Urinalysis and other Body Fluids - Semen Analysis

ANALYSIS OF URINE AND OTHER BODY FLUIDS • SEMEN ANALYSIS OUTLINE • • • • Introduction Semen o Physiology o Specimen Handling o Semen Analysis o Additional Testing • • • INTRODUCTION • • • • • • • Semen is a complex body fluid composed mainly of sperm cells and fluids contributed by accessory male reproductive organs - the prostate and seminal vesicles. It is routinely analyzed to determine male infertility and also to evaluate the effectiveness of vasectomy. Other tests are also available for other reasons for performing semen analysis such as for forensic investigations. • • • SEMEN SEMEN ANALYSIS PHYSIOLOGY Normal Values for Semen Analysis Volume 2-5 ml Viscosity Pours in droplets pH 7.2-8.0 Sperm concentration 20-160 millions/ml Sperm count >40 million/ejaculate Motility >50% within 1 hr Quality >2.0 Morphology >30% normal forms(strict criteria) >50% normal forms(routine criteria) White blood cells <1.0 million/ml Four Fraction of Semen: o Testes and Epididymis o Seminal vessels o Prostate o Bulbourethral Glands The mixing of all four fraction during ejaculation is essential for the production of a normal semen specimen. Spermatozoa (5%) o Produced in the seminiferous tubules of the testes. o They mature and are stored in the epididymis. o Spermatozoa and fluid from the epididymis contribute about 5% of the semen volume. Seminal Vessel (60-70%) o Produce the majority of the fluid (60%) present in the semen. o The fluid contains high content of fructose that the spermatozoa readily metabolize. o Spermatozoa do not become motile until they are exposed to the fluid from the seminal vessel Prostate Gland (20-30%) o 20 to 30% of the semen volume is acidic fluid. o The fluid contains high concentrations of acid phosphatase, citric acid, zinc and proteolytic enzymes responsible for both the coagulation and liquefaction of the semen following ejaculation Bulbourethral Glands (5%) o Contribute about 5% of fluid volume in the form of a thick, alkaline mucus that helps to neutralize acidity from the prostate secretions and the vaginal acidity. SPECIMEN HANDLING • • When performing fertility testing, 2 or 3 samples are usually tested at 2-week intervals with 2 abnormal samples considered significant The laboratory should provide warm sterile glass or plastic containers. Specimens should be kept at room temp. and deliver to the laboratory within 1 hour of collection A fresh specimen is clotted and should be liquefy within 30-60min after collection Specimen awaiting analysis should be kept at 37°C Specimen should be collected by masturbation. If this is not possible, only nonlubricant-containing polymeric silicone (Silastic) condoms should be used. Ordinary condoms are not acceptable because they contain spermicides All semen specimens are potential reservoirs of human immunodeficiency and hepatitis viruses and standard precautions must be observed at all times during the analysis. Specimen are discarded as biohazardous waste. Majority of the sperm are contained in the first portion of the ejaculate. Specimens are collected following a period of sexual abstinence of at least 3 days and not longer than 5 days Specimens collected following prolonged abstinence tend to have higher volumes and decreased motility. APPEARANCE • • • • • • • Normal semen has a gray-white color, appears translucent, and has a characteristic of musty odor. Increased white turbidity indicates the presence of WBC and infection within the reproductive tract. WBCs must be differentiated from immature sperm(spermatids) Leukocyte esterase reagent strip test may be useful to screen the presence of WBCs Varying degrees of red coloration are associated with the presence of RBC and are abnormal. Yellow coloration may be caused by urine contamination, specimen collection following prolonged abstinence, and medications. Urine is toxic to the sperm, thereby affecting the evaluation of motility. VOLUME • • • Semen can be measured by pouring the specimen into a clean graduated cylinder calibrated in 0.1-ml increments. Increased volume may be seen following periods of extended abstinence. Decrease volume is associated with infertility and may indicate improper functioning of one of the semenproducing organs. VISCOSITY • • • • Incompletely liquefied specimens will be clumped and highly viscous. Normal semen specimen should be easily drawn into a pipette and form droplets that do not appear clumped or stringy when discharged from the pipette. Ratings of 0 (watery) to 4(gel like) can be assigned. Increased viscosity and incomplete liquefaction will impede sperm motility • • pH • • • • Increased pH is indicative of infection within the reproductive tract. Decreased pH is associated with increased prostatic fluid. Semen for pH testing can be applied to the pH pad of a urinalysis reagent strip and the color compared to the manufacturer’s chart. Dedicated pH testing paper also can be used. SPERM CONCENTRATION COUNT • • • • • • • • • • • • Sperm concentration is usually performed using the Newbauer counting chamber. The sperm are counted in the same manner as cells in the CSF cell count, that is, by diluting the specimen and counting the cells in the Newbauer chamber. The most commonly used dilution is 1:20 prepared using a mechanical rather than a Thoma pipette. Dilution of the semen is essential because it immobilizes the sperm before counting. The traditional diluting fluid contains sodium bicarbonate and formalin, which immobilize and preserve the cells; however good results can also be achieved using tap water. The Makler counting chamber provides a method for counting undiluted specimen using a counting chamber with 1mm² grid divided into 100 squares engraved in the cover plate Sperm are immobilized by heating part of the specimen prior to charging the chamber. Sperm motility using the unheated portion of the specimen can also be evaluated in the chamber Using the Newbauer hemocytometry, sperms are usually counted in the four corner and center squares of the large center square. Both sides of the hemocytometer are loaded and counted and the counts should agree within 10%. Only fully developed sperms should be counted. Immature sperms and WBCs often referred to as ‘round cells’ must not be included. The presence of round cells may be significant and they may need to be identified and counted SPERM MOTILITY • • The presence of sperm capable of forward, progressive movement is critical for fertility, because once presented to the cervix, the sperm must propel themselves through the cervical mucosa to the uterus, fallopian tubes and ovum. Assessment of sperm motility should be performed in well mixed, liquefied specimen within 1hr of semen collection. Sperm Motility Grading Grade WHO Criteria Sperm Motility Action 4.0 a Rapid, straight-line motility Slower speed, some lateral 3.0 b movement Slow forward progression, 2.0 b noticeable lateral movement 1.0 c No forward progression 0 d No movement Alternative Sperm Motility Grading Progressive Sperm moving linearly or motility (PM) in a large circle Nonprogressive Sperm moving with an motility (NP) absence of progression Immotility (IM) No movement SPERM MORPHOLOGY • • • • • • • • • • • It is evaluated with respect to both head & tail appearance. Abnormalities in head morphology are associated with poor ovum penetration, whereas tail abnormalities affect motility. A normal sperm has an oval-shaped head approximately 5µm long and 3µm wide and a long, flagellar tail approximately 45µm long. The head and the tail are connected by the neck and the middle piece, which contains mitochondria that provide energy for flagellar tail motion. Critical to ovum penetration is the enzyme containing acrosomal cap located at the tip of the head Normal Spermatozoa Structure Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion. Staining can be performed using Wright’s, Giemsa, or Papanicolaou Stain. Air-dried slides are stable for 24 hr. At least 200 sperm should be evaluated and the percentage of abnormal sperm reported. Abnormalities of Sperm Heads and Tails ADDITIONAL TESTING Abnormal Result SPERM VIABILITY • • • • • Decreased sperm viability may be suspected when a specimen has a normal sperm concentration with markedly decreased motility. Viability is evaluated by mixing the specimen with eosinnigrosin stain, preparing a smear, and counting the no. of dead cells in 100 sperm. Living cells-bluish-white color Dead cells- red against the purple background. Normal viability- 75% living cells SEMINAL FLUID FRUCTOSE • • • Decreased motility with clumping Normal analysis with continued infertility • • ANTISPERM ANTIBODIES • • • • • • • • • • • It can be present in both men and women. May be detected in semen, cervical mucosa, or serum and are considered as possible cause of infertility. Male anti-sperm antibodies are more frequently encountered. When the blood-testes-barrier is disrupted, as can occur following surgery, vasectomy reversal, trauma and infection, the antigens of the sperm produce an immune response that damages the sperm. (+)Ab in male partner- clumps of sperm are observed during routine semen analysis. (+)Ab in female partner- normal semen analysis accompanied by continued infertility. It may be demonstrated by mixing the semen with the female cervical mucosa or serum and observing for agglutination. Two Frequently Used Tests: MAR & Immunobead test MAR test o Is a screening procedure used primarily to detect the presence of IgG antibodies. o (+)microscopically visible clumps of sperm and particles or cells. o Less than 10% of the motile sperm attached to the particles is considered normal. Immunobead test o More specific procedure. o Can be used to detect the presence of IgG, IgM and IgA and will demonstrate what area of the sperm is affected. o Presence of beads on less than 20% of the sperm is normal. ADDITIONAL TASTING FOR ABNORMAL SEMEN ANALYSIS Abnormal Result Decreased Motility with normal count Decreased count Possible Abnormality Viability Lack of seminal vesicle support medium Test • Eosin-nigrosin stain • Fructose level Test • MAR & Immunobead test • Sperm agglutination with male serum Female anti-sperm • Immunobead test antibodies • Sperm agglutination with female serum MICROBIAL AND CHEMICAL TESTING • Specimens can be screened for the presence of fructose using Resorcinol test. A normal quantitative level of fructose is equal to or greater than 13µmol/ejaculate. Specimens for fructose levels should be tested within 2 hours or frozen to prevent fructolysis Possible Abnormality Male anti-sperm antibodies • • Presence of more than 1 million leukocytes/mm indicates infection within the reproductive frequently the prostate. Routine aerobic and anaerobic cultures and tests for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum are most frequently performed. Additional chemical testing preformed on semen may include determination of levels of neutral aglucosidase, zinc, citric acid, and acid phosphate Decreased fructose level are associated with lack of seminal fluid. Decreased neutral a-glucosidase suggest a disorder of epididymis. Decreased zinc, citrate, and acid phosphatase indicate a lack of prostatic fluid. Normal Semen Chemical Values Neutral a-glucosidase ≥20mU/ejaculate Zinc ≥2.4µmol/ejaculate Citric acid ≥52µmol/ejaculate Acid Phosphatase ≥200µ/ejaculate POSTVASECTOMY SEMEN ANALYSIS • • • • Only concerned in the presence or absence of spermatozoa Specimens are routinely tested at months interval, beginning at 2 months postvasectomy and continuing until 2 consecutive monthly specimens shows no spermatozoa. Examination of wet preparation using phase microscopy for the presence of motile and non-motile sperm A negative wet preparation is followed by centrifugation of the specimen for 10 mins and examination of the sediment SPERM FUNCTION TEST Test Hamster egg penetration Cervical mucus penetration Hypo-osmotic swelling In vitro acrosome reaction Description Sperms are incubated with speciesnonspecific hamster eggs and penetration is observed microscopically Observation of sperm penetration ability of partner’s mid cycle cervical mucus Sperm exposed to low sodium concentrations are evaluated for membrane integrity and sperm viability Evaluation of the acrosome to produce enzymes essential for ovum penetration.