Semen Analysis PDF

Semen Analysis MT104 Analysis of Urine and Body Fluids Lawrence R. Santiago, RMT Introduction Advances in the field of andrology and assisted reproductive technology (ART), and increased concern over fertility, particularly by couples choosing to have children later i...

Semen Analysis MT104 Analysis of Urine and Body Fluids Lawrence R. Santiago, RMT Introduction Advances in the field of andrology and assisted reproductive technology (ART), and increased concern over fertility, particularly by couples choosing to have children later in life, have resulted in increased emphasis on semen analysis. Patients with abnormal results on the routine semen analysis performed in the clinical laboratory often are referred to specialized andrology laboratories for further testing to determine the need for in vitro fertilization (IVF) physiology: testes Epididymis Seminal vesicle Prostate gland bulbourethral glands testes Paired glands in the scrotum – for the secretion of sperm the external location of the scrotum contributes to a lower scrotum temperature that is optimal for sperm development. Semen Production Starts in the epithelial cells of the seminiferous tubules. Specialized Sertoli cells provide support and nutrients for the germ cells as they undergo mitosis and meiosis (spermatogenesis) When the spermatogenesis is complete the immature (non-motile) enter the epididymis. In the epididymis the sperm mature and develop flagella The sperm remain stored in the epididymis until ejaculation. Propelled through the vas deferens to the ejaculatory  The ejaculatory ducts receive both sperm from the vas deferens and fluid from the seminal vesicles (which produces most of the fluid present in semen 60% to 70%) Seminal fluid contains high concentration of fructose and flavin used as a medium for transport Spermatozoa utilizes fructose as energy needed for the flagella to propel them through the female reproductive organ Prostate gland aids in propelling the sperm through the urethra Bulbourethral glands – 5% volume contribution, thick alkaline mucus aids in the neutralization of vaginal wall acidity  In the absence of fructose, sperm do not display motility in the semen analysis. Flavin is responsible for the gray appearance of semen The muscular prostate gland, located just below the bladder, surrounds the upper urethra and aids in propelling the sperm through the urethra by contractions during ejaculation. Approx 20% to 30% of the semen volume is acidic fluid produced by the prostate gland. The milky acidic 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.  The bulbourethral glands, located below the prostate, contribute about 5% of the fluid volume in the form of a thick, alkaline mucus that helps to neutralize acidity from the prostate secretions and the vagina. It is important for semen to be alkaline to neutralize the vaginal acidity present as a result of normal bacterial vaginal flora. Without this neutralization, sperm motility would be diminished. Specimen collection most of the sperm are contained in the first ejaculate Complete collection is essential uncollected first ejaculate results to falsely decreased sperm count, pH falsely increased and the specimen will not liquefy Uncollected last portion results to falsely increased sperm count, pH falsely decreased and the specimen will not clot. Abstinence of 2 days and not longer than 7 days Prolonged abstinence results to higher volumes and decreased motility.  2 or 3 samples are recommended to be collected not less than 7 days or more than 3 weeks apart. Warm sterile glass or plastic containers. Collected in a room provided by the laboratory through masturbation If not possible, use non-lubricated condoms (rubber or polyurethane) Delivered to the lab within 1 hour Patient’s name and birth date must be recorded, including the period of sexual abstinence completeness of the sample Specimen handling Standard precautions must be observed as all semen specimens are potential reservoirs for HIV Semen Analysis Parameters reported 1. Appearance 2. Volume 3. Viscosity 4. pH 5. Sperm concentration and count, motility and morphology appearance Normal – gray white color, translucent and has a characteristic musty odor Abnormal – appears almost clear due to low sperm concentration - increased white turbidity indicates presence of WBCs and infection within the reproductive tract Microscopic examination WBCs must be differentiated from immature sperm (spermatids) Leukocyte esterase can be useful to detect presence of WBCs Red discolorations are indications of RBCs present and considered abnormal Yellow discoloration may be caused by urine. Urine is toxic to sperm. liquefaction Fresh specimen is clotted and liquefies within 30 to 60 minutes after collection. Failure to liquefy within 60 minutes may be caused by prostatic enzymes deficiency – should be reported Examination of specimen should be done after liquefaction If no liquefaction occurs after 2 hours, addition of alpha- chymotrypsin or bromelain may be added to induce liquefaction volume Normal volume – 2mL to 5mL Decreased volume is frequently associated with infertility Viscosity Droplets that form threads longer than 2 cm are considered highly viscous and are reported as abnormal Ratings 0 – watery 4 – gel like pH Prostatic fluid – acidic Seminal vesicle – alkaline measured within 1 hour Normal range 7.2 to 8.0 Sperm Concentration and Sperm Count Normal range – 20 million per mL to 250 million per mL Borderline – 10 to 20 million per mL Neubauer chamber is used With dilution ratio of 1:20 Dilution is essential for immobilization of sperm Diluent – sodium bicarbonate or saline or distilled water Sperm count Counted in four corner and center squares of the large center square similar to a manual RBC count Both sides of hemocytometer are loaded and allowed to settle for 3 to 5 minutes then they are counted. Fully developed sperm should be counted, immature sperm and WBCs are not included. (significant, excluded count) example Dilution 1:20 60 sperm counted x 1,000,000 = 60,000,000 sperm/mL 60,000,000 sperm/mL x 4mL = 240,000,000 sperm/ejaculate Motility Presence of sperm capable of forward progressive movement is critical for fertility 10 uL semen (undiluted) under a 22x22 cover slip allow to settle for 1 minute. 20 high power fields are evaluated with 0 to 4 grade. 0 = no movement. 4 = rapid straight line movement Minimum 50% motility with a rating of 2.0 after 1 hr is considered normal Sperm Motility Grading 4.0 rapid straight line motility 3.0 slower speed, some lateral movement 2.0 slow forward progression, noticeable lateral movement 1.0 no forward progression 0 no movement Example field 1 = 4.0 fied 2 = 3.0 field 3 = 2.0 field 4 = 1.0 etc... >2.0 = motility 66% Sperm morphology Oval head shape 5um long and 3um wide and long Flagellar tail 45um long Acrosomal cap vital for ovum penetration located at the tip of the head of sperm Acrosomal cap is half of the head and cover approx 2/3 of the sperm nucleus Midpiece 7.0um long – thickest part of the tail – produces energy for tail motility  Seminiferous tubules – spermatogenesis Epididymis – sperm maturation Vas deferens – propel sperm to the ejaculatory ducts Seminal vesicles – provide nutrients for sperm and fluid Prostate gland – provide enzymes and proteins for coagulation and liquefaction Bulbourethral glands – add alkaline mucus to neutralize prostatic acid and vaginal acidity Sperm Morphology Head – oval shaped approx. 5um long and 5um wide and long Acrosomal cap – vital for ovum penetration Tail – or flagellum approx. 45um long for sperm motility Midpiece – approx. 7.0um long thickest part, covered with mitochondrial sheath that produces energy required by the tail for motility Nucleus contains the genetic material Additional Testing for Abnormal Semen Analysis Vitality: Eosin-Negrosin stain Vitality is evaluated by mixing the specimen with an eosin- nigrosin stain, preparing a smear, and counting the number of dead cells in 100 sperm using a brightfield or phase- contrast microscope. Living cells are not infiltrated by the dye and remain bluish white, whereas dead cells stain red against the purple background. Normal vitality requires 50% or more living cells and should correspond to the previously evaluated motility. The presence of a large proportion of vital but immobile cells may indicate a defective flagellum, whereas a high number of immotile and nonviable cells may indicate epididymal pathology. Seminal Fructose Low sperm concentration may be caused by lack of the support medium produced in the seminal vesicles, which can be indicated by a low to absent fructose level in the semen. low levels are caused by abnormalities of the seminal vesicles, bilateral congenital absence of the vas deferens, obstruction of the ejaculatory duct, partial retrograde ejaculation, and androgen deficiency. Specimens can be screened for the presence of fructose using the resorcinol test that produces an orange color when fructose is present Continued A normal quantitative level of fructose is equal to or greater than 13 µmol per ejaculate. can be determined using spectrophotometric methods. Specimens for fructose levels should be tested within 2 hours of collection or frozen to prevent fructolysis Antisperm antibodies Detected in semen, cervical mucosa or serum Blood-testes barrier separates sperm from the male immune system Barrier disruption due to vasectomy reversal, trauma and infection may lead to the formation of antibodies against sperm antigens, damaging the sperm Damaged sperm may lead to the production of antisperm antibodies in the female partner. detection – male subject Can be suspected when clumps of sperm are observed during semen analysis. Graded as “few” “moderate” or “many” on microscopic examination Detection – female subject Results in normal semen analysis accompanied by continued infertility Presence of antisperm antibodies in females may be demonstrated by mixing semen with female cervical mucosa or serum and observing for agglutination. Mixed Agglutination Reaction (MAR) Test Detects the presence of IgG antibodies Semen containing motile sperm is incubated with IgG AHG and a suspension of latex particles (or treated with RBCs covered with IgG) AHG binds simultaneously on the latex particles or RBCs forming microscopically visible clumps of sperm and particles or cells. >10% of sperm attached to the particles is considered normal Immunobead test More specific – detects IgG, IgM and IgA Demonstrates what area of the sperm the antibodies are affecting Head directed antibodies interfere with penetration Tail directed antibodies affect movement through cervical mucosa  Sperm + polyacrylamide beads (coated with anti-IgG / anti- IgM / anti-IgA) Beads attach to sperm at particular areas Depending on the beads used test could be reported as “IgM tail antibodies” or “IgG head antibodies” Presence of beads on sperm at less than 50% is considered normal Microbial testing >1 million WBCs per milliliter indicates infection within the reproductive system. (frequently prostate) Routine culture studies look for the presence of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum. Rape cases Specimen enhancement with xylene and examining under phase microscopy. Motile sperm can be detected up to 24 hours after intercourse. Non motile sperm can persist for 3 days Heads remain and may be present for 3 days after intercourse. Seminal fluid contains high concentrations of prostatic acid phosphatase. Detection of this enzyme can aid in determining the presence of semen in the sample. Seminal glycoprotein p30 (prostatic specific antigen) – specific, present even in the absence of sperm. Postvasectomy Semen Analysis The only concern is the presence or absence of spermatozoa Determines if complete sterilization has taken place Care should be taken not to overlook even a single sperm Routinely tested at monthly intervals beginning at 2 months post vasectomy and continuing until two consecutive monthly specimens show no protozoa Examination of wet preparation… if negative, followed by specimen centrifugation for 10 minutes and examining the sediment.