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Andrology lab

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1. Semen analysis according to WHO 2010
2. The importance of andrology laboratory inside IVF unit

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Andrology lab

  1. 1. Andrology Laboratory….. Is it important ? Dr. Yasmin Magdi Abd-Elkreem
  2. 2. Andrology Laboratory • Andrology: The science of male reproduction including sperm production and diseases and abnormalities thereof. • The Andrology Laboratory offers a full range of specialized tests and services for the evaluation and treatment (IUI) of male infertility by highly skilled laboratory personnel, all of whom have completed specialized training in infertility laboratory techniques.
  3. 3. Andrology Laboratory • convenient, same-site andrology services in an atmosphere that is attractive, private, and comfortable. 1. Semen analysis. 2. specialized tests (Sperm Morphology Classification using Kruger’s Strict Criteria, Sperm Viability Testing, Hypo-osmotic Swelling Test (HOS) , Testing for Anti-sperm antibodies, Testing for Leukocytospermia. 3. Sperm function test (Sperm-mucus Interaction Tests, Capacitation, Acrosome Reaction, Zona Binding assays). 4. Semen cryopreservation. 5. Sperm DNA fragmentation. 6. Intrauterine inseminations. 7. Sperm preparation for in vitro fertilization (IVF) procedures and intracytoplasmic sperm injection (ICSI). 8. provide counseling and options for cryopreservation before initiating chemotherapy, radiation, ICSI or surgery. 9. Testicular and epididymal sperm processing and cryopreservation.
  4. 4. What is semen analysis??!! • Is a test on the fluid that is released when a man has an orgasm. • Inflects the quality and quantity of spermatogenesis, spermiogensis, sperm transportation and maturation process. • is usually one of the first tests done to help determine whether a man has a problem fathering a child (infertility). • Modern approach is to interpret with regard to: • diagnosis of specific lesions; and • indicators of dysfunctional and/or functional potential. • Requires understanding of the relevance of sperm patho-physiology. • In any case, the results must be accurate and reliable.
  5. 5. Why perform semen analysis? • Diagnosis of sterility • Diagnosis of infertility (as part of a couple's infertility investigation) • Prognosis for fertility • Effectiveness of vasectomy • Identify treatment options: • surgical treatment • medical treatment • assisted conception treatment • Cryopersvation Therefore = a screening test to help direct management.
  6. 6. How is semen analysis done? • Analysis is done through two steps -Sample collection -Sample analysis According to World Health Organization: Department of Reproductive Health and Research WHO laboratory manual for the examination and processing of human semen. 5th edition. 2010. • International minimum standards are, by consensus, the World Health Organization’s Lab Manual. • Focus is on standardization with expanded section on quality control. • Basic infertility work-up.
  7. 7. 1. Sexual abstinence - 2- 7 days -No “ejaculation” not just “No intercourse” 2. Specimen collection (Ways to collect semen) - Masturbation (Optimal specimen for analysis) - Coitus interrupts (often lost a part) - Split ejaculate (2 containers) - Accepted lubricants (non sperm toxic) 3. Specimen containers -non sperm toxic ( containers, condoms) only provided by the laboratory. -Tested for every type or lot. 4. Specimen Transport - In the laboratory location (Ideal) - Off site ( within 1 hour), avoid excess heat or container damage, instruct in the semen report, patient assignment Semen collection
  8. 8. Semen collection Retrograde ejaculation • In some men, the semen passes back into the bladder at ejaculation - this is confirmed by examination of a sample of post-ejaculatory urine. • The man must take sodium bicarbonate the day before, and the day of, his appointment – to alkalinize his urine or FNA is recommended. • Before collection, he should pass urine and then wait until he feels there is some urine in his bladder before masturbating • Assess volume and pH of the urine. • Centrifuge, resuspend pellets Perform a standard semen analysis with this suspension
  9. 9. Semen sample analysis Microscopic AnalysisMacroscopic analysis Sperm countLiquefaction Sperm motilityViscosity Sperm kineticsVolume Sperm morphologyColor and Turbidity AggregationpH Agglutination Vitality Determination of Immature germ cells and Leukocytes RBCs
  10. 10. Macroscopic examination Liquefaction: • Liquefaction is the breakdown of the gel portion of the seminal plasma – the enzymes for this are in the prostatic fluid • Semen is normally ejaculated as a coagulum and liquefied within 20 minutes after ejaculation (37 °C). • Non liquefied semen is a fairly rare occurrence, may indicate prostatic dysfunction, and should be noted. • Aspirate the specimen into plastic pasture pipette and observe it If homogeneous and quite watery Liquefaction is complete If heterogeneous mixture Liquefaction is not complete • Note: Normal liquefied semen samples may contain jelly-like granules (gelatinous bodies) which do not liquefy. • If after 2 hours the specimen has not liquified proteolytic enzymes such as alpha-chymotrypsin may be added to allow the rest of the analysis to be performed.
  11. 11. Viscosity : Measured by drawing and releasing semen from a pipet and noting viscosity using a subjective scale. - If viscosity is high repeat gently passage of specimen into pipette several times. - If viscosity is very high add equal volume of sperm media following by repeating pipetting. Macroscopic examination Leaves the Pipette in small discrete drops.Normal Sample The drop will form a thread about 2 cm long.( + ) The drop will form a thread more than 2 cm long.( ++ ) No drops formed, shows a continuous filament.( +++ ) Not aspirated into the plastic pasture pipette.( + +++ )
  12. 12. Volume: • Measured using a graduated cylinder or centrifuge tube. • 1.5-5.5 mL (with no dilution). • Decreased volume (i.e., <1.5 mL; hypospermia) may indicate a collection error, reduced abstinence, or ejaculatory dysfunction. • Increased volume (>5.5 mL; hyperspermia) may be indicative of extensive abstinence. • Aspermia (no semen) may indicate a retrograde ejaculation and should be followed by a postejaculation urinalysis to check for the presence of sperm in the urine. Macroscopic examination
  13. 13. Color: -The presence of gross macroscopic particles or debris should be noted. pH: - Secretions from the prostate and seminal vesicles contribute to seminal pH. - Measured using litmus paper. - The pH of the semen is normally (7.2-7.8) - Abnormal pH may be indicative of secondary sex gland dysfunction). Macroscopic examination Grey-opalescentNormal Less OpaqueLow sperm Count Red-brown or pinkishRBCs present YellowDrugs or vitamins
  14. 14. Microscopic examination  In Case of Presence of Sperm: • Sample should be examined to determine the motility classification, aggregation, agglutination and the dilution required for accurate assessment of sperm concentration. Aggregation • adherence either of immotile spermatozoa to each other. • or of motile spermatozoa to mucus strands, non-sperm cells or debris is considered to be nonspecific aggregation. Agglutination • Motile spermatozoa are attached to each other Head-to-Head, Tail-to-Tail or in a mixed way. (Immunological cause of infertility) isolated <10 spermatozoa per agglutinate, many free spermatozoa Grade 1(+): moderate 10–50 spermatozoa per agglutinate, free spermatozoa Grade 2(++): large agglutinates of >50 spermatozoa, some spermatozoaGrade 3(+++):
  15. 15. Head-to-Head Tail-to-Tail Tail-tip-to-tail-tip Mixed Tangle
  16. 16. Microscopic examination Motility Different categories of sperm motility: •Progressive motility (PR): Spermatozoa moving actively, either linearly or in a large circle, regardless of speed. (Represents the quality of sperm). a. Rapid b. Slow •Non-progressive motility (NP): all other patterns of motility with an absence of progression, e.g: swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed. • Immotility (IM): no movement. If the motility is less than 50% then vital test must be done. If motility is Zero , then pentoxyphillene stimulation test is required
  17. 17. Microscopic examination Vitality - Stained (red=Dead) sperm and unstained (Vital) sperm. -Viable do not take up the stain - Eosin +/- Nigrosin Immature germ cells and Leukocytes - Peroxidase positive cells appear with yellow to brown stained cells while pink stained cells are other cells. - Formation of small air-bubbles is normal (The higher the concentration of peroxidase positive cells, the more air bubbles will form).
  18. 18. Microscopic examination RBCs (Red blood cells) - Should not be present. - Increased number may indicate a reproductive tract infection or damage to a small capillary during sample production. Concentration • Varity of counting chambers are available for determining sperm concentration. (Hemocytometer, Makler counting chamber, MicroCell). • Homogenous, mixed semen sample is used. • Glass coverslip used, allows the sperm to distribute evenly in a very thin layer. • The total sperm count for the ejaculate can be calculated by multiplying the sperm concentration by the specimen volume.
  19. 19. Microscopic examination  If No Sperm seen: • First scan the whole slide and record if there are any motile and immotile spermatozoa • If no sperm ,then add to semen specimen equal volume of Ham ̀s F10 media and centrifuge it at 1500 rpm for 10 minutes. • Concentrate the sample in 250 micron and mix it well then Scan 10 micron at x400 magnification ……. record presence of any sperm. • Second sample is useful. • If azospermia: fructose level must be ordered to verify the integrity of the vas and seminal vesicles
  20. 20. Microscopic examination Morphology • Because of the small size of the human sperm head, must use an air-dried smear which has been stained • Prepared samples are assessed using a 100× oil-immersion objective under bright field optics • The WHO recommends that 200 spermatozoa are counted per sample • Fields for counting must be selected at random Dark greenAcrosome= Stained Red=Nucleus pale greenEquatorial region = GreenMidepiece and tail=
  21. 21. Computer Aided Semen Analysis - CASA Advantages: 1. Increased objectivity and consistency of measurements. 2. Increased accuracy and precision of analysis. 3. Provides a description of vigor ( velocity and tail beat frequency) and pattern of motion (Linearity, and amplitude of lateral head displacement) Disadvantages: 1. Can overestimate or underestimate sperm count. 2. Sperm count should be between 20-50 million/mL for accurate analysis. 3. Requires extensive QC to demonstrate accuracy and precision.
  22. 22. Computer Aided Semen Analysis - CASA Count and motility estimation by CASA • High Resolution camera attached to Microscope takes video of semen sample for a prefixed time, till 200 sperms are imaged. • The video is then examined through software for sperms with respect to their number, their motion characters with in the frame. • User feeds the data on chamber depth, volume of sample loaded.
  23. 23. Count and motility estimation by CASA • The sperm tracks are analyzed and a number of kinematic parameters are derived, including: – Velocity (VCL, VSL and VAP) – Velocity ratios (expression of the path shape and regularity) – Amplitude of lateral head displacement – Beat/cross frequency • The proportion of sperm in a sample which meet particular kinematic criteria is used to predict (failure) of: – Mucus-penetrating ability – Hyperactivation (a marker of sperm function) • Able to assess the kinematics of hundreds of sperm in a couple of minutes Computer Aided Semen Analysis - CASA
  24. 24. Morphometry by CASA • Stained semen smear/ unstained sample using AAV Technology (Negative phase Contrast in 40x) is exposed to the M’scope and image is grabbed through camera • 100/ 200 sperm images are grabbed • Then the images are analyzed after super imposing with standard normal/ abnormal sperm templates by the software • Results are expressed as percent normal/ Abnormal. Computer Aided Semen Analysis - CASA 2-3 µM <1 µM 3-5 µM 1.5 X Head 45 µm
  25. 25. Reference ranges WHO 2010 Lower Reference limitParameter 1.5Sperm Volume (mL) 15Sperm concentration (106/mL) 39Total sperm number (106/ejaculate) 32Progressive motility (PR, %) 40Total motility (PR+NP, %) 58Vitality (Live sperm, %) 4Sperm morphology (NF, %) >/=7.2pH <1Leucocytes (106/mL) <50MAR/immunobead test (%) NoneRBCs
  26. 26. Semen nomenclature -Spermia Refers to Semen -Aspermia -Hyperspermia -Hypospermia -Leucocytospermia -Hematospermia -Pyospermia -Azoospermia -Cryptozoospermia - Oligozoospermia - Asthenozoospermia - Teratozoospermia -Normozoospermia -Globozoospermia - No Ejaculate Volume. -Increased Ejaculate Volume. -Decreased Ejaculate Volume. -Increased Pus cells in Ejaculate. -Presence of RBCs in semen. -Presence of WBCs in semen. -No Sperm present. -No Sperm in wet Prepration…. Found Sperm after Conc. -If Count <15 million/ml -If PR motile Sperm is <35% -If Abnormal Forms >4% -If Sample with Normal Parameters of Count, Motility and Normal forms. -Sperm with Rounded Head
  27. 27. Thank You!

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