17. 17. Sample Collection: Preparation:  The man should be given clear written and spoken instructions concerning the collection of the semen sample. These should emphasize that the semen sample needs to be complete and that the man should report any loss of any fraction of the sample.  The following information should be recorded on the report form: the man’s name, birth date and personal code number, the period of abstinence, the date and time of collection, the completeness of the sample, any difficulties in producing the sample, and the interval between collection and the start of the semen analysis. 3/13/2016 8:18:33 PM DR. FURQUAN ALAM 17
  18. 18. Sample Collection for Diagnostic/ Research purpose:  The sample should be obtained by masturbation and ejaculated into a clean, wide-mouthed container made of glass or plastic, from a batch that has been confirmed to be non-toxic for spermatozoa.  The specimen container should be kept at ambient temperature, between 20 °C and 37 °C, to avoid large changes in temperature that may affect the spermatozoa after they are ejaculated into it. It must be labelled with the man’s name and identification number, and the date and time of collection.  The specimen container is placed on the bench or in an incubator (37 °C) while the semen liquefies.  Note in the report if the sample is incomplete, especially if the first, sperm-rich fraction may be missing. If the sample is incomplete, a second sample should be collected, again after an abstinence period of 2–7 days. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 18
  19. 19. Sterile collection of semen for assisted reproduction:  This is performed as for diagnostic collection but the specimen containers, pipette tips and pipettes for mixing must be sterile. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 19
  20. 20. Sterile collection of semen for microbiological analysis:  In this situation, microbiological contamination from non-semen sources (e.g. commensal organisms from the skin) must be avoided. The specimen containers, pipette tips and pipettes for mixing must be sterile.  The man should:  Pass urine.  Wash hands and penis with soap, to reduce the risk of contamination of the specimen with commensal organisms from the skin.  Rinse away the soap.  Dry hands and penis with a fresh disposable towel.  Ejaculate into a sterile container.  Note: The time between collection of the semen sample and the start of the investigation by the microbiological laboratory should not exceed 3 hours. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 20
  21. 21. Collection of semen at home:  A sample may be collected at home in exceptional circumstances, such as a demonstrated inability to produce a sample by masturbation in the clinic or the lack of adequate facilities near the laboratory.  The man should be given clear written and spoken instructions concerning the collection and transport of the semen sample. These should emphasize that the semen sample needs to be complete, i.e. all the ejaculate is collected, including the first, sperm-rich portion, and that the man should report any loss of any fraction of the sample. It should be noted in the report if the sample is incomplete.  The man should be given a pre-weighed container, labelled with his name and identification number.  The man should record the time of semen production and deliver the sample to the laboratory within 1 hour of collection.  During transport to the lab., the sample should be kept between 20 °C - 37 °C.  The report should note that the sample was collected at home or another location outside the laboratory. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 21
  22. 22. Collection of semen by condom:  A sample may be collected in a condom during sexual intercourse only in exceptional circumstances, such as a demonstrated inability to produce a sample by masturbation.  Only special non-toxic condoms designed for semen collection should be used; such condoms are available commercially.  The man should be given information from the manufacturer on how to use the condom, close it, and send or transport it to the laboratory.  The man should record the time of semen production and deliver the sample to the laboratory within 1 hour of collection.  During transport to the lab., the sample should be kept between 20 °C and 37 °C.  The report should note that the sample was collected by means of a special condom during sexual intercourse at home or another location outside the laboratory. Note: Ordinary latex condoms must not be used for semen collection because they contain agents that interfere with the motility of spermatozoa (Jones et al., 1986). 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 22
  23. 23. Safe handling of specimens:  Semen samples may contain dangerous infectious agents (e.g. human immunodeficiency virus (HIV), hepatitis viruses or herpes simplex virus) and should therefore be handled as a biohazard. If the sample is to be processed for bioassay, intra-uterine insemination (IUI), in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), or if semen culture is to be performed, sterile materials and techniques must be used. Safety guidelines as outlined in Appendix 2 should be strictly followed; good laboratory practice is fundamental to laboratory safety (WHO, 2004). 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 23
  24. 24. Precautions  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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 24
  25. 25. Storage  The semen specimen should be examined immediately after collection.  It should be kept at room temperature. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 25
  26. 26. Indications For Analysis:  To determine the fertility of the man.  After a male has undergone vasectomy to check the completeness of the procedure (retrograde ejaculation).  In medico-legal situations such as disputes about the paternity of a child.  After reversal of vasectomy to confirm the success of the procedure. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 26
  27. 27.  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 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 27
  28. 28. Sample Form For Semen Analysis 3/13/2016 9:12:39 PM DR. FURQUAN ALAM 28
  29. 29. 3/13/2016 9:17:25 PM DR. FURQUAN ALAM 29
  30. 30. NOTE: Prolonged abstinence will lead to increased volume, but reduced motility. The patient should evacuate his bladder before specimen collection. If retrograde ejaculation is suspected, a post- ejaculate urine sample is collected and examined for presence of sperms. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 30
  31. 31. Assessment Of Semen:(according To WHO)  Standard tests: 1. Volume 2. pH 3. Sperm concentration 4. Total sperm count 5. Motility 6. Morphology 7. Vitality 8. White blood cells 9. Immunobead test 10.MAR test 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 31
  32. 32.  Optional tests:  Alpha galactosidase (neutral): 20mU or more.  Zinc (total): 2.4 micromol or more  Citric acid(total): 52 micromol or more  Acid phosphatase: 200U or more  Fructose: 13 micromol or more 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 32
  33. 33. Volume: Measured by aspirating into a pipette or by using a syringe(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 collection High volume: >10ml: Dilutional Oligozoospermia Aspermia: Absence of ejaculate Retrograde ejaculation Anejaculation B/l ejaculatory duct obstruction 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 33
  34. 34. Colour: Homogenous grey opalescent appearance (Normal). 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. Tumor of the genital tract. Increased turbidity indicates inflammatory process in some part of the reproductive tract. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 34
  35. 35. 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 prostatic secretion. To liquefy, add bromeline, plasmin or chymotrypsin. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 35
  36. 36. Normal liquefaction time: 20-60min Viscosity of liquefied semen can be estimated by: Gentle aspiration into a 5ml pipette, then allowing the semen to drop by gravity. Observe the length of the thread. Normal semen leaves as small discrete drops. Increased viscosity is associated with poor invasion of cervical mucus in post-coital studies as well as decreased ability to fertilize ovum. Absence of viscosity points to reduced cell content. The semen from males with b/l congenital absence of vas deferentia & seminal vesicles fails to coagulate due to absence of substrate. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 36
  37. 37. pH: Measured by using a pH meter or pH paper. Normal: 7.2-8 Semen is the strongest buffer in the body. Seminal vesicle & vas deference secretions alkaline Prostatic secretion acidic(due to citric acid, proteolytic enzymes, acid phosphatase) Motility is reduced in acidic medium. pH<7 is associated with largely prostatic secretions due to congenital aplasia of vas & seminal vesicles and when contaminated with urine. pH>8 is associated with acute infection of prostate, seminal vesicles or epididymis. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 37
  38. 38. Sperm Concentration: WBC Micropipette Semen-0.5mark Diluting Fluid- 11 mark Charge in counting chamber. Count the number in four corner squares. Sperm/ml = Nx10x20x1000 = Nx50,000 4 If it is very viscid, add mucolytic agent in 1:1 dilution and multiply by 2. If count is high(>100m), then use higher dilution. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 38
  39. 39. 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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 39
  40. 40. Normal count : 15 to 150 million/ml. Oligozoospermia : < 15million/ml. Causes:  Mumps: orchitis  Prostatitis  Hypopituitarism  Hypogonadotropic hypogonadism  Estrogen secreting tumors  Hypo/Hyperthyroidism  Drugs: Sulfasalazine, Cimetidine, Estrogen, Nitrofurantoin, Caffeine, Alcohol, Cocaine, Smoking tobacco, Herbal medications, Chemotherapeutic agents. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 40
  41. 41. Motility: Routinely used technique:  Place a drop of liquefied semen on a glass slide.  Cover with cover slip 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: Cover slip- 22mm x 22mm Semen- 10µl , depth of 20µm. Phase contrast microscopy – 400x/600x at 37 °C. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 41
  42. 42. Grading: Rapid progressive motility Slow/Sluggish progressive motility Non - progressive motility Immotility 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 42
  43. 43. 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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 43
  44. 44. Normal Motility: > 32% If less than this, asthenozoospermia. Causes:  Cold  Radiation  Spermicides, Pesticides  Prolonged heat exposures  Prolonged abstinence  Autoimmunity Progressive loss of motility by 5%/hr after 3hrs. After liquefaction or within 60mins after ejaculation. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 44
  45. 45. Sperm Morphology:  Thin smears similar to blood smears are made feathering technique.  Before staining, mucoid material is removed by gentle washing with semen dilution fluid. Then wash gently with buffered distilled 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. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 45
  46. 46. 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 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 46
  47. 47. Computer Aided Sperm Analysis: Automation seeks to establish standard methods of analysis and set acceptable levels of accuracy in measuring various parameters to promote interlaboratory comparisons. Advantages: Subjective errors avoided Motility can be assessed quantitatively Capable of handling many samples without undergoing fatigue Morphological defects can be made out 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 47
  48. 48. Agglutination Of Sperms: Motile sperms stick to each other in various orientations like- head to head, midpiece to midpiece, tail to tail or in combinations depending on the specificity of antisperm antibodies. Agglutination points to immunological cause of infertility. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 48
  49. 49. 3/13/2016 9:37:54 PM DR. FURQUAN ALAM 49
  50. 50. Antisperm Antibodies: Can occur in the: a)Serum of male or female b)Seminal plasma c)Spermatozoa Effects: a)Lowered progressive motility. b)Decreased ability to penetrate cervical mucus. 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 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 50
  51. 51. Antisperm antibodies are found in following conditions: Testicular disease Autoimmune azoospermatogenesis Following vasectomy Repeated infections Obstruction of ducts Cryptorchidism Varicocele Testicular biopsy Trauma Torsion Genetic predisposition 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 51
  52. 52. Techniques of detecting antibodies: Agglutination Immobilization Precipitation Complement fixation Passive hemagglutination Cytotoxicity For screening, Mixed antiglobulin reaction (MAR) test Immunobead method 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 52
  53. 53. 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 deficiency Epithelial cells from urogenital tract. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 53
  54. 54. Biochemical Assays:  Prostate gland function  Zinc  Citric acid  Acid phosphatase  Seminal vesicles  Fructose  Prostaglandins  Epididymis  Alpha glucosidase  L-carnitine  Plycerophosphocholine 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 54
  55. 55. Determination of fructose: Procedure: Pipette 5 ml of resorcinol reagent in a test tube. [Resorcinol reagent: 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 colored ppt. in 30 seconds. In quantitative assays, this is compared with a known fructose standard at 490nm. Normal level of fructose: 150-300mg/dl. Reduced levels: Seminal vesicle dysfunction. High sperm count 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 55
  56. 56. 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 & anaerobic organisms.  If >1000CFUs/ml, then do antibiotic sensitivity tests. E. Coli can cause sperm agglutination and immobilization. This is mediated by mannose and mannose binding cell surface structures present on both cell types. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 56
  57. 57. 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-MB )  abnormal activities of mid piece. The tests include: a. Sperm penetration assay (SPA) b. Hemizona assay c. Acrosin assay d. Hypo osmotic saline test e. Cervical mucous penetration test 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 57
  58. 58. a. Sperm penetration assay: Uses zona denuded golden hamster eggs as hosts for the penetration of human sperms. This measures Sperm capacitation Sperm oocytes fusion Sperm incorporation in oocytes Decondensation of sperm chromatin b. Hemizona assay: Utilizes unfertilized oocytes 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 58
  59. 59. c. Acrosin assay: Measures acrosin, a trypsin like serine protienase specific to sperm acrosome. It is responsible for sperm penetration of the zona pellucida, after its release is triggered by the binding of sperm to zona. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 59
  60. 60. D. 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 assessment of membrane function is a useful indicator of fertilizing ability of spermatozoa. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 60
  61. 61. E. Cervical mucous penetration test/ Post coital 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, the endocervical mucous is collected in a Luer syringe. The volume, colour and viscosity (Spinbarkeit ) of the mucous noted. A drop of mucous is placed on a slide and examined for the presence of sperms. Atleast 10 motile sperms should be present per hpf. The material should be examined for leucocytes, erythrocytes and trichomanads. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 61
  62. 62. 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 centrifugation Stain with H & E. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 62
  63. 63. 3/13/2016 9:16:24 PM DR. FURQUAN ALAM 63
  64. 64. 2) Determination of acid phosphatase: More sensitive 2500 king armstrong units 3) Blood group substances 4) Florence test: screening method Depends on presence of cholines 5) Precipitin test: Using specific antiserum by capillary tube reaction. 6) Determination of sperm specific LDH isoenzyme. 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 64
  65. 65. TYPICAL HEAD DEFECTS 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 65
  66. 66. TYPICAL MID PIECE DEFECTS Bent Flat implantation Too thin Irregular Too thick 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 66
  67. 67. TYPICAL TAIL DEFECTS Hairpin Cytoplasmic drop Double Coiled Stumped 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 67
  68. 68. THANK YOU 3/13/2016 8:00:38 PM DR. FURQUAN ALAM 68

×