SEMINAL FLUID EXAMINATION

Darshan Trivedi Msc MLT (PREVIOUS)

CONTENT
1. Introduction 2. Specimen collection 3. Storage & Transport 4. Important Terminologies 5. Semen Examination (a)Physical examination (b)Chemical examination (c)Microscopic examination (d)Microbiological examination (e)Immunological examination 6. Medico Legal Aspects 7. Model report

INTRODUCTION
DEFINATION:The seminal fluid is a composite solution containing basically of spermatozoa , suspended in seminal plasma made by seminal vesicle and accessory sex glands .

WHY IT IS DONE?
Semen examination is an integral part of evaluation of INFERTILITY. As a result of it s relative simplicity, semen examination is often requested before the more complicated and expensive examination of female. Repeat examination should be done if once it is found to be abnormal. Follow up of patients who have undergone vasectomy. Artificial insemination, invitro fertilization, and for intrauterine insemination. For detection of future infertility due to radiotherapy and chemotherapy for cancer treatment . For congenital abnormalities. Finally for MEDICOLEGAL purposes purposes.

SPECIMEN COLLECTION
The specimen collection should be collected after a period of 3 days ABSTINENCE from coitus. Bladder should be evacuated prior to semen collection. Ask the patient not to drink alcohol prior to test for a few days. Preferably should be collected in a clinical pathology laboratory. The best sample is the 1 collected in morning. Best method of collection is MASTURBATION. The sample collected by masturbation is complete and uncontaminated.

Producing a semen sample does not cause any discomfort. However the patient may feel embarrassed about the method, so the alternate method for collection is COITUS INTERREPTUS for subject who cannot perform masturbation. If performed in laboratory the room should be quiet, reduced and one which will guarantee total privacy. Should not be collected using CONDOMS. If collected outside the laboratory the sample should be delivered to lab within 2 hrs.

STORAGE AND TRANSPORT

Semen examination should be performed immediately but if not possible store at room temperature, DO NOT REFRIGERATE . If collected outside the lab the sample should not be transported in extremes of temperature (less than 20 c and more than 40 c). Rather it should be transported near body temperature and it should never be exposed to direct sunlight for any length of time.

EQUIPMENT FOR COLLECTION:COLLECTION:Wide mouth, clean dry bottle (50ml). The container should be free of detergents.

TERMINOLOGIES IMPORTANT TO REMEMBER

ASPERMIA: ASPERMIA:- It is total absence of ejaculate. AZOOSPERMIA: AZOOSPERMIA:- Absence of spermatozoa in semen. OLIGOZOOSPERMIA: OLIGOZOOSPERMIA:- It is reduction in volume of ejaculate and total sperm count less than 20 million cells/ml. ASTHENOZOOSPERMIA: ASTHENOZOOSPERMIA:- Characterizes by spermatozoa less than 50% actively motile.(category {a} and {b} ). TERATOZOOSPERMIA: TERATOZOOSPERMIA:- 50% spermatozoa with normal morphology. HYPERSPERMIA: HYPERSPERMIA:- Increase in semen volume and count more than 300 million/ml. NECROSPERMIA: NECROSPERMIA:- Normal count of sperm but non motile spermatozoa. HAEMOSPERMIA: HAEMOSPERMIA:- Plenty of RBC and pus cells in semen.

EXAMINATION
PHYSICAL EXAMINATION:EXAMINATION:1.VOLUME:1.VOLUME:Generally 2-6 ml of semen is obtained after 2-5 days of abstinence. 22It is measured by using a 10 ml graduated tube or a 10 ml measuring cylinder. Less volume may be due to non-abstinence. non2. APPEARANCE:APPEARANCE:Normal semen is a thick, mucoid, viscous, opaque fluid which is generally white or whitish grey in color. Color may change due to:to:(a)Presence of urine:urine:May be present in man with disturbances of bladder neck function and ejaculation. A high urea content may confirm presence of urine.

(b)Presence of blood:- Traces of fresh blood imparts pink blood:color to semen. Lager amounts imparts brighter color to semen. (c)Presence of bilirubin:- Presence of bilirubin may impart bilirubin:yellow color to semen just as it dose to other body fluids. 3. LIQUIFICATION TEST:TEST:Normal :- 20-30 min. 20Generally the semen coagulates after coming out and liquefy due to activity of enzymes secreted from prostate gland on fibrinogen like precursor secreted by seminal vesicle. It is assessed visually.

4. VISCOSITY:- Normally semen is viscous in nature. VISCOSITY:It is measures using a pasture pipette. If drop by drop falls viscosity is normal 5.ODOUR:5.ODOUR:- Normal semen has fishy pungent sweet odour. It may change due to bacterial growth. 6. AUTOAGGLUTINATION:AUTOAGGLUTINATION:Agglutination of spermatozoa is noted when motile sperm stick to each other in various orientation such as head to head , tail to tail etc. This depend on specificity of sperm antibodies directed against this sperm structures. If fibrinogen like precursor is high, auto agglutination is suggestive of an immunological cause of infertility.

CHEMICAL EXAMINATION:1. PH:- NORMAL:- 7.9 - 8.1 PH:- NORMAL:Detected by PH paper strip of 6-9 range. 6Becomes slightly alkaline on standing at R.T. May be slightly acidic in APLASIA of vasa defrentia and seminal vesicle.

2.FRUCTOSE:2.FRUCTOSE:- Main carbohydrate supplying energy for the movement of sperm Present in high concentration in seminal plasma secreted by seminal vesicle. Normal:-150Normal:-150-300mg/dl Decreased condition:- 1>T.B of seminal vesicle. condition:2>Androgen deficiency. 3>Testosterone therapy. 4>Obstruction of ejaculatory duct.

PROCEDURE:PROCEDURE:-

5ml resorcinol reagent + 0.5 ml semen HEAT Orange red ppts

RESORCINOL REAGENT:- resorcinol :-50 mg REAGENT::con.HCl ::- 33 ml D/W ::- 100 ml 3. ACID PHOSPHATASE:- Secreted by prostate gland. PHOSPHATASE:Mostly used by forensic laboratory as a test for the presence of semen. 4. CITRIC ACID:- Secreted by prostate gland. ACID:Is a marker for prostatic function.

5.ZINC:5.ZINC:- Secreted by prostate gland and stabilized seminal protein. It can also be used as a prostatic marker. 6.PROSTAGLANDIN F2:-Stimulate Contraction of urethral smooth muscle F2:and aid sperm transport in female. 7.LDH L4:- This enzyme is specific to sperm. It may be released into semen L4:when sperm die and disintegrate. 8.TRANSFERRIN:8.TRANSFERRIN:- It is produced and released into the testicular component of SERTOLI CELLS. It may be useful marker for sertoli cell only syndrome. 9.INHIBIN:9.INHIBIN:- It s a group of substance produced by sertoli cells. The main action of INHIBIN is to feedback on to the pituitary and suppress FSH(Follicle stimulating hormone) secretion . Its determination may be used as sertoli cells marker.

MICROSCOPIC EXAMINATION:EXAMINATION:1. MOTILITY:- Normally actively motile. 60% or more motile in normal sample. METHOD:Place a drop of liquefied semen on glass slide and cover with cover slip. Examine in low power & high dry power objective using low light by adjusting diaphragm. Grading For Motility Observation:Grade 0:- Non-motile Grade 1:- sluggish forward progression Grade 2:- Defining forward Grade 3:- Good forward movement Grade 4:- Vigorous and rapid forward progression Check motility after 2,4,6 hrs interval.

2. Sperm count:count:Normal:-40Normal:-40-300 million cells/cumm cells/cumm DECREASED COUNT:- Oligospermia:- due to COUNT:- Oligospermia:Suppression of endogenous gonadotropin Estrogen secreting tumor Hypo & hyper thyroidism Mumps leprosy Irradiation orchitis Hypopituitarism Varioceles Hypogonadism Azoospermia:Azoospermia:Ductal obstruction Klinefelter syndrome Sertoli cell only syndrome Post mumps INCREASED COUNT:- more than 300 million cells / cumm is known as COUNT:POLYSPERMIA results in infertility.

METHOD:METHOD:Mix the specimen. Draw semen upto 0.5 mark in WBC pipette. Draw semen diluting fluid upto 11 mark & mix well. SEMEN DILUTING FLUID:FLUID:NaHCo3= 5 gm Formalin neutral= 1 ml D/W = 100 ml Used to immobilize sperm and preserve them. Load it in improved Neubaeurs chamber & allow to settle for 5 min. Count the sperm in four end W square.

CALCULATION:CALCULATION:n= sperm counted Multiplication factor= 50000 Diluting factor= 20 Sperm/ml= n*50000 =n*10 million Total count=n*10 million* volume of ejaculation

ABNORMAL FORMS:- Normal: 0-20% FORMS:0More than 20% indicative of infertility. Small pin head sperm most encountered abnormal forms. 1. Abnormal shaped head:head:Too small, too large, double head, pointed head, ragged head. 2. Middle piece:piece:Absent , bifurcated, or swollen 3. Tail:Tail:Rudimentary, double, curved, or absent, more than one tail. 4. Vacuoles in chromatin

MORPHOLOGY:After liquefaction, make a thin smear of semen on a glass slide just as blood film is prepared. Let it air dry & heat gently for fixation. If necessary remove mucus by dipping the smear in semen diluting fluid. Staining could either be done by Leishman staining, Papanicolau stain or carbol fuschin staining method. We will go with Leishman staining & after completing observe under oil immersion objective.

OBSERVATION:OBSERVATION:Head= light blue Vacuoles= Dark blue Body & tail= pinkish or red red.

OTHER FINDINGS:FINDINGS:1. Pus cells:cells:Normal:- 0/hpf Normal:- 1-0/hpf Increased due to inflammation due to infection in some part of reproductive system. Also seen in infection of seminal vesicle 2. Epithelial cells:cells:Normal:- 0/hpf Normal:-1-0/hpf Always present in semen. They are never associated with presence of any pathology. 3. RBC:RBC:Not usually present in semen. Present due to manifestation of infection or any malignancy in genital tract. T.B of seminal vesicle, rupture of blood vesicle, infection of prostate, vitamin c deficiency. 4. Protozoa & Bacteria:- Presence of Trichomonas & other bacteria. Bacteria:-

MICROBILOGICAL EXAMINATION:EXAMINATION:It may be necessary to culture semen in order to exclude microbiological cause of infertility. Cultures should be aimed at isolation of Neisseria gonorrhoeae, gonorrhoeae, Chlamydia trachomatis, Ureaplasma spps. trachomatis, spps. gonorrhoeae:= 1. Neisseria gonorrhoeae:= Stained preparations:- Gram negative diplococci in neutrophols. preparations:neutrophols. Cultured on: (a) Modified Thayer-Martin {MTM} medium. Thayer(b) Modified New York city {MNYC} medium. 2. Chlamydia trachomatis:=trachomatis:=Detected by iodine preparation & immnoflouresecence. In laboratory can be cultured only on Egg or tissue culture.

OTHER ORGANISMS WHICH MAY BE FOUND ARE:= 1.Candida albicans 1. Candida 2. staphylococcus epidermidis 3. streptococcus viridans 4. E.coli 5. mumps virus 6. small pox virus 7. genital herpes

IMMUNOLOGICAL EXAMINATION:EXAMINATION:Usually done to detect antibodies against spermatozoa present in cervical mucus. IgG and IgA type of antibodies are found. IgA type is most significant. TEST DONE FOR IMMUNOLOGICAL EXAMINATION ARE:ARE:1. Franklin and Duke s test 2. mixed agglutination reaction 3. immunobead assay These tests are not usually performed in routine seminal fluid examination

MEDICO LEGAL ASPECTS:ASPECTS:Detection of semen is of MEDICOLEGAL IMPORTANCE in case of ALLEGED RAPE or SEXUAL ASSAULT . The following test are done from the obtained material ::1. 2. 3. 4. 5. 6. 7. Demonstration of sperm indirect smear . Florence test Barberio s test CPK (creatine phoshpokinase ) (creatine Acid phosphatase Precipitation test DNA detection

MODEL REPORT

BIBLIOGRAPHY:BIBLIOGRAPHY:1. Bhatacharya 2. Godkar 3. Ochei & Kohlatkar 4. Ramnik sood 5. Henry todd 6. Tortora 7. www.google.com ( for images)