Seminal Fluid Analysis: Composition, Objectives - PDF

OTHER BODY FLUIDS SEMINAL FLUID  OBJECTIVES At the end of this module, the students will be able to: Describe the function of reproductive structures for seminal fluid formation. Discuss the composition of seminal fluid. Describe the performance of seminal flui...

OTHER BODY FLUIDS SEMINAL FLUID  OBJECTIVES At the end of this module, the students will be able to: Describe the function of reproductive structures for seminal fluid formation. Discuss the composition of seminal fluid. Describe the performance of seminal fluid analysis from collection to physical, chemical and microscopic evaluation. Identify and describe morphologic appearance of normal and abnormal forms of spermatozoa. Discuss the origin and clinical significance of cells other than sperm in the seminal fluid.  CLINICAL SIGNIFICANCE Reasons for Seminal Fluid Analysis: Fertility testing Postvasectomy semen analysis Forensic analyses (medicolegal / alleged rape) Others: Selection of donors for therapeutic insemination, in-vitro fertilization or embryo transfer Exposure screening to reproductive toxicants Cryopreservation/sperm banking MALE REPRODUCTIVE SYSTEM MALE REPRODUCTIVE SYSTEM  DEFINITION OF TERMS Seminiferous tubules – site of spermatogenesis; spermatozoa production (regulated by the Sertoli cells) Sertoli cells- play a role in supplying nutrients, hormones, and other necessary substances for normal spermatogenesis; nurse cells for developing sperms Epididymis - storage and sperm maturation; where sperm gain motility until ejaculation. Interstitial cells of Leydig – produces and secretes testosterone Vas / Ductus deferens– propels sperm to the ejaculatory duct Seminal vesicle (60-70%) – provide nutrients (fructose) for sperm; alkaline fluid; fructose, prostaglandins & flavin Fructose – energy source for sperm Flavin - imparts the characteristic gray or opalescent to light yellow; green-white fluorescence under UV Prostaglandin- assumed to lower the female immune response to the semen Phosphorous and potassium - contributes to sperm motility Proteins (e.g., semenogelin) - play a role in coagulation of the ejaculate, forming a gel-like protective layer around the sperm  DEFINITION OF TERMS Prostate gland (20-30%) Prostatic fluid secretions – for coagulation and liquefaction -principal components of this milky, slightly acidic fluid are: a. Citric acid b. Enzymes (ACP and proteolytic enzymes) – ACP - can be used to identify semen Proteolytic enzyme - liquefaction c. Proteins - coagulation d. Zinc - used to evaluate prostate function and a decreased level is associated with prostate gland disorders. Bulbourethral gland (5%) – add alkaline mucus to neutralize prostatic acid and vaginal acidity  DEFINITION OF TERMS AZOOSPERMIA refers to the total absence of sperm cells that results from bilaterally small underdeveloped testes; ejaculatory obstruction from the previous traumatic or operative procedures and possibly secondary to gonorrhea OLIGOSPERMIA characterized by the presence of few motile cells and low sperm count resulting from a bilateral or unilateral hypotrophic testes and possibly secondary to hypothyroidism NECROSPERMIA condition in which there is a low percentage of live and a high percentage of immotile or dead spermatozoa in semen ASTHENOZOOSPERMIA condition characterized by inability of vital sperm cells to move (slow moving sperm cells or “lazy” sperm cells) FACTORS AFFECTING SEMEN QUALITY A. Sample collected First portions of semen: mainly prostatic secretions rich in sperm cells When a portion of the initial ejaculate is not collected: volume sperm concentration pH (alkaline) Semen coagulum will fail to liquefy Later fractions / last portion: dominated by seminal vesicular fluid When the last portion of an ejaculate is missing: semen volume sperm concentration pH Coagulum will not form Note: Losing the first portion of the ejaculate has more influence on the results of semen analysis than losing the last portion FACTORS AFFECTING SEMEN QUALITY B. Activity of the accessory sex glands volume of seminal fluid = enhances sperm motility = dilution may occur (underestimate sperm concentration) C.Time since the last sexual activity Abstinence Minimum: 2 days Under: without a minimum of two (2) days abstinence may affect sperm number and quality Maximum: 7 days Prolonged abstinence: volumes and motility ( flavin – yellowish semen color) Absence of ejaculation: spermatozoa accumulate in the epididymis then overflow into the urethra and have the tendency to be flushed out in the urine FACTORS AFFECTING SEMEN QUALITY D. Penultimate abstinence period A single ejaculation will not clear all sperm cells as the epididymis are not completely emptied by one ejaculation, some spermatozoa remain from the time of the previous ejaculation. this influences the range of age and quality of spermatozoa in the ejaculate E. Size of the testis influences the total number of spermatozoa per ejaculate reflects the level of sperm activity which also affects sperm morphology In a journal study of Takihara, it was found out that the size of the testis bears a direct correlation with testicular function F. Manner of collection Product of masturbation: Lower quality (collected into containers in a room near the laboratory) Product of sexual activity: High quality (collected from non-spermicidal condoms used during intercourse) due to sexual arousal  SP ECIMEN CO L L ECTIO N A. PREPARATION clear written and spoken instructions concerning the collection of the sample  emphasize that the semen sample needs to be complete  patient should report any loss of any fraction of the sample 1. Abstinence minimum of 2 days and a maximum of 7 days of sexual abstinence 2. Collection site 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 3. Report Form name of the patient date of birth period of abstinence date and time of collection completeness of the sample any difficulties in producing the sample time interval between collection and the start of semen analysis (TF; TR) SP ECIMEN CO L L ECTIO N  SP ECIMEN CO L L ECTIO N B. COLLECTION PROPER Complete collection EMPTY BLADDER before ejaculation (urine is toxic to sperm) Fertility test: 2-3 samples within a 3-month period and at least 7 days apart should be examined 1. Container clean wide mouthed container made of glass or plastic that has been confirmed to be non-toxic for spermatozoa pre-weighed container labeled with his name and identification number 2.Transport & Preservation transported to the laboratory within one hour of collection ( ASAP / 30 MINS – 1 HR) kept at ambient temperature between 20 and 37 degrees Celsius or close to body temperature during transport or while waiting analysis Under armpit, between thighs or in the breasts inside the bra, pocket Artificial insemination: preserved in frozen state and stored for one year at -85 degrees Celsius (seminal banks)  SP ECIMEN CO L L ECTIO N B. COLLECTION PROPER 3. Manner of collection Masturbation – best method Condom – nonlubricated rubber or polyurethane condom should be used Ordinary latex condoms: not acceptable because they have spermicidal content (interfere with the motility of spermatozoa). Coitus interruptus – withdrawal method not a reliable means of semen collection because the first portion of the ejaculate, which contains the highest number of spermatozoa, may be lost and the low pH of the vaginal fluid may affect sperm motility Vaginal vault aspiration aspiration of seminal fluid from the vaginal vault after coitus  SP ECIMEN CO L L ECTIO N C. SAFE HANDLING OF SPECIMENS Semen samples may contain infectious agents such as: HIV HBV Herpes simplex virus Handled as biohazard (specimen are discarded as biohazardous waste) Sterile materials and techniques must be used if the sample is to be processed for bioassay, intra-uterine insemination, in-vitro fertilization or intracytoplasmic sperm injection  P HYSICAL EXAMINATIO N Semen analysis should begin within 30 to 60 minutes right after liquefaction A. Liquefaction Immediately after ejaculation, semen is typically a semi-solid coagulated mass. Within a few minutes at room temperature, the semen usually begins to liquefy (become thinner), at which time a heterogeneous mixture of lumps will be seen in the fluid. A normal liquefied semen samples may contain jellylike granules which do not liquefy. The presence of mucus strands, however, may interfere with semen analysis.. Failure of liquefaction to occur may be caused by a deficiency in prostatic enzymes and should be reported. In these cases, additional treatment, mechanical mixing or enzymatic digestion may be necessary: Addition of an equal volume of Dulbecco’s phosphate -buffered saline, followed by repeated pipetting. Inhomogeneity can be reduced by repeated (6-10 times) gentle passage through a blunt gauge 18 or gauge 19 needle attached to a syringe. Digestion by bromelain, a proteolytic enzyme, may help to promote liquefaction P HYSICAL EXAMINATIO N  P HYSICAL EXAMINATIO N B.Viscosity Normal = small discrete drops Abnormal = thread >2 cm long viscosity = can interfere with sperm motility, sperm concentration, detection of antibody-coated spermatozoa and measurement of biochemical markers Reporting: 0 = watery 4 = gel-like Alternate reporting: normal, slightly viscous (thick), moderately viscous, and extremely viscous (unable to be aspirated into the pipette)  P HYSICAL EXAMINATIO N C. Color and Appearance Normal = homogeneous, grey-opalescent appearance; translucent Abnormal Turbid = WBCs (infection) Rusty red or red-brown = RBCs (indicates hemospermia) or due to drug pyridium Yellowish = flavin due to prolonged abstinence; contamination with first morning urine, jaundice (e.g., in cases of Hepatitis), multivitamins Clear = infertility  P HYSICAL EXAMINATIO N D. Volume Normal = 1.5 – 5 mL Low (hypospermia) = infertility, obstruction of the ejaculatory duct, congenital bilateral absence of the vas deferens, poorly developed seminal vesicles, loss during collection, partial retrograded ejaculation or androgen deficiency (hypogonadism) High (hyperspermia) = active inflammation of the accessory glands, prolonged abstinence Measured by: = weighing the sample in the pre-weighed container = collecting the sample into a clean graduated centrifuge tube/measuring cylinder P HYSICAL EXAMINATIO N  P HYSICAL EXAMINATIO N E. Odor Normal = distinct musty, bleach-like odor Abnormal = infections CHEMICAL EXAMINATIO N A. pH Normal = 7.2 – 8.0 pH = infections in reproductive tract (bacteria=alkaline) pH = ejaculatory duct obstruction or congenital bilateral absence of the vas deferens or an increase prostatic secretion  MICRO SCO P IC EXAMINATIO N phase contrast microscope is recommended for all examinations of unstained preparations of fresh semen at a total magnification of x400 (at x1000 if stained preparation) Microscopic examination of semen involves: Sperm motility Vitality Concentration Morphology  MICRO SCO P IC EXAMINATIO N A. INITIAL MICROSCOPIC EXAMINATION Examined at a total magnification of x100; LPO provide an overview of the sample, especially the number of spermatozoa assess the presence of mucus strands, sperm agglutination or aggregation determine cells other than spermatozoa such as epithelial cells and round cells (leukocytes and immature germ cells) allow the microscopist to determine the appropriate dilution and chambers to use when performing sperm number determination  MICRO SCO P IC EXAMINATIO N A. INITIAL MICROSCOPIC EXAMINATION 1. Making A Wet Preparation Mix the semen sample well. This will allow uniform distribution of the semen components especially the spermatozoa Do not mix vigorously (and in a vortex mixer at high speed) – this will create bubbles and damage the spermatozoa. Mix by aspirating the sample 10 times using a disposable plastic pipette. Remove an aliquot of semen (10 ul) immediately after mixing. Transfer the aliquot onto a clean slide and cover it with a coverslip (22 mm x 22 mm). The weight of the coverslip will spread the sample. A fixed depth of about 20 um allows the spermatozoa to swim freely. 50% of the sperm should be motile in categories a, b, and c (within 1 hour) 25% or more should show progressive motility (a and b)  MICRO SCO P IC EXAMINATIO N B. Sperm Motility  MICRO SCO P IC EXAMINATIO N B. Sperm Motility  MICRO SCO P IC EXAMINATIO N B. Sperm Motility INTERPRETATION  MICRO SCO P IC EXAMINATIO N C. Sperm Concentration  MICRO SCO P IC EXAMINATIO N C. Sperm Concentration  MICRO SCO P IC EXAMINATIO N C. Sperm Concentration  MICRO SCO P IC EXAMINATIO N C. Sperm Concentration  MICRO SCO P IC EXAMINATIO N C. Sperm Concentration  MICRO SCO P IC EXAMINATIO N C. Sperm Number SPERM NUMBER / SPERM COUNT = SPERM CONCENTRATION X SEMEN VOLUME  MICRO SCO P IC EXAMINATIO N D. Sperm Morphology  MICRO SCO P IC EXAMINATIO N D. Sperm Morphology